JP2001169999A - Fluorescence yield measuring method and instrument - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enhance the measurement accuracy of a fluorescence yield in a fluorescence yield measuring instrument and method for determining the fluorescence yield by measuring the fluorescence generated from vital tissue by irradiation with exciting light. SOLUTION: In determining the fluorescence yield which is a ratio of the photodetecting intensity of the exciting light Le received in the vital tissue 1 by irradiation with the exciting light Le and the intensity of the fluorescence generated from the vital tissue 1 by the photodetection of this exciting light Le from both, >=2 kinds of reference light having wavelengths regions different from the wavelength region of the exciting light and having the wavelength regions different from each other are emitted from a light source unit 100 and are cast to the vital tissue 1. The intensity of each of the reflected reference light obtained by detecting the reflected reference light reflected by the vital tissue 1 is inputted to a fluorescence yield computation unit 400 and is substituted into the proximate operation expression stored in the previously formed fluorescence yield computation unit 400, by which the proximate value of the photodetecting intensity of the exciting light received by the vital tissue 1 is calculated and the fluorescence yield is determined by using this proximate value.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring fluorescence yield, which measures fluorescence generated from a living tissue by irradiation with excitation light as an image.

[0002]

2. Description of the Related Art Conventionally, fluorescent light generated from a living tissue is detected by irradiating the living tissue with light in a wavelength region on the shorter wavelength side of visible light as excitation light, and the intensity of the fluorescent light is analyzed. Fluorescence yield measuring devices for measuring changes in tissue properties of living organisms due to various diseases have been studied.

[0003] For example, the intensity of an image of the living tissue (hereinafter referred to as a fluorescent image) due to the fluorescence generated from the living tissue due to the irradiation of the exciting light is determined by the intensity of the living tissue formed by the reflection of the exciting light by the living tissue. Japanese Patent Application Laid-Open No. 62-247232 discloses a method of normalizing by dividing by the intensity of a tissue image (hereinafter referred to as an excitation light image) and measuring the tissue properties of a living body based on the normalized fluorescence image. Has been proposed.

In this method, the ratio of the intensity of the excitation light received by a part of the living tissue to the intensity of the fluorescence generated from the part due to the reception of the excitation light, that is, the fluorescence yield between the normal tissue and the diseased tissue is different. It measures changes in the tissue properties of the living body due to various diseases based on the differences, and it is difficult to directly measure the intensity of the excitation light received by the living tissue. By performing detection, the intensity of the excitation light received by the living tissue is substituted for measurement. Since this fluorescence yield is a value represented by the ratio of the intensity of the excitation light to the intensity of the fluorescence, it is not affected by the distance and angle between the irradiation point irradiated with the excitation light and the measurement site of the living tissue. Measurement of tissue properties of a living body can be performed.

[0005] As an alternative to the intensity of the excitation light received by the living tissue, instead of using the excitation light image reflected by the living tissue, the living tissue is irradiated with near-infrared light and reflected by the living tissue. The fluorescence yield is determined by the ratio of the intensity of the fluorescent image to the intensity of the near-infrared light image using an image of the living tissue (hereinafter, referred to as a near-infrared light image) obtained by the reflected light of the near-infrared light. Techniques are also being considered.

[0006]

However, light in the wavelength region on the shorter wavelength side of visible light is easily absorbed by the living tissue, and its absorptance also varies depending on the tissue properties of the living tissue. The intensity of the excited light may not correctly reflect the intensity of the excited light received by the living tissue. In addition, when the fluorescence yield is determined using near-infrared light, light absorption by living tissue as described above does not occur, but near-infrared light has a wavelength that is significantly different from excitation light, and It is difficult to irradiate infrared light with the same light distribution characteristics through the same optical system, and when the light distribution characteristics are different from each other, there is a problem that the measurement accuracy of the fluorescence yield decreases.

[0007] This type of problem involves fluorescence (auto-fluorescence) generated when a living tissue is irradiated with excitation light and fluorescence generated when a living tissue that has previously absorbed a fluorescent diagnostic agent is irradiated with the excitation light. (Drug fluorescence).

The present invention has been made in view of the above circumstances, and has as its object to obtain a highly accurate fluorescence yield to more accurately measure the tissue properties of a living body.

[0009]

According to the present invention, there is provided a method for measuring a fluorescence yield, comprising the steps of: measuring the intensity of fluorescence generated from a living tissue by irradiating excitation light; and the intensity of the excitation light received by the living tissue. A fluorescence yield measuring method for determining a fluorescence yield that is a ratio of the excitation light to the living tissue by irradiating the living tissue with two or more types of reference light having wavelength regions different from the excitation light and having wavelength regions different from each other. The intensities of the respective reflected reference lights reflected by the tissue are obtained, and the approximate intensities are calculated by substituting the intensities of the reflected reference lights into a previously created approximation equation. The method is characterized in that the fluorescence yield is obtained as intensity.

