JP2002350350A - Biochemical inspection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biochemical inspection method capable of investigating the state of a biochemical reaction in each probe array element exactly. SOLUTION: The inspection method comprises the steps of capturing an optical image as a reference one by illuminating a reference array containing luminescent molecules with an excitation light, capturing an optical image as a sample one by illuminating a biochemical inspection array with the same excitation light as the above light, and making a correction to the sample optical image with the reference one.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biochemical testing method for testing the status of a biochemical reaction.

[0002]

2. Description of the Related Art Recently, for "sequencing by hybridization" (SBH), arrayed hybridization (arrayed hybridization) has been proposed.
on) Several methods of reaction have been developed. This includes, for example, SBH using a method of stepwise hybridization between different oligonucleotide probes arranged in a grid on a membrane and an array of DNA samples.

[0003] Conventionally, a "geno sensor (genos)"
The term "sensor" has been used to describe a method in which an oligonucleotide as a recognition element for a target nucleic acid sequence and a complementary sequence is bound to the surface of a two-dimensional array. In addition, the concept of a genosensor includes a microfabrication device that can rapidly detect hybridization and has microelectronic components at each test site.

For such a two-dimensional array, a novel flow-through genosensor as described below has recently been provided. There, spots and tightly packed holes or channels are spread across the wafer of solid support material,
The nucleic acid recognition element is immobilized. Known microfabrication techniques can be used to produce micro- or nano-channel glass and porous silicon useful as a support wafer. The flow-through genosensor utilizes a variety of known detection methods, including microfabricated optical and electronics detection components, films, charged coupled element arrays, camera systems, and phosphorescence storage technology. This flow-through device offers the following advantages over known planar surface designs.

(1) The detection sensitivity is improved because the surface area is greatly increased.

(2) The time required for the hybridization reaction is reduced (the time required for the average target molecule to encounter the probe bound to the surface is reduced from several hours to several milliseconds), and the hybridization is speeded up. In addition, mispairing can be identified in both forward and reverse reactions.

(3) Since the solution can flow gradually through the porous wafer, a dilute nucleic acid solution can be analyzed.

(4) The rapid drying of small droplets of probe solution on planar surfaces exposed to the atmosphere promotes chemical bonding of the probe molecules to the surface in each separation region.

This flow-through device having the above advantages is hereinafter referred to as a three-dimensional array. As a prior art relating to such a three-dimensional array, Japanese Patent Application Laid-Open No. 9-504864 is cited, and its configuration and operation will be briefly described with reference to FIG.

FIG. 27 is a diagram showing a tapered sample well array forming a three-dimensional array. In FIG. 27, a porous glass wafer 101 has a plurality of tapered holes 10.
2, and a tapered well 103 is embedded in each of the holes 102. Well 103 with taper
Constitutes a binding region of a biomolecule immobilized thereon.
A channel 104 of 1-10 μm diameter is included at the bottom. Each channel 104 has a number of minute through holes 1 as shown.
05. Using this well array, detection is performed in the following steps.

(1) A solution of 4 ml of 3-glycidoxypropyl-trimethoxysilane, 12 ml of xylene, and 0.5 ml of N, N-diisopropylethylamine (Hunig base) is allowed to flow into the holes of the wafer. The wafer is immersed in the solution at 80 ° C. for 5 hours, then flushed with tetrahydrofuran and dried at 80 ° C. to make the wafer an epoxysilane-derivatized glass.

(2) A plurality of oligonucleotide probes having 5′- or 3′-alkylamine (introduced during chemical synthesis) are dissolved in water at 10 μM to 50 μM, each of which is made of a porous glass wafer 101. (Silica wafer). After overnight reaction at 65 ° C., the surface is briefly flushed with 65 ° C. water and then with 10 mM triethylamine to remove unreacted epoxy groups on the surface. The amine-derivatized oligonucleotide is then bound to the epoxysilane-derivatized glass by flushing again with 65 ° C. water and air drying.

(3) The amplification product is 5'-labeled by the polymerase chain reaction, which incorporates [ ³² P] nucleotides into the product during amplification, or using gamma- ³² P [ATP] + polynucleotide kinase. By
Prepare target DNA (analyte). Unincorporated label is removed by Centricon filtration. Preferably, if one PCR fragment is labeled with 5'-biotin, a single strand can be prepared by streptavidin affinity chromatography. A concentration of at least 5 nM (5 fmol / μl),
A hybridization buffer (50 mM Tris-HC) with a specific activity of at least 5,000 cpm / fmol
Dissolve the target DNA in 1, pH 8, 2 mM EDTA, 3.3 M tetramethylammonium chloride). PCR fragments of several hundred bases in length are suitable for hybridization with oligonucleotides tethered to a surface of at least octamer length.

(4) The target DNA sample is poured into the porous region of the chip and incubated at 6 ° C. for 5 to 15 minutes to perform hybridization. Then, at 18 ° C
The washing is performed by flowing the hybridization solution through the porous chip for the same time. Alternatively, instead of tetramethylammonium chloride, 1
Hybridization can also be performed with a buffer containing MKCL or NaCl or 5.2M betaine.

(5) The hybridization intensity is detected and quantified using a CCD genosensor device.
The CCD genosensor device has a high resolution and a high sensitivity, and is prepared for chemiluminescent, fluorescent or radioactive labeling.

[0016]

Conventionally, the same area sensor or line sensor such as a CCD is used as the luminous intensity of each biochemical reaction state in the probe array element without being limited to the above-mentioned prior art. , It is necessary to uniformly illuminate all the probe array elements. This is because the light emitting state of the light emitting molecule depends on the intensity of the excitation light.

Therefore, it is easily conceivable to monitor the intensity of the excitation light and correct the detection value based on the spatial distribution of the intensity. However, even in this case, the relationship between the intensity of the excitation light and the emission intensity is complicated, and the relationship changes depending on the density, temperature, time, etc. of the luminescent molecules, so that biochemical phenomena in the probe array element are caused. However, there is a disadvantage that it is not possible to accurately inspect each reaction state.

Further, noise due to excitation light noise and probe dispensing error is not sufficiently removed. Furthermore, since the luminescent properties of the luminescent molecules are not taken into account, there is a disadvantage that each reaction state cannot be accurately inspected.

An object of the present invention is to provide a biochemical inspection method capable of accurately inspecting each biochemical reaction state in a probe array element.

[0020]

Means for Solving the Problems In order to solve the problems and achieve the objects, the biochemical inspection method of the present invention is configured as follows.

(1) The biochemical inspection method of the present invention
A biochemical solution is supplied to a biochemical test array having on its surface a second biochemical substance that specifically reacts with the first biochemical substance, and excited by light. By detecting the emission intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light, the first biochemical substance and A biochemical inspection method for inspecting, for each probe array element, a reaction state with a second biochemical substance that specifically reacts with the first biochemical substance, wherein the luminescent molecule is retained. The excitation light is applied to the reference array to take a light image as a reference light image, and the same excitation light is applied to the biochemical test array to take a light image as a sample light image. Les It is corrected by Arensu light image.

(2) The biochemical test method of the present invention is the method described in (1) above, wherein the first biochemical substance is labeled with a plurality of different light-emitting molecules excited by light. Using a mixed solution of a plurality of samples as a solution of a sample to be tested, and using the reference array in which the plurality of different luminescent molecules are mixed and held at a fixed ratio, a probe in each probe array element and The reaction state with each of the samples is inspected.

(3) The biochemical inspection method of the present invention is the method according to the above (1) or (2), and the reference array has a structure in which luminescent molecules are held in a uniform distribution on a substrate. I have.

(4) The biochemical testing method of the present invention
A biochemical solution is supplied to a biochemical test array having on its surface a second biochemical substance that specifically reacts with the first biochemical substance, and excited by light. By detecting the emission intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light, the first biochemical substance and A biochemical inspection method for inspecting, for each probe array element, a reaction state with a second biochemical substance that specifically reacts with the first biochemical substance; The light image of the biochemical test array after supplying the solution of the substance is divided into a plurality of divided images by the probe array element unit, and a measurement region and a local background region are respectively defined for each of the divided images. Specify the local server Detecting the local background from click ground region, it corrects the detected amount from the measurement region in the detected amount.

(5) The biochemical inspection method of the present invention is the method according to any one of the above (1) to (3), and further comprises dividing an optical image into the probe array element units to obtain a plurality of divided images. Then, a measurement area and a local background area are designated for each of the divided images, a local background is detected from the local background area, and the detection amount from the measurement area is corrected by this detection amount.

(6) The biochemical test method of the present invention
Is supplied to an array for biochemical examination, on the surface of which are each held a second biochemical substance that specifically reacts with the first biochemical substance, The first biochemical substance and the first biochemical substance are detected by detecting the luminescence intensity of the luminescent molecule for each probe array element of the biochemical test array using a label according to A biochemical inspection method for inspecting a reaction state with a second biochemical substance specifically reacting with a chemical substance for each probe array element, wherein a non-linear coefficient is set as a parameter for determining a luminescence function. The number of luminescent molecules present in each probe array element is determined using the nonlinear coefficient.

(7) The biochemical examination method of the present invention
A biochemical solution is supplied to a biochemical test array having on its surface a second biochemical substance that specifically reacts with the first biochemical substance, and excited by light. By detecting the emission intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light, the first biochemical substance and A biochemical inspection method for inspecting, for each probe array element, a reaction state with a second biochemical substance that specifically reacts with the first biochemical substance; An abnormal area designated on the optical image of the array for biochemical inspection after supplying the solution of the substance is defined as a non-target area.

(8) The biochemical test method of the present invention
A biochemical solution is supplied to a biochemical test array having on its surface a second biochemical substance that specifically reacts with the first biochemical substance, and excited by light. By detecting the emission intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light, the first biochemical substance and A biochemical inspection method for inspecting, for each probe array element, a reaction state with a second biochemical substance that specifically reacts with the first biochemical substance, wherein the accumulation time is the same. An average image of a plurality of light images of the array type detector is set as a target image.

(9) The biochemical test method of the present invention
Is supplied to an array for biochemical examination, on the surface of which are each held a second biochemical substance that specifically reacts with the first biochemical substance, The first biochemical substance and the first biochemical substance are detected by detecting the luminescence intensity of the luminescent molecule for each probe array element of the biochemical test array using a label according to A biochemical inspection method for inspecting a reaction state with a second biochemical substance that specifically reacts with a chemical substance for each of the probe array elements, wherein a light image obtained by the array-type detector is On the other hand, a smoothing process is performed using an averaging operator.

(10) In the biochemical testing method of the present invention, the solution of the first biochemical substance is coated on the surface with the second biochemical substance that specifically reacts with the first biochemical substance. Is supplied to the array for each biochemical test held therein, and the intensity of the luminescent molecules is arrayed for each probe array element of the array for biochemical test using a label with a luminescent molecule that emits light by excitation with light. The first type is detected by the type detector.
A biochemical test method for testing a reaction state between a biochemical substance of the present invention and a second biochemical substance that specifically reacts with the first biochemical substance for each probe array element. The minimum detection amount in the target area of the light image obtained by the array-type detector is defined as the fluorescent background, and the light image obtained by subtracting the fluorescent background from the light image is defined as the target image.

(11) The biochemical inspection method of the present invention is the method described in (4) above, and the shape of the divided image is a parallelogram.

(12) The biochemical inspection method of the present invention is the method according to any one of the above (6) to (8) or (11), wherein the boundary between the measurement area and the local background area is The boundaries are the same.

(13) The biochemical inspection method of the present invention is the method described in (4) or (5) or (11), and is a binary image of a light image obtained by the array-type detector. Detecting the width of the dark region of the compressed image of the array row or array column that forms a gradient with respect to the x-axis or y-axis of the array, and calculating the gradient of the array row or the gradient of the array column when the dark region width is the largest. It is assumed that each of the divided images is a gradient of a row dividing line or a column dividing line.

(14) The biochemical inspection method of the present invention is the method described in (13) above, wherein the binary value is a median value of the output in the target area of the light image obtained by the array type detector. And the binarized image is obtained.