A model operation formula is prepared by combining a variable into which the intensities of the two or more types of reflected reference light are respectively substituted and unknown coefficients, and the variable of the model operation expression is used as a reference sample for the two or more types. The intensity obtained when substituting the intensity of each standard reflection reference light reflected by the standard sample by irradiating the reference light, the standard sample received when the excitation light was irradiated on the standard sample By determining the unknown coefficient so as to be closest to the received light intensity of the excitation light, the approximation equation can be created.

The fluorescence yield measuring apparatus according to the present invention comprises: an excitation light irradiating means for irradiating a living tissue with excitation light; a fluorescence detecting means for detecting the intensity of fluorescence generated from the living tissue by the irradiation of the excitation light; A fluorescence yield measuring device comprising: a calculation unit for calculating a fluorescence yield which is a ratio of a received light intensity of the excitation light received by the living tissue by the irradiation of the excitation light and an intensity of the fluorescence generated from the living tissue. Wherein the reference light irradiating means for irradiating two or more types of reference light having different wavelength regions from each other with the excitation light and having different wavelength regions from each other, and the two or more types of reference irradiated by the reference light irradiating means. Reference light detecting means for detecting the intensity of each of the reflected reference lights reflected by the living tissue, and wherein the calculating means comprises the reflected reference light obtained by the reference light detecting means. Intensity is substituted into the previously prepared approximation calculation equation to calculate the approximate intensity of the received light intensity of the excitation light,
The fluorescence yield is obtained by using the approximate intensity as the received light intensity of the excitation light.

[0012] The fluorescence yield measuring device includes a jig for obtaining the approximate operation expression used by being attached to the fluorescence yield measuring device, and the jig is irradiated with the excitation light and the reference light. The standard sample can be mounted at a position.

The jig may be capable of applying a part of a human body such as a palm as the standard sample.

The two or more types of reference light include at least two or more of light in a blue wavelength region, light in a green wavelength region, light in a red wavelength region, and light in a near-infrared wavelength region. It can be.

[0015] The approximation formula may be created for each pixel of the image.

The approximate calculation expression may express the received light intensity of the excitation light by one expression.

The approximation equation is expressed by an equation in which r is a variable in r-θ coordinates with the origin being the center of rotation of the intensity distribution of the excitation light or the reference light applied to the standard sample. be able to.

The standard sample means a homogeneous sample having light absorption characteristics, reflection / scattering characteristics and fluorescence emission characteristics equivalent to those of normal tissue of a living body.

The fluorescence yield means a ratio between the intensity of the excitation light received by the living tissue and the intensity of the fluorescence generated from the living tissue by receiving the excitation light.

[0020]

According to the fluorescence yield measuring method and apparatus of the present invention, two or more different types of reference light having different wavelength ranges from the excitation light are used as the intensity of the excitation light to obtain the fluorescence yield. Since the approximate intensity calculated by appropriately combining the intensities of the respective reflected reference lights reflected by the living tissue due to the irradiation is used, the absorption of the reference light by the living tissue is smaller than when only one type of reference light is used. Alternatively, a measurement error due to a difference in light distribution is small, and an approximate intensity closer to the actual received light intensity of the excitation light can be obtained. As a result, a value of the fluorescence yield can be obtained with higher accuracy.

Further, a model operation formula is prepared by combining a variable into which the intensities of the two or more types of reflected reference light are respectively substituted and unknown coefficients. The intensity obtained when substituting the intensity of each standard reflected reference light reflected by the standard sample by irradiating the above-described reference light, the standard sample is irradiated when the standard sample is irradiated with the excitation light. If the above-mentioned unknown coefficient is determined so as to be closest to the received light intensity of the received excitation light, and the approximate calculation formula is created, the value of the fluorescence yield can be obtained with higher accuracy.

A jig for obtaining the approximate arithmetic expression used by being attached to the fluorescence yield measuring apparatus;
If the standard sample can be attached to the jig at a position where the excitation light and the reference light are irradiated, the value of the fluorescence yield can be obtained with higher accuracy.

Further, if the jig can apply a part of a human body such as a palm as the standard sample, the value of the fluorescence yield can be obtained with higher accuracy.

Further, the two or more types of reference light are at least two or more of light in a blue wavelength region, light in a green wavelength region, light in a red wavelength region, and light in a near infrared wavelength region. Is included, the value of the fluorescence yield can be obtained with higher accuracy.

Further, if the approximation formula is created for each pixel of the image, the value of the fluorescence yield can be obtained with higher accuracy.

Further, the approximation equation is expressed by an equation in which r is a variable in r-θ coordinates having the origin at the center of rotation of the intensity distribution of the excitation light or the reference light applied to the standard sample. In this case, the operation can be executed faster, and the load on the devices such as the arithmetic unit and the memory can be reduced.

[0027] Further, if the approximate calculation expression represents the received light intensity of the excitation light by one expression, the calculation can be executed more quickly, and the burden on devices such as a calculator and a memory can be reduced. Can be.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing a schematic configuration of a fluorescence endoscope apparatus to which the fluorescence yield measuring method and apparatus of the present invention are applied. As shown in the figure, the fluorescent endoscope device 800 includes a white light Lw, a near infrared light Ls, and an excitation light L
light source unit 100 including three light sources that respectively emit light e, and excitation light Le emitted from the light source unit 100.
Is irradiated to the living tissue 1 through the irradiation optical fiber 21, and an endoscope unit 200 that captures an image of fluorescence generated from the living tissue 1 by the irradiation of the excitation light Le, converts the image into an electric image signal, and outputs the image signal. A relay unit 30 that inputs an image signal output from the endoscope unit 200, converts the image signal into two-dimensional image data composed of digital values, and outputs the two-dimensional image data.
0, storing and calculating the two-dimensional image data output from the relay unit 300 to obtain image data representing the fluorescence yield (hereinafter referred to as fluorescence yield image data Dks), and displaying this fluorescence yield image data Dks It comprises a fluorescence yield calculation unit 400 which converts the signal into a signal and outputs the signal, and a display unit 500 which inputs and displays the display signal output from the fluorescence yield calculation unit 400.

The light source unit 100 has an end face 21a of a branch optical fiber 21A branched from the irradiation optical fiber 21.
And a filter (not shown) that is connected to the end face 21b of the branch optical fiber 21B branched from the irradiation optical fiber 21 and that is rotatably attached to the rotating shaft of the motor 12 and that transmits the three primary colors of red, green and blue. A disc-shaped rotary filter 13 having an R filter, a G filter, and a B filter) as shown in FIG. 2 is disposed between the white light source 10 and the white light focusing lens 11. By being rotated, the white light Lw emitted from the white light source 10 is separated into red light, green light, and blue light (hereinafter, red light, green light, and blue light are collectively referred to as surface-sequential light Lm). , The end face 21 of the branch optical fiber 21A through the white light focusing lens 11 sequentially.
a.

Further, a GaAs-LD made of 780 nm
The near-infrared light Ls emitted from the near-infrared light source 14 that generates light having a wavelength of 400 nm reflects light having a wavelength of 410 nm and 780 n
The light passes through a dichroic mirror 15 that transmits light having a wavelength of m, is condensed by a condenser lens 16, and is incident on an end face 21b of a branch optical fiber 21B.

Further, 410 n of InGaN-LD
The excitation light Le emitted from the excitation light source 17 that generates light having a wavelength of m is reflected by the reflection mirror 18 at a substantially right angle,
Further, the light is reflected by the dichroic mirror 15 at a substantially right angle, is condensed by the condensing lens 16, and is incident on the end face 21b of the branch optical fiber 21B.

Note that, as shown in FIG.
A light blocking portion 13a that does not transmit the white light Lw is provided in a region other than the R filter, the G filter, and the B filter of No. 3, and the light blocking portion 13a when the near infrared light Ls or the excitation light Le is emitted. Is inserted between the white light source 10 and the white light condenser lens 11, and the near-infrared light Ls and the excitation light Le
Are emitted at different timings, so that these lights do not enter the irradiation optical fiber 21 at the same time.

The endoscope unit 200 includes a bendable end portion 201, a light source unit 100 and a relay unit 3.
00 and an operation unit 202 connected thereto, and the excitation light Le and the near-infrared light Ls emitted from the light source unit 100.
And an irradiation optical fiber 21 for propagating the plane-sequential light Lm;
A cable 27 for transmitting an electrical image signal captured and converted by the image sensor 26 is laid from the distal end 201 to the operation unit 202. And
The near-infrared light Ls and the surface-sequential light Lm that have entered the branch optical fiber 21 are emitted from the end face 21 c at the other end of the branch optical fiber 21, and are irradiated on the living tissue 1 as reference light through the irradiation lens 22.

An image (hereinafter referred to as a near-infrared light image Zs) of near-infrared light, which is reflected reference light reflected by the living tissue 1 irradiated with the near-infrared light Ls, passes through the objective lens 23 and is prism-shaped. 24, the optical path of the light is changed to a substantially right angle by the prism 24, an image is formed on the image sensor 26, the image is captured by the image sensor 26, converted into an electric image signal, and transmitted to the relay unit 300 by the cable 27. Transmitted.

On the other hand, plane-sequential light Lm which is reflected reference light reflected by the living tissue 1 irradiated with the plane-sequential light Lm
, The red light image Zr, the green light image Zg, and the blue light image Zb (hereinafter, the red light image Zr, the green light image Zg, and the blue light image Zb are collectively referred to as a plane-sequential light image Zm)
As in the case where the living tissue 1 is irradiated with the near-infrared light Ls, the image is captured by the image sensor 26, converted into an image signal, and transmitted to the relay unit 300 via the cable 27.