(15) The biochemical inspection method of the present invention is the method according to the above (13) or (14), wherein each of the dark areas or each of the bright areas of the compressed image of the array rows or arrays is read. An interval between the row dividing lines or an interval between the column dividing lines is obtained from a plurality of points formed at the center position.

(16) The biochemical inspection method of the present invention is the method according to the above (15), wherein each of the continuous bright regions is compared with the arrangement condition of the probe array element, and When there is an invalid area, the invalid area is replaced with a dark area based on the arrangement condition of the probe array elements, and then the interval between the row dividing lines or the column dividing lines is obtained.

(17) The biochemical inspection method of the present invention is the method according to the above (13) or (14), and between the row dividing lines or the column dividing lines depending on the arrangement condition of the probe array elements. Find the interval of.

(18) The biochemical examination method of the present invention is the method according to the above (13) or (14), wherein the center position of the position detecting array element provided in the biochemical examination array is determined. The initial position of the row dividing line or the column dividing line is determined based on the detected center position.

(19) The biochemical inspection method of the present invention is the method according to the above (4), (5) or (11), wherein the divided image is divided into two values by the median of the outputs in the divided image. A rectangle obtained by binarizing the rectangle circumscribing the binarized image by a predetermined amount is defined as a boundary of the local background area, and the outside of the boundary in the divided image is defined as the local background area.

(20) The biochemical inspection method of the present invention is the method according to the above (4), (5) or (11), and further comprises dividing the divided image by a median of outputs in the divided image. A rectangle obtained by binarizing the rectangle and contracting a rectangle inscribed in the binarized image by a certain amount is defined as a boundary of the measurement region, and the inside of the boundary is defined as the measurement region.

(21) The biochemical inspection method of the present invention is the method according to the above (4), (5) or (11), and further comprises the step of determining the local light intensity from the average luminous intensity per unit area of the measurement area. The intensity obtained by subtracting the average emission intensity per unit area of the ground region is detected as the reaction intensity with each probe.

(22) In the biochemical test method of the present invention, a solution of a test sample containing a first biochemical substance is supplied to a biochemical test array, and the solution is labeled with a luminescent molecule. The first biochemical substance and the first biochemical substance are specifically detected by detecting the luminescence intensity of the luminescent molecule for each probe array element of the array for biochemical test with an array-type detector. In the biochemical inspection method for inspecting a reaction state with a reacting second biochemical substance for each of the probe array elements, the luminescence intensity or the average luminescence per unit area corresponding to a standard area of the probe array element Detect intensity.

(23) The biochemical test method of the present invention is the method according to any one of the above (1) to (10) or (22), and the biochemical test array is porous or fiber. Supplying a probe solution to different positions on the surface of a three-dimensional reaction support composed of a quality substance or a molded product,
A solution of a test sample containing the first biochemical substance in the biochemical test array, which comprises holding a probe contained in the solution inside the porous or fibrous substance or molded article. Supply.

[0044]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a diagram showing a configuration of a microscope apparatus for performing a biochemical inspection method according to a first embodiment of the present invention. In FIG. 1, a mercury light source or the like is used as the excitation light source 11. On the optical path of the excitation light emitted from the light source 11, a shutter 12, a lens 13, an excitation filter unit 140 capable of selectively switching between two excitation filters 14, and a dichroic mirror 15 are arranged. On the 15 reflected light paths,
The objective lens 16 and the later-described biochemical test array 1 or reference array 4 are arranged. Further, a plurality of N light beams are provided on the transmitted light path of the dichroic mirror 15.
An ND filter unit 170 that can selectively switch the D filter 17, an imaging lens 18, and a CCD camera 19 are arranged. An image processing unit 20 is connected to the CCD camera 19, and a display unit 21 is connected to the image processing unit 20.

FIGS. 2A and 2B are diagrams showing a schematic configuration of an array for a biochemical test used in the first embodiment. FIG. 2A shows the format of the upper surface. Diagram,
FIG. 2B is a partial side view. The biochemical test array 1 is a three-dimensional array shown in FIG. As shown in FIG. 2A, probe array elements 3 are two-dimensionally arranged on the biochemical test array 1, and array elements 2 for position detection are arranged at four corners. These probe array element 3 and position detecting array element 2
Consists of a tapered well 103 as shown in FIG. 27 and a channel 104 of 0.1-10 μm diameter at the bottom.
Each channel 104 has many minute through-holes 105 as shown in FIG. 2B. The probe 31 is connected to the wall surface of each through hole 105.

That is, in the biochemical test array 1, a probe solution is preferably supplied to each of the tapered wells 103 on the surface of the porous glass wafer 101 in order to ensure the probe binding reaction. Is preferably held in each through hole 105 of each channel 104 provided inside the porous glass wafer 101, but the probe is directly provided in each through hole 105 without providing the porous glass wafer 101. A small amount of the solution may be dispensed and held. The member including the through-hole 105 is configured as a three-dimensional reaction carrier made of a porous or fibrous substance or a molded product.

FIG. 3 is a diagram showing the format of the upper surface of the reference array used in the first embodiment. The reference array 4 is a three-dimensional array capable of holding fluorescent molecules. As shown in FIG. 3, the fluorescent molecule holding elements 5 are two-dimensionally arranged in the reference array 4, and the position detecting array elements 2 are arranged at four corners, respectively.

As shown in FIGS. 2A and 3, the biochemical test array 1 and the reference array 4 have the same format. The four position detection array elements 2 located at the four corners are located at the same position in the biochemical examination array 1 and the reference array 4, and are placed on the stage (not shown) under the microscope. And the reference array 4 are used for positioning the two. Therefore, the position detecting array element 2 does not necessarily need to be at the position shown in FIGS. 2A and 3, and may be at any position as long as they are at the same position. Of course, the format itself of the biochemical test array 1 and the reference array 4 may be any format as long as these conditions are satisfied.

The outline of the biochemical test according to the first embodiment will be described below.

[0051] The first inspector, a fluorescent molecule the same n _RM pieces of fluorescent molecules each fluorescent molecule retaining element 5 to be used as a label to a substrate for the array, each position detecting fluorescent molecules labeled with different emission wavelengths The reference array 4 is manufactured by spotting on the array element 2 for use. Where R
Is a suffix indicating a reference, and M is a suffix corresponding to a fluorescent substance.

Next, excitation illumination is applied to the reference array 4 positioned and arranged under microscopic observation, and a fluorescent image is taken by the CCD camera 19 to be a reference fluorescent image. The reference fluorescence image is captured by the number of types of the fluorescent substance used for the label. In order to prevent the deterioration of the fluorescent substance, the shutter
It is preferable to shut off the excitation illumination for example.

Next, the examiner checks the reference array 4
Is discharged, and the biochemical test array 1 is arranged at the same position. In the biochemical test array 1, fluorescent molecules having an emission wavelength different from that of the label are previously immobilized on the array element 2 for position detection of the array substrate, and the corresponding probe solution is immobilized on the probe array element 3 in advance.

Next, the examiner supplies a solution of the test sample labeled with a fluorescent molecule to the biochemical test array 1 to cause a binding reaction between each probe and a substance in the sample solution. The unbound material is removed from the probe array element 3.

Next, excitation light is applied to the biochemical test array 1 and a fluorescent image is taken by the CCD camera 19 to be a sample fluorescent image. The sample fluorescence image is captured in the same number as the number of types of the fluorescent substance used for the label similarly to the reference fluorescence image. In order to prevent deterioration of the fluorescent substance, it is preferable to shut off the excitation illumination by the shutter 12 or the like immediately after the image is captured.

Next, the reference fluorescence image and the sample fluorescence image are each divided into element units, and a plurality of divided images (the divided images are obtained by multiplying the number of types of fluorescent substances used for labels × the number of probe array elements × 2) Exists).
The division condition at this time is determined based on the coordinates of the center of gravity of the four position detection array elements 2 or the coordinates of the centroid of the binarized image, and a known array format.

By the way, when an image is formed on a light receiving element having a γ value of 1 with a stigmatic lens, a binarization point at a level of 最大 of the maximum signal is considered as an intersection between the principal ray and the optimum image plane. good. Therefore, all the divided images are binarized at a level of １ ／ of the maximum signal to set an area of the binarized image, and the maximum x coordinate, minimum x coordinate, maximum y coordinate and minimum y coordinate of this area are set. Find coordinates.

For the reference binarized image, the maximum
x coordinate_RMkmaxAnd the minimum x coordinate is x _RMkmin, Maximum y coordinate
To y_RMkmaxAnd the minimum y coordinate is y_RMkminAnd sample
For a binarized image, the maximum x coordinate is x_SMkmax, Minimum x
Coordinate x_SMkmin, The maximum y coordinate is y_SMkmaxAnd minimum y
Mark y_SMkminAnd At the same time, the sample fluorescence image
Calculate the actual area of the binarized image only for the divided image
And this is S_MkAnd

FIG. 4 shows the reference fluorescence image and
FIG. 5 is a view showing a divided image of the sample fluorescence image, and FIG.
Is the output from the CCD on the line segment AB shown in FIG.
It is a figure showing a signal. Binarized area 41 and the maximum of this area
x coordinate x_max, Minimum x coordinate x _min, Maximum y coordinate y_maxYou
And the minimum y coordinate y_minIs shown in FIGS. 4 and 5.
(Note that the subscripts RMk and SMk are indistinguishable in the figure.
Omitted because it is important).

Next, two coordinates giving a rectangular area for determining the measurement area 42 and the noise sampling area 43 are represented by (x ′ _min , y ′) as shown in FIGS.
_min ) and (x ' _max , y' _max )
_min , y ′ _min , x ′ _max and y ′ _max are given by the following equations.

[0061]

(Equation 1) Here, β is the image magnification of the optical system, and δ is given by the following equation.

[0062]

(Equation 2)

Here, λ is the fluorescence wavelength of the label M to be detected,
a is the numerical aperture on the sample side, and Δ is the defocus amount. The first term on the right side represents the distance from the center of the diffraction image to the valley between the second and third order diffraction peaks (blur amount due to diffraction), and the second term is the blur caused by the focusing error. Indicates the amount (the amount of blurring due to defocus). That is, equation (5) may be considered as an equation that gives the maximum blur amount when the focusing error is considered.

As shown in FIG. 5, the divided image is formed of a composite signal of the signal 51 to be detected and the noise signal 52. Although the noise depends on the device configuration and the measurement environment, generally, the excitation light noise (noise caused by the excitation light) is dominant, and sometimes stray light noise (noise caused by a light source other than the excitation light) cannot be ignored. . Further, the excitation light noise includes reflection light noise of the excitation light on the substrate, self-emission noise of the substrate due to the excitation light, and the like.

FIG. 6 is a diagram showing a signal obtained by subtracting the aforementioned noise signal from the output signal. By performing the subtraction processing, the signal 61 to be detected can be extracted. Although FIGS. 4 to 6 are modeled and represented for easy explanation, actually, the signal to be detected and the noise signal are not flat, and are uneven depending on the position.
In particular, the excitation light noise mentioned above reflects uneven illumination,
This is the biggest cause of noise signal unevenness.

In the present embodiment, even when the binarized area is not circular as shown in FIG. 4 due to the unevenness of the signal to be detected, or when a plurality of binarized areas exist in one divided image, x ′ _min , y ′ _min ) and (x ′ _{min
By using a} rectangular area defined by _max , _y'max ) as the measurement area, it is guaranteed that all signals to be detected are included in this measurement area.

Therefore, in the present embodiment, the average noise signal in the noise sampling area 43 is subtracted from the signal in the measurement area 42 on the assumption that the noise signal is uneven.
The detection signal shown in FIG. 6 is obtained. The average noise signal is obtained by dividing the sum of the signals in the noise sampling area by the area of the noise sampling area. Next, the sum of the subtracted detection signals in the measurement area 42 is defined as the signal strength of each array element.

According to such a method, since the noise signal can be sampled near the signal to be detected, the detection accuracy of the signal to be detected is improved. In addition, since the measurement area is set in consideration of the defocus as described above, it is apparent that defocus noise does not occur.

As described above, the signal intensity of the number of types of fluorescent substance used for labeling × the number of probe array elements × 2 is obtained. Here, the signal intensities of the probe array element 3 and the fluorescent molecule holding element 5 are represented by P _RMk and P _SMk , respectively. Here, R and S are suffixes indicating a reference and a sample, respectively, M is a suffix corresponding to a fluorescent substance, k
Is the number of the fluorescent molecule holding element 5 and the number of the corresponding probe array element 3 at the same time.

Next, the signal intensity P of the probe array element 3
_SMk is corrected by the area of the probe array element 3 to obtain P _Mk . P _Mk is obtained by the following equation.

[0071]

(Equation 3)

Here, S _O is the standard area of the probe array element 3, and S _Mk is each probe array element 3 previously calculated.
Is the actual area of the binarized image. By this correction, noise due to a probe dispensing error can be removed.

Next, the intensity signal P _{Mk of} each probe array element 3 of the sample fluorescence image is represented by the number n _Mk of fluorescent molecules (or chemiluminescent molecules) of the label M present in the probe array element 3 as follows. become.

[0074]

(Equation 4)

Here, ρ is a non-linear coefficient of the emission function of the fluorescence and is given by a constant. I _Mk can be expressed by the following equation.

[0076]

(Equation 5)

Here, n _RM is the number of fluorescent molecules (or chemiluminescent molecules) of the label M existing in the fluorescent molecule holding element 5 of number k of the reference array 4.

Therefore, I _Mk can be obtained by the equation (8), and the number n _Mk of fluorescent molecules (or chemiluminescent molecules) of the label M existing in the probe array element 3 can be obtained by using the equation (7). That is, for each probe array element 3, equations are created by the number of detection values of the signal intensity P _Mk of the number of labeling substances, and by solving these equations, fluorescent molecules of the label M present in the probe array element 3 ( Or chemiluminescent molecule) number n _Mk is determined. From equations (7) and (8),

(Equation 6) Becomes

If the nonlinear coefficient ρ can be regarded as 0 in the equation (8), I _Mk is a fluorescent molecule (or a chemiluminescent molecule).
This is the signal strength per signal. At this time, equation (9) becomes a very simple equation, and n _Mk is obtained by the following equation.

[0080]

(Equation 7)

Next, a method of obtaining n _{Mk by} the successive approximation method when the nonlinear coefficient ρ cannot be regarded as 0 will be described.
In equation (9), the approximate value of the i-th n _Mk is

(Equation 8)

, And n _Mk on the right-hand side for which this is obtained is i−
The first approximation of n _Mk is used. This is expressed by the following equation.

[0083]

(Equation 9) Here, i = 1, 2, 3,.... Also, as the initial value of n _Mk

[0084]

(Equation 10) , And the following equation may be given as a convergence condition.

[0085]

[Equation 11]

That is, when equation (13) is satisfied,

(Equation 12) _Is determined as n _Mk .

FIGS. 7 and 8 are flowcharts showing the inspection procedure according to the first embodiment. Hereinafter, the inspection method and its operation will be described with reference to FIGS.

(1) Step S1: First, the examiner prepares a solution of two kinds of biochemical substances labeled with fluorescent molecules of two colors. In this case, the examiner prepares solutions of the two biochemical substances to be compared at the same concentration, and labels one with FITC and the other with rhodamine. These labeling substances may be other substances as long as they are combinations of substances having different fluorescence wavelengths. Then, the prepared solutions of the two biochemical substances were mixed in the following manner:
Mix at a ratio of 1 and stir to obtain a mixed solution of biochemical substances. The mixing ratio may be changed depending on the solution of the two biochemical substances and the properties of the labeling substance.

(2) Step S2: The inspector manufactures a reference array 4 having fluorescent molecules of two colors. In this case the examiner, the respective FITC and rhodamine by the same fluorescent molecules number n _RM fluorescent molecules retaining element 5 used as a fluorescent label substrate for the array, labeled different emission respectively for position detection array fluorescent molecules wavelength The reference array 4 is manufactured by spotting on the element 2. At this time, the spotting position is a position corresponding to the probe array element 3. Here, R is a suffix indicating a reference, and M is a suffix corresponding to a fluorescent substance.

In the spotting method, the number of fluorescent molecules to be used is specified from the weight and the molecular weight of the fluorescent molecules to be used, a solution of the fluorescent molecules having the number of fluorescent molecules is prepared so as to have a predetermined amount, and this solution is prepared. The ink is discharged onto an array substrate by an ink jet method. The number of fluorescent molecules n _RM per element can be calculated from these various amounts (the amount of the solution and the number of fluorescent molecules in the solution) and the discharge amount of the solution. When a solid phase reagent is used as a method for holding a fluorescent molecule, reliable holding is possible, but this is not always necessary.

(3) Step S3: The examiner takes the fluorescent image of the reference array 4 with the CCD camera 19 for each color while switching the ND filter 17 on the observation optical path a. In this case, the reference array 4 is excited and illuminated by the excitation light source 11, and the image of the fluorescence generated by the fluorescent molecules in each fluorescent molecule holding element 5 is captured by the CCD camera 19. At this time, the examiner switches the excitation filter unit 140 to operate the excitation filter 1 corresponding to the color of the desired fluorescent molecule.
The ND filter 4 is arranged on the illumination optical path b, and the ND filter unit 170 is further switched to arrange the desired ND filter 17 on the observation optical path a.

The excitation light from the excitation light source 11 is
3. Dichroic mirror 1 via excitation filter 14
The light is reflected by 5 and irradiates the entire upper surface of the reference array 4 via the objective lens 16. Reference array 4
The fluorescent light generated from the object lens 16, the dichroic mirror 15, the ND filter 17, and the imaging lens 18
Through the CCD camera 19, and a fluorescent image is captured for each color of fluorescent molecules. Therefore, at this stage, 2 colors x
As many fluorescent images as the number of ND filters can be obtained. The fluorescent image captured by the CCD camera 19 is sent to the image processing unit 20.

In this embodiment, the light receiving element is C
We use a CD, but we do not specify a CCD,
Other area sensors may be used. Therefore, the "CCD" described below is also valid for the "area sensor".

(4) Step S4: The examiner switches the ND filter 17 on the observation optical path a and converts the background image of the reference array 4 into CCs for each color of fluorescent molecules.
Take a picture with the D camera 19. In this case, the excitation illumination is shut off by the shutter 12 immediately after the fluorescence image of the reference array 4 is captured, and the background image is captured by the CCD camera 19. The number of the background images is the same as the number of the fluorescent images. This background image is composed of dark current noise and stray light noise unnecessary for the detection intensity. The background image captured by the CCD camera 19 is sent to the image processing unit 20.

(5) Step S5: The examiner supplies a mixed solution of biochemical substances to the biochemical test array 1 and causes it to react specifically with each probe. In this case, the inspector arranges the biochemical test array 1 shown in FIG. 2A on the sample surface under the observation of the fluorescence microscope shown in FIG. 1, and uniformly places the biochemical substance on the surface. Supply the mixed solution. As a result, a specific binding reaction occurs between the probes in the probe array elements 3 on the biochemical test array 1 and the biochemical substances contained in the mixed solution. As a result, each probe array element 3
Within, the number of fluorescent molecules corresponding to the strength of the reaction will be indirectly bound to the probe.

(6) Step S6: The examiner removes unreacted biochemical substances from the biochemical test array 1. In this case, the inspector removes unbound biochemical substances from each probe array element 3 of the biochemical test array 1 after the above-described binding reaction. Generally, a method of washing using a washing solution is employed, but when the reaction carrier has a three-dimensional structure, the solution may be removed with a pump or the like without using the washing solution. However, it is needless to say that the use of the cleaning liquid ensures removal.

(7) Step S7: The examiner takes the fluorescent image of the biochemical test array 1 with the CCD camera 19 for each color while switching the ND filter 17 on the observation optical path a. In this case, the biochemical test array 1 is excited and illuminated by the excitation light source 11, and an image of the fluorescence generated by the fluorescent molecules in each probe array element 3 is captured by the CCD camera 19. At this time, the examiner switches the excitation filter unit 140 to operate the excitation filter 1 corresponding to the color of the desired fluorescent molecule.
The ND filter 4 is arranged on the illumination optical path b, and the ND filter unit 170 is further switched to arrange the desired ND filter 17 on the observation optical path a.

The excitation light from the excitation light source 11 is
3. Dichroic mirror 1 via excitation filter 14
The light is reflected at 5 and irradiates the entire upper surface of the biochemical test array 1 via the objective lens 16. Array for biochemical testing 1
The fluorescent light generated from the object lens 16, the dichroic mirror 15, the ND filter 17, and the imaging lens 18
Through the CCD camera 19, and a fluorescent image is captured for each color of fluorescent molecules. This biochemical test array 1
Are the same as the number of background images and the number of fluorescent images of the reference array 4. The fluorescent image captured by the CCD camera 19 is sent to the image processing unit 20.

(8) Step S8: The examiner switches the ND filter 17 on the observation optical path a while changing the background image of the biochemical examination array 1 for each color of the fluorescent molecule.
Take a picture with the D camera 19. In this case, the excitation illumination is shut off by the shutter 12 and the background image is captured by the CCD camera 19 immediately after capturing the fluorescence image of the biochemical test array 1 described above. The number of background images in the biochemical test array 1 is the same as the number of fluorescent images. This background image is composed of dark current noise and stray light noise unnecessary for detection intensity, similarly to the background image of the reference array 4.
The background image captured by the CCD camera 19 is sent to the image processing unit 20.

(9) Step S9: The image processing section 20 subtracts each background image from the reference and each fluorescent image of the sample to obtain a corrected image for each color and each ND filter. Generally, the output of the light receiving element includes DC noise such as dark current noise and stray light noise. In order to remove this DC noise, the background image is subtracted from the fluorescence image for each of the reference array 4 and the biochemical test array 1, and this is referred to as a corrected image and is subjected to subsequent processing. Accordingly, at this stage, corrected images of the number of 2 colors × ND filters are obtained. Note that the fluorescence image and the background image are no longer necessary.

(10) Step S10: The image processing unit 20
The center of gravity of the image corresponding to the position detection array element 2 in each corrected image is detected. As shown in FIGS. 2A and 3, array elements formed in the biochemical test array 1 and the reference array 4 are two-dimensionally arranged, and four array elements are located at four corners. Are used as array elements 2 for position detection. Other array elements are used as the probe array element 3 or the fluorescent molecule holding element 5.

Since the position detecting array element 2 is for outputting a position signal, any substance in the element may be used as long as it is a luminescent substance or a reflective substance. It is better to use a fluorescent substance having a fluorescent wavelength different from the fluorescent wavelength. The reason is that the possibility that the fluorescence generated from the position detection array element 2 acts as noise can be eliminated. In this case, illumination is performed at the excitation wavelength for position detection, and the position is detected.

(11) Step S11: The image processing unit 20
Each corrected image is rotated and moved to be a rotated image for each color and each ND filter. In this case, the position coordinates are corrected such that each quadrilateral formed by the four position coordinates obtained for the reference array 4 and the biochemical test array 1 is a rectangle with the minimum deviation, and Each corrected image is rotationally moved around the center of each image so that each side is parallel to the coordinate axis. Each of the images after the movement is called a rotated image. Note that each corrected image is no longer required.

(12) Step S12: The image processing unit 20
In this case, the rotated image is divided into images for each of the fluorescent molecule holding element 5 and the probe array element 3 and divided into images for each color, each ND filter, and each element. A division condition is determined from the four position coordinates of each rotation image of the biochemical test array 1 and the arrangement conditions of the fluorescent molecule holding element 5 and the probe array element 3, and all the rotation images are subjected to fluorescence under this division condition. The image is divided into images for each of the molecule holding elements 5 or the probe array elements 3 to be divided images. Note that the divided image of the rotated image of the reference array 4 is referred to as a reference divided image,
The divided image of the rotated image of the biochemical test array 1 is called a sample divided image. Therefore, at this stage, divided images corresponding to 2 × the number of ND filters × the number of probe array elements are obtained. Note that each rotated image is no longer required.