The excitation light Le incident on the branch optical fiber 21 is emitted from the end face 21c at the other end of the branch optical fiber 21 and passes through the irradiation lens 22 as reference light.
An image (hereinafter, referred to as a fluorescent image Zk) generated by the living tissue 1 that has received the excitation light Le is also captured by the image sensor 26 and converted into an image signal and relayed by the cable 27 in the same manner as described above. Transmitted to unit 300.

The image sensor 26 has n × m rows and columns of pixels, and has a wavelength of 410 nm on the light receiving surface.
An excitation light cut filter 25 that transmits light in a wavelength region exceeding 10 nm is provided, and the excitation light Le incident on the objective lens 23 at the same time as the fluorescence image Zk is cut off.

Further, since the fluorescence generated from the living tissue by the irradiation of the excitation light is weak, the fluorescence image Zk, the red light image Zr, the green light image Zg, the blue light image Zb, and the near infrared light image Z
In order to receive s with one image sensor, the irradiation intensity of each light and the transmission characteristics of each filter are adjusted so that the intensity of each image has an appropriate value.

A cable 27 is connected to the relay unit 300, and the red light image Zr, the green light image Zg, the blue light image Zb, and the near-infrared light image Z transmitted by the cable 27.
s and the image signal carrying the fluorescent image Zk are subjected to noise suppression, defect correction, video signal processing, and the like by the process circuit unit 31, and further converted to a digital signal by the A / D converter 32, and are converted into an n × m matrix. Is output as two-dimensional image data.

Each of the two-dimensional image data output from the relay unit 300 is converted into red light image data Dr, green light image data Dg, blue light image data Db, and near-infrared light image data in the fluorescence yield calculation unit 400. Ds and a red light image memory 4 for storing as fluorescent image data Dk
1a, green light image memory 41b, blue light image memory 41
c, near-infrared light image memory 42, and fluorescent image memory 4
3 are provided. Each of the two-dimensional image data stored in each of the image memories and the approximate calculation formula previously obtained by using a standard sample measuring jig to be described later and stored in the approximate calculation formula memory 45 are the received light excitation light intensity calculator. The calculation is performed to obtain an approximate value of the received light intensity of the excitation light received by the living tissue 1 and the result is stored in the received light image memory 47 as received light image data Dej.

The fluorescence image data Dk stored in the fluorescence image memory 43 and the light-excitation light image data Dej stored in the light-excitation light image memory 47 are input to a fluorescence yield calculator and operated, and the result is obtained. Is output as fluorescence yield image data Dks, and the fluorescence yield image data Dks is input to a video signal processing circuit 49, converted into a display signal, and output.

The display unit 500 includes the video signal processing circuit 4
The display signal converted by 9 is input and displayed as a visible image.

It should be noted that the fluorescence endoscope apparatus 800 has a standard sample measuring jig 600 as shown in FIG. 3 which is necessary for obtaining a coefficient of an approximation equation for approximating the intensity of the excitation light received by the living tissue 1. Comes with. This standard sample measuring jig 60
Reference numeral 0 denotes a positioning holder 610 provided with an insertion port 611 for mounting the distal end portion 201 of the endoscope unit 200, and a homogenous material having light absorption characteristics, reflection / scattering characteristics, and fluorescence emission characteristics equivalent to those of normal tissue of a living body. Disc-shaped standard sample 6
20 and the distal end portion 20 of the endoscope unit 200.
1 is attached to the sample holder 612 of the positioning holder 610 to which the device 1 is attached.
The standard sample 620 is applied to the standard sample 620, and the light emitted from the light source unit 100 is irradiated to the standard sample 620 via the endoscope unit 200.
The light reflected by 0 and the fluorescence generated from the standard sample 620 are measured, and the calculation is performed by the fluorescence yield calculation unit 400, whereby the coefficients of the above approximate calculation expression are obtained.

In place of the standard sample 620, a similar measurement can be performed by applying a part of the human body previously diagnosed as a normal tissue, for example, a hand 630 as shown in FIG. .

Next, the operation of the above embodiment will be described. First, a method of using the standard sample measuring jig 600 to obtain an approximate arithmetic expression for obtaining an approximate value of the intensity of the excitation light received by the living tissue as two-dimensional image data will be described.

As shown in FIG. 3, the distal end portion 201 of the endoscope unit 200 is inserted into the insertion portion 611 of the positioning holder 610.
To the sample holder 6 of the positioning holder 610.
12 is applied with a standard sample 620. In this state, the excitation light Le is emitted from the light source unit 100 and is emitted to the standard sample 620 via the irradiation optical fiber 21 and the irradiation lens 22. The standard fluorescence image of the standard sample 620 due to the fluorescence generated from the standard sample 620 by the irradiation of the excitation light is incident on the prism 24 through the objective lens 23, and the optical path of the standard sample 620 is changed to a substantially right angle by the prism 24.
An image is formed on the image, captured, and converted into an electric image signal. This image signal is further input to the relay unit 300 via the cable 27, and is input to the A /
The signal is converted into a digital signal by the D converter 32, output as standard fluorescent image data DHk, and stored in the fluorescent image memory 43 of the fluorescent yield calculation unit 400. Since the standard sample 620 is a homogeneous sample having the same fluorescence generation characteristics as normal tissue of a living body, it generates fluorescence at an intensity corresponding to the intensity of the received excitation light Le, and the standard fluorescence image data DHk is The two-dimensional image data reflects the intensity of the excitation light Le received by the sample 620.