(13) Step S13: The image processing section 20
Each divided image corresponding to the optimum ND filter 17 is extracted as a measurement image for each color and each element.

In this case, the image processing unit 20 determines that the maximum signal intensity is within the dynamic range from the reference divided images of the number of ND filters of the fluorescent molecule holding element 5 of the same color and at the same position. One of the reference divided images having the highest signal strength is extracted. This is called a measurement reference image. Similarly, the image processing unit 20
Is a sample divided image in which the maximum signal strength is within the dynamic range and the signal intensity is the maximum among the sample divided images of the number of ND filters of the probe array element 3 at the same position in the same color. Extract one. This is called a measurement sample image.

(14) Step S14: The image processing section 20
Each of the extracted measurement images is binarized to obtain a binarized image for each color and each element. For each of the measurement reference image and the measurement sample image, 1 /
Binarization is performed at two levels, which are referred to as a reference binary image and a sample binary image, respectively. For the sample binarized image, the area S _Mk of the binarized region 41 is calculated.

(15) Step S15: The image processing unit 20
The measurement area 42 and the noise sampling area 43 are set by each binarized image.

In this case, the image processing unit 20 sets the maximum x-coordinate x for each of the reference binarized images.
_RMkmax , minimum x-coordinate _xRMkmin , maximum y-coordinate _yRMkmax, and minimum y-coordinate _yRMkmin are obtained. Similarly, for all of the sample binarized images, a maximum x coordinate x _SMkmax , a minimum x coordinate x _SMkmin , a maximum y coordinate y _SMkmax and a minimum y coordinate y _SMkmin are obtained, and then a noise sampling area is set. Coordinates x ' _{RM kmax} , x' _RMkmin , y '
_{RMkmax, y 'RMkmin, x'} SMkmax, x 'SMkmin, y' SM
Calculate _kmax and _y'SMkmin .

A rectangle defined by the point (x ′ _RMkmin , y ′ _RMkmin ) and the point (x ′ _RMkmax , y ′ _SMkmax ) is set as a measurement area of the measurement reference image, and the outside of the rectangle is the noise of the measurement reference image. This is a sampling area.
Similarly, the point (x ′ _SMkmin , y ′ _SMkmin ) and the point (x ′
_The inside of the rectangle determined by _SMkmax , _y'SMkmax ) is the measurement region of the measurement sample image, and the outside of the rectangle is the noise sampling region of the measurement sample image.

(16) Step S16: The image processing section 20
The average noise signal is subtracted from the signal in the measurement area 42 on the measurement image.

In this case, the image processing section 20 obtains noise per unit area from the signal of the noise sampling area 43 of the reference image for measurement, and subtracts this signal from the signal of the measurement area 42 of the reference image for measurement to obtain the noise of the reference image. This is a detection signal. Similarly, noise per unit area is obtained from the signal of the noise sampling area 43 of the measurement sample image, and this signal is subtracted from the signal of the measurement area 42 of the measurement sample image to obtain a detection signal of the sample image.

(17) Step S17: The image processing section 20
The signal intensity per standard area of the probe array element 3 is calculated for each color.

In this case, the image processing section 20 calculates the sum of the detection signals in the measurement area 42 of each fluorescent molecule holding element 5 as P
_Calculated as _RMk . Similarly, each probe array element 3
_Is calculated as P _SMk . Next, the standard area S _O of the probe array element 3
The signal intensity per hit is calculated for each color using the equation (6).
These signal intensities are obtained as intensities from which excitation light unevenness, excitation light noise, and intensity errors caused by probe dispensing errors have been removed.

(18) Step S18: The image processing unit 20
The number of fluorescent molecules (or chemiluminescent molecules) existing per standard area of the probe array element 3 is calculated for each color. In this case, the image processor 20 from the signal intensity per standard area S _O of the probe array elements 3, (11), (12) and (13) the standard area S _O per probe array element 3 using equation _Is calculated for each color.

(19) Step S19: The image processing section 20
A scatter diagram is displayed with the number of fluorescent molecules (or chemiluminescent molecules) calculated for each color as both axes. In this case, the image processing unit 20
Shows a scatter diagram in which the number of fluorescent molecules of each of the probe array elements 3 of the two-color fluorescent substances is set on the x-axis and the y-axis, respectively, in the display unit 2.
1 is displayed.

FIG. 9 shows an example of the scatter diagram, in which the x-axis is the number of fluorescent molecules of sample A and the y-axis is the number of fluorescent molecules of sample B on a logarithmic memory. FIG. 9 schematically shows each probe of the m probe array elements 3, and the balance of the number of fluorescent molecules of the sample A and the sample B is within the range 91 (between the two broken lines) except for the "probe 3". is there. In this case, the binding reaction of Sample A and Sample B with the probe was determined to be substantially the same,
For, it is determined that the reaction of sample B is clearly stronger than the reaction of sample A. Incidentally, the x-axis and y-axis are both logarithmic axes, two broken lines determining the range 91, y = m _n x and _{y = (1 / m n)} x, solid line y
= X (three straight lines are mathematically parallel). When setting a range of ± 10% with respect to y = x, _mn = 1.1, and this value can be appropriately set and displayed by the image processing unit 20. Such a scatter diagram can accurately represent the reaction state between the probe of each probe array element and the sample.

According to the first embodiment, since the reference fluorescence image is introduced, the output can be properly corrected even when the excitation illumination has spatial unevenness. Furthermore, since the method takes into account the emission characteristics of the fluorescent molecules used and their temporal variations, the number of fluorescent molecules n _Mk of the label M present for each probe array element, ie, the reaction state between the probe and the target substance, can be accurately determined. Can be sought. In this case, the label is not limited to a fluorescent molecule, but may be any luminescent molecule such as a chemiluminescent molecule. The capture of the reference fluorescence image and the capture of the sample fluorescence image
There is no problem even if the order is reversed.

FIG. 10 is a diagram showing the format of the upper surface of the array for biochemical examination used in the second embodiment. The biochemical test array 6 shown in FIG. 10 has a configuration in which the four position detection array elements 2 in the biochemical test array 1 shown in FIG. ing.

FIG. 11 is a diagram showing the format of the upper surface of the reference array used in the second embodiment. The reference array 7 shown in FIG. 11 is composed of a three-dimensional array capable of holding fluorescent molecules, and is provided with a fluorescent molecule holding region 8 on one surface. The fluorescent molecule holding region 8 is set as a region wider than the region occupied by all the probe array elements 3 shown in FIG. The biochemical test array 6 and the reference array 7 are used in place of the biochemical test array 1 and the reference array 4 shown in the first embodiment.

Hereinafter, the outline of the biochemical test according to the second embodiment will be described.

First, the inspector supplies the same fluorescent molecule as the label to the array substrate with a uniform distribution to the fluorescent molecule holding region 8 to manufacture the reference array 7.

Next, excitation illumination is applied to the reference array 7 positioned and arranged under microscope observation, and a fluorescent image is captured by the CCD camera 19 to be a reference fluorescent image. The reference fluorescence image is captured by the number of types of the fluorescent substance used for the label. In order to prevent the deterioration of the fluorescent substance, the shutter
It is preferable to shut off the excitation illumination for example.

Next, the inspector checks the reference array 7
Is discharged, and the biochemical test array 6 is arranged at the same position. In the biochemical test array 6, the probe solution is previously immobilized on the probe array elements 3 respectively.

Next, the examiner supplies a solution of the test sample labeled with a fluorescent molecule to the biochemical test array 6 to cause a binding reaction between each probe and a substance in the sample solution. The unbound material is removed from the probe array element 3.

Next, excitation light is applied to the biochemical test array 6, and a fluorescent image is taken by the CCD camera 19 to be a sample fluorescent image. The sample fluorescence image is captured in the same number as the number of types of the fluorescent substance used for the label similarly to the reference fluorescence image. In order to prevent deterioration of the fluorescent substance, it is preferable to shut off the excitation illumination with a shutter or the like immediately after the image is captured. The signal intensity of each probe array element 3 of the sample fluorescence image is represented by P _Mk .
Here, M is a subscript corresponding to the fluorescent substance, and k is the number of the probe array element.

Next, the image processing section 20 detects the position and the actual area S _Mk of each probe array element 3 by image processing, calculates P _Mk using equation (6), and calculates the position from the reference fluorescence image. The signal strength corresponding to the standard area S _O of the probe array element 3 at the same position as is extracted from the probe array element 3, and this signal strength is represented by P _RMk . Here, R is a subscript meaning a reference.

Next, if the signal intensity P _{Mk of} each probe array element 3 of the sample fluorescence image is expressed by the number n _Mk of fluorescent molecules of the label M present in the probe array element 3, the same equation (7) as described above is obtained. Become.

The subsequent processing is the same as in the first embodiment. However, unlike the first embodiment, the _nRM is unknown in the second embodiment, and therefore, generally only the composition ratio of the fluorescent substance is obtained. However, if I _Mk is obtained by previously calibrating the reference array 7 with the reference array 4 used in the first embodiment, the absolute quantity of the fluorescent substance can be obtained.

In the second embodiment, it is preferable to use a reference substrate having no autofluorescence action.
This is because the reference array 7 cannot secure a sampling area for removing autofluorescence noise. However, the same substrate as reference array 7 and as second reference array, a region corresponding to each probe array element in the detecting the self luminous intensity as a sampling region, the signal strength P _Mk this
, The auto-fluorescence noise can be removed even for a reference substrate having an auto-fluorescence effect. In this case, there is an advantage that other noises can be removed at the same time. An inherent effect of the second embodiment is that the reference array can be easily manufactured. Note that, as in the first embodiment, there is no problem in taking the reference fluorescence image and taking the sample fluorescence image even if the order is reversed.

According to the present invention, the following operations are provided.

(1) According to the biochemical inspection method of the present invention, by correcting the sample light image with the reference fluorescence image, even if the illumination is not uniform, the probe and the biochemical substance in each probe array element can be used. The reaction state with the can be accurately inspected.

(2) When a reaction carrier having a three-dimensional structure is used,
Although the reaction system tends to be inaccurate due to its complexity, according to the biochemical inspection method of the present invention, since the actual comparison can be made, the effect of the above (1) increases.

(3) According to the biochemical inspection method of the present invention, in addition to the effects of the above (1), even if there is a difference in the state of the probes held in each probe array element, the reference sample and the From the comparison of the reaction state of the test sample, the reaction state between the probe and the test sample for each probe array element can be accurately inspected.

(4) According to the biochemical inspection method of the present invention, it is possible to accurately inspect the reaction state between the probe and the test sample for each probe array element.

(5) According to the biochemical inspection method of the present invention, a reference array can be easily manufactured.

(6) According to the biochemical inspection method of the present invention, the excitation light noise in the vicinity of the probe array element can be removed, so that the reaction state in each probe array element can be inspected accurately. it can.

(7) According to the biochemical inspection method of the present invention, when a reaction carrier having a three-dimensional structure is used, since the optical characteristics on the surface are complicated, noise near the probe array element can be corrected. The effect of the above (6) increases.

(8) According to the biochemical inspection method of the present invention, in addition to the effect of the above (6), even if there is a difference in the state of the probes held in each probe array element, the reference sample and the From the comparison of the reaction state of the test sample, the reaction state between the probe and the test sample for each probe array element can be accurately inspected.

(9) According to the biochemical inspection method of the present invention, the excitation light noise near the array element can be removed, so that the reaction state of each array element can be inspected accurately.

(10) According to the biochemical inspection method of the present invention, when a reaction carrier having a three-dimensional structure is used, the amount of reaction is proportional to the area of the probe array element. Note that errors will be removed. Therefore, it is possible to accurately compare the reaction state between the probe and the test sample for each probe array element.