Next, the near-infrared light L
s is emitted, the standard sample 620 is irradiated through the endoscope unit 200 in the same manner as described above, and the standard sample 620 is reflected by the standard sample 620 irradiated with the near-infrared light Ls. A standard near-infrared light image is captured, and an image signal obtained by the capturing is stored in the near-infrared light image memory 42 as standard near-infrared light image data DHs via the relay unit 300.

Further, a standard surface sequential light image of the standard sample 620 by the surface sequential light reflected by the standard sample 620 irradiated with the surface sequential light Lm emitted from the light source unit 100 via the endoscope unit 200 That is, the standard red light image, the standard green light image, and the standard blue light image are also picked up in the same manner as described above, and the image signals obtained by the picked up images are output via the relay unit 300 to the standard red light image data DHr and the standard green light image. Data DHg and standard blue light image data D
Hb is stored in the red light image memory 41a, the green light image memory 41b, and the blue light image memory 41c.

The standard red light image data DHr and the standard green light stored in the red light image memory 41a, the green light image memory 41b, the blue light image memory 41c, the near-infrared light image memory 42, and the fluorescent image memory 43, respectively. The image data DHg, the standard blue light image data DHb, the standard near-infrared light image data DHs, and the standard fluorescent image data DHk are input to the approximation coefficient calculator 44, and the external input terminal 40
Is input to the approximation coefficient calculator 44 and stored in the approximation coefficient calculator 44 into the model calculation expression including the unknown coefficient, and the value of the unknown coefficient of the model calculation expression is calculated. The model calculation formula in which the value of the unknown coefficient is determined is used as an approximation calculation formula memory 4 as an approximation calculation formula for approximating the intensity of the excitation light received by the living tissue 1.
5 is stored.

Here, a detailed description will be given of the case where the unknown coefficient of the model operation expression is obtained from each of the two-dimensional image data.

The fluorescence yield is a value represented by the ratio of the intensity of the excitation light received by the living tissue by the irradiation of the excitation light to the intensity of the fluorescence generated from the living tissue receiving the excitation light. It is difficult to directly measure the intensity of the excitation light received by the tissue, and the excitation light in the wavelength region on the short wavelength side of visible light has a different absorption rate depending on the tissue properties of the living body and is not uniformly absorbed by the living tissue. On the other hand, light in the wavelength region on the long wavelength side of visible light has significantly different light distribution characteristics because the wavelength region is significantly different from the wavelength region of the excitation light. A large error will occur even if you try to measure.

However, if the light receiving intensity of the excitation light is approximated by using four types of light having different wavelength ranges, the above-mentioned disadvantage can be compensated for. By calculating an approximate operation expression based on the two-dimensional image data obtained by
A more accurate value of the two-dimensional image data that approximates the intensity of the excitation light received by the living tissue can be obtained.

The approximation equation is obtained by converting the two-dimensional image data actually obtained by actual measurement into a model operation equation including unknown coefficients input from the external input terminal 40 and stored in the approximation coefficient calculator 44. It is obtained by substituting as a value.

More specifically, red light Lr, which is plane-sequential light,
A standard red light image, a standard green light image, a standard blue light image, and a standard near-infrared light image obtained by irradiating the standard sample 620 with the green light Lg, the blue light Lb, and the near-infrared light Ls Red light image data DHr, standard green light image data DHg, standard blue light image data DHb, and standard near-infrared light image data DH which are two-dimensional image data obtained by
s and the standard fluorescence image data DHk reflecting the intensity of the excitation light Le received by the standard sample 620 as a known value, the model calculation formula Q = α × P1 + β × P2 + γ × P3 + δ × P4 + ε P1 of the formula (1) By substituting the unknown coefficients α, β, γ, δ, and ε into P2, P3, P4, and Q, respectively, the pixel positions (i,
An equation to be obtained is set for each j). That is, DHk (i, j) = αij × DHr (i, j) + βijDH
g (i, j) × + γij × DHb (i, j) + δij × DH
s (i, j) + εij where i = 1 to n, j = 1 to m DHk (i, j): value of standard fluorescence image data at pixel position (i, j) DHr (i, j): pixel Value of standard red light image data at position (i, j) DHg (i, j): value of standard green light image data at pixel position (i, j) DHb (i, j): pixel position (i, j) DHs (i, j): value of near-infrared light standard image data at pixel position (i, j) αij: unknown coefficient of standard red light image data at pixel position (i, j) βij: unknown coefficient of standard green light image data at pixel position (i, j) γij: unknown coefficient of standard blue light image data at pixel position (i, j) δij: standard near-infrared light at pixel position (i, j) Unknown coefficient of optical image data εij: unknown constant Each pixel positions so as to satisfy the above expression based on the gist (i, j) values of unknown coefficients and unknown constants as shown below for each is determined.