(11) According to the biochemical examination method of the present invention, in addition to the effect of the above (10), even if there is a difference in the state of the probes held in each probe array element, the reference sample and the From the comparison of the reaction state of the test sample, it is possible to accurately compare the reaction state of the probe and the test sample for each probe array element.

(12) According to the biochemical inspection method of the present invention, for example, when a reaction carrier having a three-dimensional structure is used, the amount of reaction is proportional to the area of the probe array element. This will eliminate dispensing errors. Therefore, it is possible to accurately compare the reaction state between the probe and the test sample for each probe array element.

(13) According to the biochemical examination method of the present invention, the number of fluorescent molecules in the probe array element is generally not proportional to the total luminescence intensity. The coefficient is given by the binding formula of the number of fluorescent molecules. The nonlinear coefficient is a constant determined by the intermolecular distance, the structure of the probe array, and the reaction conditions. Then, the number of luminescent molecules present in each probe array element can be accurately obtained by introducing a nonlinear coefficient according to the system to be adopted.

(14) According to the biochemical inspection method of the present invention, when a reaction carrier having a three-dimensional structure is used, the reaction system becomes complicated. Therefore, the above-mentioned (13) can be obtained by using the nonlinear coefficient corresponding to the system. The effect of the present invention is increased.

(15) According to the biochemical test method of the present invention, in addition to the effect of the above (13), even if there is a difference in the state of the probes held in each probe array element, the reference sample and the From the comparison of the reaction state of the test sample, the reaction state between the probe and the test sample for each probe array element can be accurately inspected.

(16) According to the biochemical testing method of the present invention, the number of luminescent molecules in the probe array element is generally not proportional to the total luminescence intensity. The coefficient and the number of luminescent molecules are given by a binding equation. The nonlinear coefficient is a constant determined by the intermolecular distance, the structure of the probe array, and the reaction conditions. Then, the number of fluorescent molecules present in each probe array element can be accurately obtained by introducing a nonlinear coefficient suitable for the system to be adopted.

(17) According to the biochemical inspection method of the present invention, the number of luminescent molecules in the probe array element is generally not proportional to the total luminescence intensity. The coefficient and the number of luminescent molecules are given by a binding equation. The nonlinear coefficient is a constant determined by the intermolecular distance, the structure of the probe array, and the reaction conditions. Then, the number of fluorescent molecules present in each probe array element can be accurately obtained by introducing a nonlinear coefficient suitable for the system to be adopted.

(18) According to the biochemical testing method of the present invention, the number of luminescent molecules in the probe array element is generally not proportional to the total luminescence intensity. The coefficient and the number of luminescent molecules are given by a binding equation. The nonlinear coefficient is a constant determined by the intermolecular distance, the structure of the probe array, and the reaction conditions. Then, the number of fluorescent molecules present in each probe array element can be accurately obtained by introducing a nonlinear coefficient suitable for the system to be adopted.

(19) According to the biochemical inspection method of the present invention, the number of luminescent molecules for each color existing in each probe array element can be obtained.

(20) According to the biochemical inspection method of the present invention, the number of luminescent molecules in the probe array element is generally not proportional to the total luminescence intensity. The coefficient and the number of luminescent molecules are given by a binding equation. The nonlinear coefficient is a constant determined by the intermolecular distance, the structure of the probe array, and the reaction conditions. Then, the number of fluorescent molecules present in each probe array element can be accurately obtained by introducing a nonlinear coefficient suitable for the system to be adopted.

FIG. 12 is a diagram showing a configuration of a microscope apparatus for performing the biochemical inspection method according to the third embodiment of the present invention. 12, the same parts as those in FIG. 1 are denoted by the same reference numerals.

In FIG. 12, a system control unit 22, a file processing unit 23, and an input unit 24 are connected to the image processing unit 20. The storage unit 25 is connected to the file processing unit 23. The input unit 24 includes a mouse and various buttons. Note that the excitation filter 14, the dichroic mirror 15, and the ND filter 17 form a cube unit. Depending on the combination of the excitation filter 14, the dichroic mirror 15, and the ND filter 17 on the observation optical path a and the illumination optical path b, a foreign substance detection cube, a reference cube, and a fluorescent M cube are configured.

FIGS. 13 to 21 are flowcharts showing an inspection procedure according to the third embodiment. Hereinafter, FIG.
The inspection method and its operation will be described with reference to FIGS.

FIG. 13 is a main flowchart showing the procedure for measuring the fluorescent luminance. In step S21, the display unit 2
1 displays an image of a system image. Step S
At 22, the image processing unit 20 reads system variables from the storage unit 25 via the file processing unit 23. The system variables are as follows.

[0156] beta; detection aperture; lateral magnification A _S of the optical system
Delta; defocus amount absolute value lambda _G; foreign object detection wavelength _{N x,} N _y; number of effective pixels of the camera P _I; pixel camera pitch B; AD conversion bit number
t ₀ ; minimum accumulation time In step S23, the format of the biochemical test array 1 is set as the chip array format. In step S24, measurement conditions are set. Step S25
In, the image processing unit _20, H G = _{0, H} S = 0, measurement start switch = 0, the measurement end switch = 0. If SW _R = 1 in step S26, in step S27,
The reference is set, and in step S28, the examiner sets the biochemical test array 1 as a sample chip. In step S26, if SW _R = 1 is not satisfied,
In step S28, the biochemical test array 1 is set. When the measurement start switch is set to 1 in step S29, the image processing unit 20 sets the measurement start switch to 0 in step S30.

If it is determined in step S31 that SW _G = 1, the system control unit 22 switches the cube unit to a foreign object detection cube in step S32.
At 33, the shutter 12 on the optical path of the excitation light is opened. In step S34, after the image processing unit 20 captures a foreign object image (dust image) from the CCD camera 19, the process proceeds to step S35.
Then, the system control unit 22 closes the shutter 12. The image processing unit 20 differentiates the foreign matter image in step S36,
In step S37, the degree of foreign matter determination is determined, and step S38
Is used to determine the foreign matter function, and in step S39, w _Gt (x,
y) = w _G (x, y).

After step S39 or step S3
If SW _G is not 1 at 1, the system control unit 22 opens the shutter 12 at step S40. Step S41
After the image processing unit 20 captures a sample image from the CCD camera 19, the system control unit 22 closes the shutter 12 in step S42.

The image processing section 20 measures the fluorescence luminance in step S43, displays an array image of the measured data on the display section 21 in step S44, and displays t in step S45.
The measurement data is stored in the storage unit 25 via the file processing unit 23. Note that the measurement data are: ρ _R ; reference foreign matter occupancy, ρ: sample foreign matter occupancy, _{ＭＢ MB} : fluorescent background luminance, (i, j); spot address, Γ "
_M (i, j); spot luminance, Γ _MC (i, j); fluorescence crosstalk luminance, _SM (i, j); spot area, R
EM _M (i, j); message.

If the measurement end switch is set to 1 at step S46, the file processing unit 23 proceeds to step S47.
The file in which the chip array format, the sample conditions, and the measurement conditions are described is stored in the storage unit 25, and the process ends. If the measurement end switch is not 1 in step S46, the processing after step S29 is performed.

FIG. 14 is a sub-flowchart showing the procedure for setting the chip array format shown in step S23. In step S101, the examiner specifies an array format setting screen by using an option button of the input unit 24. In addition, option button 'A' is "Edit existing array format file",
"B" is "edit the array format file used last", "C" is "set a new array format file", and the default is "B".

In step S102, the examiner sets the input unit 2
4 is determined by the command button. The command button “OK” is “determine the array format setting screen and proceed to the next”, and “stop” is “quit without updating the file”.

In step S103, the option button 'A' is set, and in step S104, the FD
(Floppy (registered trademark) disk) is set, and if there is a designated file in the FD in step S105, the file processing unit 23 moves the directory to the FD in step S106. The examiner inputs the step S from the input unit 24.
At 107, a file is designated by a list box, and at step S108, the designated file is opened by a command button.

When the option button 'B' is set in step S103, the file processing unit 23 opens the lease file stored in the storage unit 25 in step S109. When the option button 'C' is set in step S103, the file processing unit 23 opens a default file in step S110. Step S
After 108, S109 and S110, step S111
Then, the examiner determines the set value of the array format by using the command button of the input unit 24.

At step S112, the image processing section 20
The chip array variables determined in step S111 are read from the text box. The chip array variables are as follows:

S ₀ ; standard area of one probe P _x ,
P _y; pitch m _{x, m y} of the probe; probe sequence number N _P; at position detection probe number step S113, the image processing unit 20 calculates the _{R 0 = 0.64 + 1.44S 0 /} πP x P y , And the process ends.

FIG. 15 is a sub-flowchart showing the procedure for setting the measurement conditions shown in step S24. In step S201, the examiner specifies a measurement condition setting screen using the option button of the input unit 24. The option button 'A' is for “editing an existing measurement condition file”, 'B' is for 'editing the last used measurement condition file', and 'C' is for 'setting a new measurement condition file'. And the default is 'B'.

In step S202, the examiner sets the input unit 2
4 is determined by the command button. The command button “OK” is “determine the specification method of the measurement condition setting screen and proceed to the next”, and “stop” is “quit without updating the file”.

When the option button 'A' is set in step S203, the examiner specifies a file from the input unit 24 in the list box in step S204, and opens the specified file in step S205 using the command button. If the option button 'B' is set in step S203, the file processing unit 23 opens the lease file stored in the storage unit 25 in step S206. If the option button 'C' is set in step S203, the file processing unit 23 opens the default file stored in the storage unit 25 in step S207.

After steps S205, S206, and S207, the inspector edits the user variables using the text box on the input unit 24 in step S208. In step S209, the inspector adds and deletes a user function using the check box in the input unit 24. In step S210, the inspector edits the measurement conditions using the input unit 24. In step S211, the examiner sets the input unit 24
Confirm the measurement conditions by pressing the command button.

In step S212, the image processing unit 20 reads the user indicator variable, the user measurement variable, and the user function variable determined in step S211 from the input unit 24, and the image processing unit 20 ends the processing.

The user indicator variables are as follows:
(() In the default value) _{M max;} fluorescent label number (2) _{M A;} fluorescent label (FITC) (lambda _A autofill) Sample A _{M B;} fluorescently labeled sample B (TEXS RED) (λ
_B automatic input) (when M _max = 2) The user measured variables are as follows. (() In the default value) _{t 0;} minimum accumulation time _{(t 0)} n _max; accumulated number of layers (AUTO) _{k max;} number of measurements (10) _{N P;} position detection marker number (position detection number of probes)
(0) The user function variables are as follows. (1 for function, 0 for nothing, 0 for default value) SW _R ; Reference processing (1) SW _RG ; Foreign matter processing of reference chip (1) (SW _R
= 1) SW _B ; Background processing (1) SW _G ; Sample foreign matter processing (1) (use of option button) (SW _G = 2 in the case of combined use output with non-processing data) FIG. It is a sub flowchart which shows the procedure of the reference setting shown in step S27. Step S
In 301, the examiner specifies a reference mode using an option button of the input unit 24. In addition, the option button 'A' is 'use existing reference file', 'B' is 'use last used reference file', 'C' is 'measure new reference brightness', The default is 'B'.

In step S302, the examiner sets the input unit 2
4 is determined by the command button. Note that the command button 'NEXT' is “Next”, and the 'BAC
K 'is "return to the front".

If the option button 'A' is set in step S303, the examiner specifies a file from the input unit 24 using the list box in step S304, and in step S305, the image processing unit 20 deletes the specified file. The target file. If the option button 'B' is set in step S303, step S3
At 06, the image processing unit 20 sets the lease file as the target file. Step S305 or step S30
After 6, in step S307, the image processing unit 20 reads the _W R (x, y) from the target file, the process ends.

If the option button 'C' is set in step S303, in step S308, the inspector sets the reference array 4 as a reference chip.
Is set. If SW _RG = 1 in step S309, the system control unit 22 switches the cube unit to a foreign object detection cube in step S310, and opens the shutter 12 in step S311. Step S31
After the image processing unit 20 captures a foreign object image from the CCD camera 19 in step 2, the system control unit 22 closes the shutter 12 in step S313. The image processing unit 20 differentiates the foreign object image in step S314, and executes step S315.
The reference foreign matter determination degree is determined in step S316.
To determine the foreign matter function, and at step S317, w
_RG (x, y) = w _G (x, y).