Αij = Rij, βij = Gij, γij = Bij, δij = Sij, ε
ij = Eij As described above, when the values of the unknown coefficient and the unknown constant of the model calculation expression are determined for each pixel position (i, j), the approximate calculation expression for obtaining the received light intensity of the excitation light is determined as the following expression. be able to. That is, yij = Rij × P1ij + Gij × P2ij + Bij × P3ij + S
ij × P4ij + Eij Here, P1ij, P2ij, P3ij, and P4ij are red light image data D obtained when an actual living tissue is measured.
By substituting the values of r, green light image data Dg, blue light image data Db, and near-infrared light image data Ds, the value of received light excitation light image data Dej is obtained as yij. The approximation formula is calculated by the approximation coefficient calculator 44
And stored in the approximate calculation expression memory 45.

The above approximation equation shows the influence of light absorption of the living tissue which causes an error when the intensity of the excitation light received by the living tissue is approximated by the reflection intensity of the light on the short wavelength side of the visible light. When the light intensity of the excitation light received by the biological tissue is approximated by the reflection intensity on the long wavelength side of the visible light, the correction is performed with the intensity of the reflected reference light on the long wavelength side of the light. It is set so that the influence of the difference in light distribution characteristics due to the difference (difference in refractive index) is corrected by the reflected reference light intensity on the shorter wavelength side of visible light, and is excited using light in one wavelength region. The intensity of the excitation light received by the living tissue can be more accurately approximated as compared with the conventional method in which the light receiving intensity of light is approximated. The coefficients Rij, Gij, Bij, Sij, and Eij obtained for each pixel position (i, j) of the two-dimensional image data are determined according to the above-described purpose.

Next, the case where the fluorescence yield is obtained by using the approximate calculation formula obtained by using the standard sample measuring jig as described above will be described. The distal end portion 201 of the endoscope unit 200 is detached from the insertion portion 611 of the standard sample measuring jig 600, and is arranged at a position where the living tissue 1, which is the original measurement target, is measured. Then, similarly to when the standard sample 620 is measured, the living tissue 1 is irradiated with the excitation light Le, the near-infrared light Ls, and the plane-sequential light Lm from the light source unit 100 via the endoscope unit 200 at different timings. Then, the fluorescent image Zk generated from the living tissue 1 and the red light image Zr, the green light image Zg, the blue light image Zb, and the near-infrared light image Zs reflected by the living tissue are respectively imaged to obtain the fluorescent image data Dk. And the near-infrared light image data Ds, the red light image data Dr, the green light image data Dg, and the blue light image data Db as the fluorescent image memory 43 and the near infrared light image memory 41, the red light image memory 41a, and the green light image The data is stored in the memory 41b and the blue light image memory 41c.

The red light image data Dr, green light image data Dg, and blue light image data D stored in each image memory
b, near-infrared light image data Ds and approximate arithmetic expression memory 4
The approximate calculation formula stored in the received light excitation light intensity calculator 4
6 and the value of each pixel position (i, j) of each of the two-dimensional image data is approximated by the following equation: yij = Rij × P1ij + Gij × P2ij + Bij × P3ij + S
The values of the received light excitation light image data Dej (i, j) that are substituted into P1ij, P2ij, P3ij, and P4ij of ij × P4ij + Eij, respectively, and approximate the intensity of the excitation light received by the living tissue 1 are obtained.
That is, Dej (i, j) = Rij × Dr (i, j) + Gij × Dg
(I, j) + Bij × Db (i, j) + Sij × Ds
(I, j) + Eij where i = 1 to n, j = 1 to m Dej (i, j): value of received light excitation light image data at pixel position (i, j) Dr (i, j): pixel position Red light image data value at (i, j) Dg (i, j): green light image data value at pixel position (i, j) Db (i, j): blue light at pixel position (i, j) Image data value Ds (i, j): value of near-infrared light image data at pixel position (i, j) Rij: coefficient of red light image data at pixel position (i, j) Gij: pixel position (i, j) j) coefficient of green light image data at pixel position Bij: coefficient of blue light image data at pixel position (i, j) Sij: coefficient of near-infrared light image data at pixel position (i, j) Eij: pixel position (i, j) The constant in j) and the received excitation light image Receiving excitation light image stored in the re 47 data Dej (i,
j) and the fluorescence image data Dk (i, j) obtained when the excitation light Le is irradiated and stored in the fluorescence image memory 43.
Is input to the fluorescence yield calculator 48 and the fluorescence image data D
The value of k (i, j) is converted to the image data Dej (i,
j) to obtain the fluorescence yield image data Dk
s (i, j) is determined. That is, Dks (i, j) = Dk (i, j) / Dej (i, j) where i = 1 to n, j = 1 to m The fluorescence yield image data Dks obtained by this operation
(I, j) is converted into a display signal by the video signal processing circuit 49 and is displayed as a visible image by the display unit 500. The standard sample and the biological tissue also generate fluorescence when irradiated with blue light Lb on the short wavelength side of visible light, but the emission intensity is reduced to the intensity of the blue light reflected by the standard sample and the biological tissue. Since the intensity is extremely weak, it is not necessary to remove the fluorescence intensity value from the intensity value of the blue light image Zb detected by the image sensor.