After step S317 or step S317
If SW _G = 1 is not satisfied in 309, the system control unit 22
Switches the cube unit to a reference cube in step S318, and opens the shutter 12 in step S319. In step S320, the image processing unit 20
Captures a reference fluorescence image from the CCD camera 19, the system control unit 22 closes the shutter 12 in step S321.

[0177] The image processing section 20 corrects the reference background in step S322.
At 323, the file processing unit 23 stores the reference variable as a lease file.

At step S 324, the examiner sets the input unit 24
In the case where "Save" is designated by the option button of "1", the examiner designates a file by the list box from the input unit 25 in step S325, and proceeds to step S326
Then, the file processing unit 23 copies the lease file to the specified file, and ends the processing. Step S
If the examiner designates “not save” with the option button of the input unit 25 in 324, the image processing unit 20 ends the processing.

FIG. 17 is a sub-flowchart showing the procedure for capturing the sample image shown in step S41. The image processing unit 20 sets M = 1 in step S401, and sets n _Mmax = n _{max in} step S402.
In step S403, the examiner switches the cube unit to a fluorescent M cube. The image processing unit 20 sets n = 1 in step S404, and sets k = k in step S405.
1, Γ _Mn (x, y) = 0.

System control unit 22 and image processing unit 20
From the CCD camera 19 in the step S406, the fluorescence image n-th hierarchy fluorescence image Γ _'Mn (x, y) of the sample chip storage time ^{2 n-1} _{t 0} as uptake, in step S407, gamma _Mn (x , Y) = Γ _Mn (x, y) +
Γ ' _Mn (x, y) is calculated. If k = k _max is not satisfied in step S408, the image processing unit 20 sets k = k + 1 in step S409, and performs the processing of step S406 and subsequent steps.

If k = k _max in step S408, the image processing unit 20 determines in step S410 that Γ _Mn
An average of k _max images is calculated according to (x, y) = _{Ｍｎ Mn} (x, y) / k, and in step S411, the minimum luminance Γ _{Mnmin at} the center of the image is measured.

As described above, a plurality of fluorescent images of a sample chip having the same accumulation time are captured, and an average image of the fluorescent images is calculated.

[0183] The image processing unit 20 determines n in step S412.
= N _Mmax instead of Γ _Mnmin in step S413
If not> 2 ^B-1 , n = n + 1 in step S414
Then, the processing after step S405 is performed. If _{Ｍｎ Mnmin} > 2 ^B-1 in step S413, the image processing unit 20 sets n _Mmax = n in step S415.

After step S415 or step S415
If n = n _Max in 412, the image processing unit 20
If M is not M = M _max in step S416, M = M + 1 is set in step S417, and the processing from step S402 is performed. If M = M _max in step S416, the processing ends.

FIG. 18 is a sub-flowchart showing the procedure for measuring the minimum luminance at the center shown in step S411. The image processing unit 20 determines in step S501
And _{Mnmi n} = ² B -1, y in step S502 =
0, x = 0 in step S503. Image processing unit 20
In step S504, 0.45N _x -1 <x <0.
55N _x −1, and in step S505, 0.45N
a _{y -1 <y <0.55N y -1} , step S50
If Γ _Mn (x, y) <Γ _Mnmin in 6, it is set in step S507 that Γ _Mnmin = Γ _Mn (x, y).

After step S507 or step S507
0.45N at 504_x-1 <x <0.55N_x-1
If not, or 0.45N in step S505_y-1 <
y <0.55N_yIf not −1, or step S5
06_Mn(X, y) <Γ _MnminIf not,
The image processing unit 20 determines in step S508 that x = N_x-1
If it is, x = x + 1 is set in step S509, and step
The processing after S504 is performed.

In step S508, x = N _x −1, and
If y = N _y −1 is not satisfied in step S510, the image processing unit 20 sets y = y + 1 in step S511, and performs the processing from step S503. In step S510, y =
If it is _Ny- 1, the image processing unit 20 ends the processing.

FIG. 19 is a sub-flowchart showing the procedure of the fluorescence luminance measurement shown in step S43. The image processing unit 20 sets M = 1 in step S601, and performs stray light correction and spatial moving averaging of the sample fluorescent image in step S602. The image processing unit 20 determines in step S6
At 03, the image dividing line is determined, the sample fluorescence image is rotated at step S604, and the fluorescence background correction is performed at step S605. In this fluorescence background correction, the minimum detection amount of fluorescence in the noise sampling area (local background area) of the sample fluorescence image is set as the fluorescence background, and the fluorescence image obtained by subtracting the fluorescence background from the sample fluorescence image is used as the target image. And In this case, as shown in FIG. 4, the boundary of the measurement area 42 and the boundary of the noise sampling area 43 are the same.

In step S606, the image processing section 20 sets j
= 1, i = 1 in step S607, and n = n _{max in} step S608. The image processing unit 20 determines in step S6
09 to check the optimal image, and at step S610,

(Equation 13) If, the measurement area is determined in step S611.

The image processing section 20 determines in step S612 that
x '_min= B₂′ (I, j−1) instead of step S
At 613, x '_max= B₂’(I, j),
In step S614, y '_min= B₁’(I-1)
In step S615, y ' _max= B₁’(I)
If not, the spot luminance is measured in step S616.
You. X 'in step S612_min= B₂’(I, j-
1) or x ′ in step S613_max= B₂’
(I, j) or y 'in step S614._{mi n}=
b₁'(I-1) or y' in step S615.
_max= B₁′ (I), step S617
In the image processing unit 20, the REM_M(I, j) = spot
Abnormal.

At step S610,

[Equation 14]

If not n = 1 in step S618, the image processing unit 20 determines that n = n− in step S619.
The process from step S609 is performed. If in step S618 it is n = 1, the image processor 20, at step _{S620, REM M (i, j} ) = a not measurable by accumulation time Univ.

Step S616 or step S620
After the image processing unit 20, in step S621 i = _{m x}
If not, i = i + 1 is set in step S622, and the processing of step S608 and subsequent steps is performed. The image processing unit 20 is a i = _{m x} in step S621, if not j = _{m y} in step S623, and j = j + 1 at step S624, performs step S607 and subsequent steps. Image processing unit 2
0 is j = _{m y} in step S623, step S
If M = M _max is not satisfied in 625, M = M + 1 is set in step S626, and the processing from step S602 is performed.
If M = M _max in step S625, the image processing unit 20 ends the processing.

In step S602, the smoothing process is performed on the sample fluorescence image by spatial moving averaging. However, the smoothing process may be performed using an averaging operator.

FIG. 20 is a flowchart showing the operation in step S602.
Procedure for stray light correction and spatial moving averaging of sample fluorescence images
It is a sub-flowchart shown. The image processing unit 20
In step S701, n = 1, and in step S702, k
= 1, Γ (x, y) = 0. The image processing unit 20
At step S703, the accumulation time 2
^n-1t₀And the nth layer stray light background of the sample chip
Capture the und image as '' (x, y) and step
In S704, Γ (x, y) = Γ (x, y) + Γ ’(x,
y).

If k = k _max is not satisfied in step S705, the image processing unit 20 determines in step S706 that k = k + 1
Then, the processing after step S703 is performed. Step S
If k = k _max at 705, the image processing unit 20
In step S707, _{ｎ n} (x, y) = Γ _Mn (x,
y) -Γ (x, y) / k, calculate k _max average images, and _{let Ｍ Mnmax} = 0 and Γ _Mnmin = 2 ^B −1. The image processing unit 20 determines in step S708 that y = k _S
And then, and x = _{k S} in step S709.

The image processing section 20 determines in step S710

(Equation 15) Is calculated, and in step S711, the maximum luminance at the center of the image is calculated.

[0198]

(Equation 16)

And minimum luminance

[Equation 17] Measurement.

The image processing section 20 determines in step S712
If x is not equal to N _x -k _S −1, then in step S713 x
= X + 1, and the processing after step S710 is performed. If x = N _x −k _S −1 in step S712 and y = N _y −k _S −1 in step S714, the image processing unit 20 sets y = y + 1 in step S715, and performs processing from step S709 onward. I do. In step S714, y =
A N _y -k _S -1, at step S716, n = n
If not _Mmax , the image processing unit 20 _proceeds to step S7
In step 17, n = n + 1, and the processing after step S702 is performed. If n = n _Max in step S716,
The image processing unit 20 ends the processing.

FIG. 21 is a sub-flowchart showing the procedure for determining the image dividing line shown in step S603. In step S801, if the number of position detection markers (the number of position detection probes) N _P = 0, the image processing unit 2
0 indicates that the optimal reaction image (whole image) is binarized in step S802, and the binarized whole image is subjected to step S802.
In step 803, the no-marker row gradient is calculated. In step S804, the no-marker minimum column pitch is calculated. In step S805, the no-marker column signal is inserted.
In step S806, a row dividing line function is calculated.

Further, the image processing section 20 performs step S807 on the whole image of the binarized sample fluorescence image.
In step S80, the column gradient for no marker is calculated.
In step S809, a no-marker minimum row pitch is calculated. In step S809, a no-marker row signal is inserted. In step S810, a column dividing line function is calculated. Image processing unit 2
In step S811, “0” is determined as to whether the obtained image dividing line is appropriate.

If N _P = 1 in step S801,
The image processing unit 20 detects the position of one marker in step S812, binarizes the optimal reaction image (whole image) in step S813, and performs a binarization on the binarized whole image.
In step S814, the row gradient for no marker is calculated, in step S815, the minimum column pitch of the one-point marker is calculated, in step S816, the column signal for no marker is inserted, and in step S817, the row dividing line function is calculated. Do.

Further, the image processing section 20 calculates the no-marker column gradient in step S818 for the binarized whole image, calculates the one-point marker minimum row pitch in step S819, and executes step S820. In step S821, a row signal for no marker is inserted, and in step S821, a column dividing line function is calculated. The image processing unit 20 determines in step S8
At 22, a determination is made as to whether the obtained image division line is appropriate.

If N _P = 2 in step S801,
The image processing unit 20 performs position detection of the two markers in step S823, binarizes the optimal reaction image (whole image) in step S824, and performs binarization on the binarized whole image.
In step S825, the row gradient for no marker is calculated, in step S826, the column gradient for no marker is calculated, and in step S827, the dividing line function for two-point marker is calculated. The image processing unit 20 determines in step S832
Determine the optimal response image.

If N _P = 3 in step S801,
The image processing unit 20 detects the positions of the three markers in step S828, calculates the dividing line function for three-point markers in step S829, and determines the optimal reaction image in step S832. If it is _N P = 4 in step S801, the image processing unit 20, the marker 4 at step S830
In step S831, a four-point marker dividing line function is calculated, and in step S832, an optimal reaction image is determined.

Step S811 or step S822
Alternatively, after step S832, in step S833, the image processing unit 20 displays on the display unit 21.

(Equation 18)

Y = a ₁ x + b ₁ (k) (line dividing line), x
= A ₂ y + b ₂ (k) (column division line) is displayed.

FIG. 22 is a conceptual diagram showing the dividing lines displayed on the sample fluorescence image on the display unit 21. FIG.
Number of position detection markers (number of position detection probes) N _P =
It shows the case where it is 0. As shown in FIG. 22, the image processing unit 20 divides the sample fluorescence image into a plurality of divided images 70 for each probe array element by a plurality of row dividing lines 71 (horizontal broken lines) and column dividing lines 72 (vertical broken lines). I do. In this case, each divided image 70 has a substantially square shape. The examiner specifies a measurement area and a noise sampling area (local background area) on each divided image 70 of the sample fluorescence image enlarged and displayed on the display unit 21 by using the mouse of the input unit 25 or the like. The image processing unit 20 detects a local background from the noise sampling area, and corrects a detection amount from the measurement area by using the detection amount.