Further, P1 and P1 in the model operation expression (1)
An equation can be established so that P3 and P3 correspond to the luminance signals Y, I, and Q signals of the NTSC video signal, respectively. That is, DHk (i, j) = αij × Y (i, j) + βij × I
(I, j) + γij × Q (i, j) + δij × DHs (i, j
j) + εij where i = 1 to n, j = 1 to m Y (i, j) = 0.30DHg (i, j) + 0.59D
Hr (i, j) + 0.11DHb (i, j) I (i, j) = 0.60DHg (i, j) -0.28D
Hr (i, j) -0.32DHb (i, j) Q (i, j) = 0.21DHg (i, j) -0.52D
Hr (i, j) +0.31 DHb (i, j) DHk (i, j): value of standard fluorescence image data at pixel position (i, j) DHr (i, j): value at pixel position (i, j) Value of standard red light image data DHg (i, j): value of standard green light image data at pixel position (i, j) DHb (i, j): value of standard blue light image data at pixel position (i, j) Value DHs (i, j): value of near-infrared light standard image data at pixel position (i, j) αij: unknown coefficient of standard fluorescence image data at pixel position (i, j) βij: pixel position (i, j) ): Unknown coefficient of standard red light image data at pixel position (i, j) δij: unknown coefficient of standard blue light image data at pixel position (i, j) εij: unknown constant Based on these equations, the standard test The values of the unknown coefficients α ij, β ij, γ ij, δ ij and the unknown constant ε ij are obtained from the values of each two-dimensional image data obtained by measuring the materials, and an approximate calculation expression can be obtained in the same manner as described above.

As another method for obtaining an approximate operation expression, the value of each unknown coefficient is set at all pixel positions (i) so that the entire region of the received light excitation light image data Dej can be represented by one approximate operation expression. , J), it is also possible to obtain an approximate operation expression so as to be determined to one common value. That is, Q (i, j) = α × P1 (i, j) + β × P2 (i, j
j) + γ × P3 (i, j) + δ × P4 (i, j) + ε where i = 1 to n, j = 1 to m In this case, four unknown coefficients α, β, γ, δ, ε On the other hand, n × m equations corresponding to each pixel position of the two-dimensional image data stand, and the unknown coefficients α, β, γ,
The values of δ and ε can be determined. Then, assuming that the values of the unknown coefficients determined by the least square method are α = S, β = R, γ = G, δ = B, ε = E, the approximate calculation formula for obtaining the light receiving intensity of the excitation light is as follows: yij = R × P1ij + G × P2ij + B × P3ij + S × P4
ij + E. Then, the received excitation light image data Dej (i,
The value of j) is determined. That is, Dej (i, j) = R × Dr (i, j) + G × Dg
(I, j) + B × Db (i, j) + S × Ds (i, j)
+ E where i = 1 to n, j = 1 to m Dej (i, j): value of received light excitation light image data at pixel position (i, j) Dr (i, j): value at position (i, j) Red light image data value Dg (i, j): green light image data Dg value at position (i, j) Db (i, j): blue light image data Db value at position (i, j) Ds (I, j): value of near-infrared light image data Ds at position (i, j) R: value of coefficient of red light image data Dr G: value of coefficient of green light image data Dg B: blue light image data The value of the coefficient of Db S: The value of the coefficient of the near-infrared light image data Ds E: The value of the constant Further, the model operation expression is expressed using three types of light by the surface sequential light Lm excluding the near-infrared light Ls. Non-linear model operation expression, for example, Q (i, j) = α1 × P1 (i, j) + α2 × 1
(I, j) ** 2 + β1 × P2 (i, j) + β2 × P2
(I, j) ** 2 + γ1 × P3 (i, j) + γ2 × P3
An approximate operation expression can also be obtained using a model operation expression having a quadratic or higher order function such as (i, j) ** 2 + δ1 × ε.

When the unknown coefficients of the above equation are obtained by using the least squares method, the S / N
The image data at the pixel position with a high ratio is selected and the least squares method is applied, or the average value of each unknown coefficient obtained from a plurality of regions with a high S / N ratio is calculated as one common value for all regions. The value of the unknown coefficient may be used. In this case, the standard deviation of the intensity of the excitation light received by the living tissue corresponding to each pixel position or region is close to the standard deviation of the intensity of the fluorescence generated from the normal tissue corresponding to each pixel position or region. By selecting a pixel position or a region, the approximation accuracy of the approximation equation can be increased.