[0210] In step S834, the examiner sets the input unit 2
Confirm with the command button of No. 5. In addition, the command button 'partition line correction' is "correct the division line",
“NEXT” is “next”, and “BACK” is “setting of measurement conditions in the main flowchart (step S
Return to 24) ", and" specify abnormal area "is" recalculate in addition to the extraneous area image ".

[0211] When the "specify abnormal area" is determined, the examiner specifies an abnormal area on the sample fluorescence image displayed on the display unit 21 by using the mouse of the input unit 25 or the like. An abnormal area is an area that is regarded as abnormal but is not processed as a foreign matter image. The image processing unit 20 sets the specified abnormal area as an area not to be inspected. Further, the image processing unit 20 adds the area of the foreign substance image to the abnormal area to make the area outside the inspection target.

FIG. 23 is a conceptual diagram showing division lines displayed on the sample fluorescence image on the display unit 21. FIG.
Number of position detection markers (number of position detection probes) N _P =
The case where it is 0 is shown. As shown in FIG. 23, the image processing unit 20 divides the sample fluorescence image into a plurality of divided images 70 in units of probe array elements by a plurality of row dividing lines 71 and a column dividing line 72. In this case, each divided image 70
Has a parallelogram shape.

As shown in FIG. 23, the arrangement state of the probe array elements 3 in the array for biochemical test is continuously shifted for each row (or for each column, or for each row and each column), and for each column (or for each row). (Or every row, or every row and every column). In this case, the division as shown in FIG. 22 cannot be performed, and the divided image for each probe array element 3 cannot be made substantially square. Therefore, the image processing unit 20 determines at least one of all the row division lines 71 and all the column division lines 72 (FIG. 23) according to the arrangement state of the probe array elements 3.
Then, a process of inclining all the column dividing lines 72) by a predetermined angle is performed. Thereby, the divided image for each probe array element 3 has a parallelogram shape.

FIG. 24 is a conceptual diagram showing an example of the gradient calculation of the row dividing line and the column dividing line of the binarized sample fluorescence image. FIG. 24 shows a case where the number of position detection markers (the number of position detection probes) N _P = 0. FIG.
The portion of each probe array element 3 in the whole image is a bright area,
It is assumed that the other parts form a binarized image that is a dark area. When binarizing the sample fluorescence image, the image processing unit 20 performs binarization using the median of the output values in the measurement region to obtain a binarized image.

The image processing section 20 detects a bright area and a dark area in the whole image from the −x direction to the + x direction, and
The compression process is performed in the direction, and a bright region compressed image and a dark region compressed image of all rows are generated. In FIG. 24, as a result of this compression processing, an area detected as a bright area is indicated by an ON signal, and an area detected as a dark area is indicated by an OFF signal. And the image processing unit 20
Are the ON signals and the OF signals indicating the compressed light and dark areas.
The width (vertical width) of the F signal in the y-axis direction is detected.

The arrangement state of the probe array elements 3 in the biochemical test array may have a slope for each row. Therefore, when there is a difference in the inclination angle for each row as shown in FIG. 24, a difference occurs in the width in the y-axis direction between the ON signals and a difference occurs in the width in the y-axis direction between the OFF signals. .

The image processing unit 20 has the widest O
The inclination angle (gradient) of the row of the probe array element 3 where the FF signal (compressed dark area) has occurred, that is, the inclination angle of one row in contact with the area indicated by the OFF signal is calculated. Then, the image processing unit 20 sets the calculated inclination angles as the inclination angles (gradients) of all the row dividing lines.

Similarly, the image processing section 20 detects a bright area and a dark area in the whole image from the y direction to the -y direction and compresses the image in the -y direction, thereby obtaining a bright area compressed image of all rows. And a compressed image of a dark area is generated. As a result of this compression processing, an area detected as a bright area is indicated by an ON signal, and an area detected as a dark area is indicated by an OFF signal. Then, the image processing unit 20 detects the width (width) in the x-axis direction of each ON signal and each OFF signal indicating the compressed light area and dark area.

The arrangement state of the probe array elements 3 in the biochemical test array may have a slope for each row. For this reason, if there is a difference in the inclination angle for each column, each O
A difference occurs in the width in the x-axis direction between the N signals, and each OF
A difference occurs in the width in the x-axis direction between the F signals.

[0220] The image processing section 20 has the O
The inclination angle (gradient) of the row of the probe array element 3 where the FF signal (compressed dark area) has occurred, that is, the inclination angle of one row in contact with the area indicated by the OFF signal is calculated. Then, the image processing unit 20 sets the calculated inclination angles as the inclination angles (gradients) of all the column dividing lines.

Next, a first example of calculation of the interval between each row dividing line and the interval between each column dividing line of the binarized sample fluorescence image will be described. As shown in FIG. 24, the image processing unit 20 sets the center position of each bright region of the compressed image of all columns to p and the center position of each dark region of the compressed image of all columns to q. Then, the image processing unit 20 calculates an interval between each row dividing line based on each point indicating the center position p of each bright area. Alternatively, the image processing unit 20 calculates an interval between each row dividing line based on each point indicating the center position q of each bright area.

The image processing section 20 sets the center position of each bright region of the compressed image of all rows to p '(not shown), and sets the center position of each dark region of the compressed image of all lines to q' (not shown). And Then, the image processing unit 20 calculates the interval between each column dividing line based on each point indicating the central position p ′ of each bright region. Alternatively, the image processing unit 20 calculates the interval between each column dividing line based on each point indicating the center position q ′ of each bright region.

When a foreign substance or the like is present on the biochemical inspection array 6 during the gradient calculation of the row dividing line and the column dividing line, the width of the compressed bright region is reduced by the probe array element 3 alone. Wider than the width of the image. In this case, the image processing unit 20 compares the width of each continuous bright area with the arrangement condition of the probe array element 3 and determines whether the width of each bright area corresponds to the width of the original bright area compressed image. to decide. If there is a portion of each of the bright regions that does not correspond to the width of the original bright region compressed image, the image processing unit 20 replaces that region with a dark region based on the arrangement condition of the probe array element 3. After that, the image processing unit 20 calculates an interval between each row dividing line or each column dividing line. As a result, it is possible to perform an appropriate gradient calculation of the row division line and the column division line excluding the influence of the foreign matter or the like.

Next, a description will be given of a second example of calculation of the interval between each row dividing line and the interval between each column dividing line of the binarized sample fluorescence image. If the position is a marker for detecting the number (position detection number of probes) N P ₌ 1, 2, 3, 4, the image processing unit 20, a predetermined provided in the biochemical test array of the position detection array element 2 The center position is detected, and the initial positions of the row dividing line and the column dividing line are calculated based on the center position. Then, the image processing unit 20 calculates an interval between each row dividing line and an interval between each column dividing line based on the initial position and the arrangement condition of the probe array element 3 with respect to each position detecting array element 2.

Next, a process for determining a measurement area in a divided image will be described.

FIG. 25 is a diagram showing a divided image of the reference fluorescent image and the sample fluorescent image. 25, the same parts as those in FIG. 4 are denoted by the same reference numerals. The image processing unit 20 binarizes the divided image with the median of the output values in the divided image to obtain a binarized image. Thereafter, the image processing unit 20 generates a rectangle 412 obtained by expanding a rectangle 411 circumscribing the binarization line 40 of the binarized image by a certain amount,
The boundary is a noise sampling area (local background area) 43. Then, the image processing unit 20 sets the outside of the boundary as the noise sampling area 43 in the divided image and sets the inside of the boundary as the measurement area 42. Thus, a region including the entire binarized region 41 and its periphery can be set as the measurement region 42.

When the fluorescence wavelength for detecting λ, a is the numerical aperture on the sample side, and Δ is the amount of defocus, the image processing unit 20 calculates the amount of expansion δ as follows: δ = 1.619 × λ / a + a | Δ |

FIG. 26 is a diagram showing a divided image of the reference fluorescent image and the sample fluorescent image. 26, the same parts as those in FIG. 4 are denoted by the same reference numerals. The image processing unit 20 binarizes the divided image with the median of the output values in the divided image to obtain a binarized image. Thereafter, the image processing unit 20 converts a rectangle 412 obtained by contracting the rectangle 411 inscribed in the binarization line 40 of this binarized image by a certain amount
The boundary of the measurement area 42 is set. Then, the image processing unit 20
The inside of this boundary in the divided image is defined as the measurement area 42,
A noise sampling area 43 outside the binarization line 40
And Thereby, only the binarization area 41 can be set as the measurement area 42.

When the fluorescence wavelength for detecting λ, a is the numerical aperture on the sample side, and Δ is the defocus amount, the image processing unit 20 calculates the contraction amount δ by the following equation: δ = 1.619 × λ / a + a | Δ |

After the measurement area determination processing shown in FIG. 25 or FIG. 26, the image processing section 20 calculates the noise sampling area 43 based on the average emission intensity per unit area of the measurement area.
The intensity obtained by subtracting the average luminescence intensity per unit area from プ ロ ー ブ is detected as the reaction intensity of each probe.

The biochemical test method of the present invention has the following configuration.

(1) In the reference array, the same number of the luminescent molecules are held in the array element corresponding to each probe array element in the same format as the array for biochemical examination.

(2) A mixed solution of a plurality of samples containing a first biochemical substance labeled with a plurality of different luminescent molecules excited and emitted by light is used as a test sample solution, and a plurality of different luminescent molecules are prepared. The reaction state between the probe in each probe array element and each of the samples is inspected by using a reference array in which is mixed and held at a fixed ratio.

(3) The emission intensity corresponding to the standard area of the probe array element or the average emission intensity per unit area is detected.

(4) As a luminescence function, a mixed solution of a plurality of samples containing a first biochemical substance labeled with a plurality of different luminescent molecules excited and emitted by light is used as a solution of a test sample. Using a reference array in which different light-emitting molecules are mixed and held at a fixed ratio, the reaction state between the probe in each probe array element and each sample is inspected.

(5) A non-linear coefficient is set as a parameter for determining the luminescence function, and the number of luminescent molecules present in each probe array element is determined using the non-linear coefficient.

(6) n _Mk is the number of luminescent molecules M in the probe array element k, J is the number of types of luminescent molecules, and I _Mk is the number of luminescent molecules M in the probe array element k per molecule. The emission intensity, ρ is a nonlinear coefficient, P _Mk is the emission intensity of the luminescent molecule M in the probe array element k, and the emission function is

[Equation 19] And

(7) The I _Mk is

(Equation 20) And

(8) P _Mk is the emission intensity of the luminescent molecule M in the probe array element k, n _RM is the number of luminescent molecules M in the reference array element k, and P _RMk is the element of the reference array. and the emission intensity of the emissive molecule M in k, the quantity n _Mk luminescent molecule M of the probe array element k formula

[0240]

(Equation 21) Ask by

(9) ρ is a non-linear coefficient, n _RM is the number of light emitting molecules M in the element k of the reference array, P _RMk is light emission intensity of the light emitting molecule M in the element k of the reference array, P _Mk is the emission intensity of the luminescent molecule M in the probe array element k,

(Equation 22) Is the number n of luminescent molecules M in the probe array element k.
Initial value of the i-th operation value of _Mk

(Equation 23)

And the sequential operation expression

(Equation 24)

And an equation for judging completion of sequential operation

(Equation 25) At the end of the sequential calculation

[0244]

(Equation 26) Is the number n of luminescent molecules M in the probe array element k.
_Mk .

(10) A mixed solution of a plurality of samples containing a first biochemical substance labeled with a plurality of different luminescent molecules excited and emitted by light is used as a solution of a test sample, and a solution of the test sample is prepared. Is supplied to the biochemical test array.

(11) A foreign substance image of the biochemical test array is acquired in advance, and the extraneous area of the foreign substance image is added to the abnormal area to make it a non-target area.

(12) The fluorescence wavelength for detecting λ, a is the numerical aperture on the sample side, Δ is the defocus amount, and the amount of expansion δ is determined by the equation δ = 1.619 × λ / a + a | Δ |.

(13) The fluorescence wavelength for detecting λ, a is the numerical aperture on the sample side, Δ is the amount of defocus, and the amount of contraction δ is determined by the equation δ = 1.619 × λ / a + a | Δ |.

The present invention is not limited to the above-described embodiment, but can be implemented with appropriate modifications without departing from the scope of the invention.

[0250]

According to the present invention, it is possible to provide a biochemical testing method capable of accurately testing each biochemical reaction state in a probe array element.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a microscope apparatus that performs a biochemical inspection method according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of a biochemical test array according to the first embodiment of the present invention.

FIG. 3 is a diagram showing a format of a reference array according to the first embodiment of the present invention.

FIG. 4 is a diagram showing a divided image of a reference fluorescence image and a sample fluorescence image according to the first embodiment of the present invention.

FIG. 5 is a diagram showing output signals from a CCD according to the first embodiment of the present invention.

FIG. 6 is a diagram showing a signal obtained by subtracting a noise signal from an output signal according to the first embodiment of the present invention.

FIG. 7 is a flowchart showing an inspection procedure according to the first embodiment of the present invention.

FIG. 8 is a flowchart showing an inspection procedure according to the first embodiment of the present invention.

FIG. 9 is a scatter diagram according to the first embodiment of the present invention.

FIG. 10 is a diagram showing a format of an upper surface of a biochemical test array used in the second embodiment of the present invention.

FIG. 11 is a diagram showing a format of an upper surface of a reference array used in the second embodiment of the present invention.

FIG. 12 is a diagram illustrating a configuration of a microscope apparatus that performs a biochemical inspection method according to a third embodiment of the present invention.

FIG. 13 is a flowchart showing an inspection procedure according to a third embodiment of the present invention.

FIG. 14 is a flowchart showing an inspection procedure according to a third embodiment of the present invention.

FIG. 15 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 16 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 17 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 18 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 19 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 20 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 21 is a flowchart showing an inspection procedure according to the third embodiment of the present invention.

FIG. 22 is a conceptual diagram showing division lines displayed on a sample fluorescence image according to the third embodiment of the present invention.

FIG. 23 is a conceptual diagram showing division lines displayed on a sample fluorescence image according to the third embodiment of the present invention.

FIG. 24 is a conceptual diagram showing an example of a gradient calculation of a row dividing line and a column dividing line according to the third embodiment of the present invention.

FIG. 25 is a diagram showing a divided image of a reference fluorescent image and a sample fluorescent image according to the third embodiment of the present invention.

FIG. 26 is a diagram showing a divided image of a reference fluorescence image and a sample fluorescence image according to the third embodiment of the present invention.

FIG. 27 is a diagram showing a tapered sample well array forming a three-dimensional array according to a conventional example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Array for biochemical testing 2 ... Array element for position detection 3 ... Probe array element 31 ... Probe 4 ... Reference array 5 ... Element for fluorescent molecule holding 6 ... Array for biochemical testing 7 ... Reference array 8 ... Fluorescent molecule holding area Reference Signs List 11 excitation light source 12 shutter 13 lens 140 excitation filter unit 14 excitation filter 15 dichroic mirror 16 objective lens 170 ND filter unit 17 ND filter 18 imaging lens 19 CCD camera 20 image processing unit DESCRIPTION OF SYMBOLS 21 ... Display part 22 ... System control part 23 ... File processing part 24 ... Input part 25 ... Storage part 40 ... Binarization line 41 ... Binarization area 42 ... Measurement area 43 ... Noise sampling area 70 ... Division image 71 ... Line Parting line 72 ... Column dividing line 101 ... Porous glass wafer 102 … Hole 103… well with taper 104… channel 105… through-hole

Claims (23)

[Claims] An array for biochemical examination in which a solution of a first biochemical substance is held on a surface thereof, and second biochemical substances specifically reacting with the first biochemical substance are held on the surface. Supply to
The first biochemistry is performed by detecting the luminescence intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light by excitation with light. A biochemical inspection method for inspecting, for each probe array element, a reaction state between a target substance and a second biochemical substance that specifically reacts with the first biochemical substance; The excitation light is applied to the reference array in which is held, and an optical image is taken as a reference light image.The same excitation illumination is applied to the biochemical inspection array to take an optical image as a sample light image, and the sample light is sampled. A biochemical inspection method, wherein an image is corrected with the reference light image. 2. A mixed solution of a plurality of samples containing the first biochemical substance labeled with a plurality of different luminescent molecules excited and emitted by light is used as a solution of a test sample, and the plurality of different luminescent properties are measured. 2. The method according to claim 1, wherein a reaction state between the probe in each of the probe array elements and each of the samples is inspected using the reference array in which molecules are mixed and held at a fixed ratio. Chemical inspection method. 3. The biochemical testing method according to claim 1, wherein the reference array has light-emitting molecules uniformly distributed on a substrate. 4. An array for a biochemical test in which a solution of a first biochemical substance is held on a surface thereof, and second biochemical substances specifically reacting with the first biochemical substance are held on the surface. Supply to
The first biochemistry is performed by detecting the luminescence intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light by excitation with light. A biochemical inspection method for inspecting, for each probe array element, a reaction state between a target substance and a second biochemical substance that specifically reacts with the first biochemical substance; The light image of the biochemical test array after supplying the solution of the biochemical substance is divided into a plurality of divided images by the probe array element unit, and each of these divided images has a measurement area and a local background. A biochemical test method, wherein an area is designated, a local background is detected from the local background area, and a detection amount from the measurement area is corrected by the detection amount. . 5. A light image is divided into a plurality of divided images by the probe array element unit, a measurement area and a local background area are designated for each of the divided images, and a local background area is designated from the local background area. 4. The biochemical testing method according to claim 1, wherein a background is detected, and the detected amount is used to correct the detected amount from the measurement area. 6. An array for biochemical examination in which a solution of a first biochemical substance is held on a surface thereof, respectively, second biochemical substances which specifically react with the first biochemical substance. Supply to
The first biochemical substance and the second biochemical substance are detected by detecting the emission intensity of the light-emitting molecule for each probe array element of the biochemical test array with an array-type detector using a label with the light-emitting molecule. A biochemical inspection method for inspecting, for each probe array element, a reaction state with a second biochemical substance that specifically reacts with one biochemical substance, wherein a non-linear coefficient is used as a parameter for determining a luminescence function. And determining the number of luminescent molecules present in each of the probe array elements using the non-linear coefficient. 7. An array for biochemical examination in which a solution of a first biochemical substance is held on a surface thereof, respectively, second biochemical substances which specifically react with the first biochemical substance. Supply to
The first biochemistry is performed by detecting the luminescence intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light by excitation with light. A biochemical inspection method for inspecting, for each probe array element, a reaction state between a target substance and a second biochemical substance that specifically reacts with the first biochemical substance; A biochemical inspection method, characterized in that an abnormal area designated on a light image of the array for biochemical inspection after supplying a solution of a biochemical substance is set as a non-target area. 8. An array for a biochemical test in which a solution of a first biochemical substance is held on a surface of a second biochemical substance which specifically reacts with the first biochemical substance. Supply to
The first biochemistry is performed by detecting the luminescence intensity of the luminescent molecule for each probe array element of the array for biochemical testing using an array-type detector using a label with a luminescent molecule that emits light by excitation with light. A biochemical inspection method for inspecting, for each probe array element, a reaction state between a target substance and a second biochemical substance that specifically reacts with the first biochemical substance, wherein the accumulation times are the same. A biochemical inspection method, wherein an average image of a plurality of light images of the array type detector is used as a target image. 9. A biochemical test array in which a solution of a first biochemical substance is held on a surface thereof, and second biochemical substances specifically reacting with the first biochemical substance are held on the surface. Supply to
The first biochemical substance and the second biochemical substance are detected by detecting the emission intensity of the light-emitting molecule for each probe array element of the biochemical test array with an array-type detector using a label with the light-emitting molecule. A biochemical inspection method for inspecting, for each of the probe array elements, a reaction state with a second biochemical substance that specifically reacts with one of the biochemical substances, obtained by the array-type detector. A biochemical inspection method, wherein a smoothing process is performed on an optical image using an averaging operator. 10. An array for a biochemical test in which a solution of a first biochemical substance is held on a surface of a second biochemical substance which specifically reacts with the first biochemical substance. By detecting the emission intensity of the luminescent molecule for each probe array element of the biochemical test array using a label with a luminescent molecule that emits light by excitation with light, using an array-type detector, whereby the second A biochemical test method for testing a reaction state between one biochemical substance and a second biochemical substance that specifically reacts with the first biochemical substance for each probe array element; Biochemistry characterized in that a minimum detection amount in a target area of a light image obtained by the array-type detector is a fluorescent background, and a light image obtained by subtracting the fluorescent background from the light image is a target image. Inspection method. 11. The biochemical inspection method according to claim 4, wherein the shape of the divided image is a parallelogram. 12. The biochemical inspection method according to claim 6, wherein a boundary of the measurement area and a boundary of the local background area are the same. 13. A dark area width of a compressed image of an array row or an array column, each of which has a gradient with respect to the x-axis or the y-axis of the binarized image of the light image obtained by the array type detector, is detected. The gradient of the array row or the gradient of the array column when the dark area width is the widest is the gradient of a row dividing line or a column dividing line of the divided image, respectively. Biochemical testing method. 14. The binarized image according to claim 13, wherein the binarized image is obtained by performing a binarization using a median value of an output in a target region of the light image obtained by the array type detector. Biochemical testing method. 15. A method for determining an interval between the row division lines or the column division lines by using a plurality of points constituting a center position of each dark area or each bright area of the compressed image of the array row or array column. The biochemical inspection method according to claim 13 or 14, wherein: 16. The method according to claim 16, wherein each of the continuous light areas is compared with an array condition of the probe array element, and if there is an invalid area in each of the light areas, the invalid area is replaced with the probe array element. 16. The biochemical inspection method according to claim 15, wherein an interval between the row dividing lines or the interval between the column dividing lines is obtained after replacing with the dark area based on the arrangement condition. 17. The biochemical inspection method according to claim 13, wherein an interval between the row dividing lines or the column dividing lines is obtained based on an arrangement condition of the probe array elements. 18. The method according to claim 18, wherein a center position of a position detecting array element provided in the biochemical test array is detected, and an initial position of the row dividing line or the column dividing line is obtained based on the center position. The biochemical test method according to claim 13 or 14, wherein 19. The divided image is binarized by a median value of outputs in the divided image, and a rectangle obtained by expanding a rectangle circumscribing the binary image by a fixed amount is set as a boundary of the local background area. 5. The local background area outside the boundary in an image.
Or the biochemical examination method according to 5 or 11. 20. The divided image is binarized by a median value of outputs in the divided image, and a rectangle obtained by shrinking a rectangle inscribed in the binarized image by a predetermined amount is set as a boundary of the measurement area. The biochemical examination method according to claim 4, wherein the inside is the measurement area. 21. An intensity obtained by subtracting an average emission intensity per unit area of the local background area from an average emission intensity per unit area of the measurement area is detected as a reaction intensity with each probe. Item 14. The biochemical examination method according to item 4, 5, or 11. 22. A solution of a test sample containing a first biochemical substance is supplied to the biochemical test array, and each probe array element of the biochemical test array is labeled using a label with a luminescent molecule. The first biochemical substance and the second biochemical substance that specifically reacts with the first biochemical substance by detecting the emission intensity of the luminescent molecule with an array-type detector. A biochemical inspection method for inspecting a reaction state for each probe array element, wherein the luminescence intensity corresponding to a standard area of the probe array element or an average luminescence intensity per unit area is detected. Inspection method. 23. The array for biochemical examination supplies a probe solution to different positions on the surface of a three-dimensionally structured reaction carrier made of a porous or fibrous substance or a molded product, and comprises a probe contained in the solution. By holding the inside of the porous or fibrous substance or molded article,
The biochemical testing method according to any one of claims 1 to 10, wherein a solution of the test sample containing the first biochemical substance is supplied to the biochemical testing array.