The measurement area is divided into a plurality of areas,
For one model operation formula, the value of the unknown coefficient is calculated for each of these divided regions, and a plurality of sets of approximate calculation formulas are obtained.
Similarly, it is also possible to divide the measurement area into a plurality of areas, set model arithmetic expressions having different expression forms for each of the divided areas, and obtain an approximate arithmetic expression for each area based on these model arithmetic expressions. .

The approximation formula for approximating the standard excitation light image data DHe is not limited to the case where it is obtained based on the two-dimensional image data obtained by irradiating four types of light having different wavelength ranges. Even when the approximation formula is obtained based on the two-dimensional image data obtained by irradiation with light having different wavelength regions as described above, the effect of reducing the approximation error can be obtained as compared with the conventional method.

In addition, the above arithmetic expression is not limited to the method of obtaining the X-Y orthogonal coordinates corresponding to the matrix pixels. For example, the rotation center position of the intensity distribution of the irradiation light, which is substantially rotationally symmetric, is determined. If a model operation expression is created using r-θ coordinates as the origin, the θ coordinate can be omitted, and the approximate operation expression can be expressed as a variable of only the r coordinate.

The near-infrared light included in the white light source can be reduced by adding a filter that transmits only the near-infrared light wavelength region to the rotary filter 12 without emitting the near-infrared light from a separate light source. If separated, the near-infrared light source 14 can be omitted.

The image representing the fluorescence yield obtained by the light irradiation is not limited to the display as a moving image of 30 frames per frame, but may be a smooth moving image such as 15 frames per frame or 10 frames per frame. Can be displayed as an image, but can be displayed as an image from which the tissue properties of the living body can be continuously observed, or as a still image.

As described above, according to the present invention, a highly accurate fluorescence yield can be obtained, and the tissue properties of a living body can be measured more accurately.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram of a fluorescence yield measuring device according to an embodiment of the present invention.

FIG. 2 is a diagram showing details of a rotary filter.

FIG. 3 is a diagram showing a structure of a standard sample measuring jig.

FIG. 4 is a diagram showing an arrangement when a part of a human body is applied to a standard sample measuring jig;

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Living tissue 21 Irradiation optical fiber 26 Image sensor 100 Light source unit 200 Endoscope unit 300 Relay unit 400 Fluorescence yield operation unit 500 Display unit 800 Fluorescence endoscope apparatus Lw White light Lm Surface sequential light Ls Near infrared light Le Excitation light

Continued on front page F term (reference) 2G043 AA04 EA01 FA06 GA06 GB18 HA05 HA09 HA12 JA02 KA01 KA02 KA05 LA03 MA01 NA05 4C061 AA00 BB01 CC06 DD03 HH54 LL01 MM03 NN01 QQ04 RR04 RR14 SS09 WW08 WW15

Claims (9)

[Claims] 1. A fluorescence yield measuring method for determining a fluorescence yield, which is a ratio between the intensity of fluorescence generated from a living tissue by irradiation with excitation light and the intensity of the excitation light received by the living tissue. And determining the intensity of each reflected reference light reflected by the living tissue by irradiating the living tissue with two or more types of reference light having different wavelength ranges from the excitation light and having different wavelength ranges from each other. Calculating the approximate intensity by substituting the intensities of these reflected reference lights into a previously created approximate arithmetic expression, and calculating the approximate yield as the received light intensity of the excitation light to obtain the fluorescence yield. Yield measurement method. 2. A model calculation formula is prepared by combining a variable into which the intensities of the two or more types of reflected reference light are respectively substituted and unknown coefficients, and the variable of the model calculation formula is used for the standard sample. The intensity obtained when substituting the intensity of each standard reflected reference light reflected by the standard sample by irradiating the above-described reference light, the standard sample is irradiated when the standard sample is irradiated with the excitation light. 2. The fluorescence yield measuring method according to claim 1, wherein the approximate calculation expression is created by determining the unknown coefficient so as to be closest to the received light intensity of the received excitation light. 3. Exciting light irradiating means for irradiating the living tissue with excitation light, fluorescence detecting means for detecting the intensity of fluorescence generated from the living tissue by irradiating the exciting light, and irradiating the living tissue with the exciting light. A fluorescence yield measuring device comprising: a calculation unit that calculates a fluorescence yield that is a ratio of a received light intensity of the excitation light received by the tissue and an intensity of the fluorescence generated from the living tissue. Reference light irradiating means for irradiating two or more types of reference light having different wavelength regions and having mutually different wavelength regions, and the two or more types of reference light irradiated by the reference light irradiating means are reflected by the living tissue. Reference light detecting means for detecting the intensity of each reflected reference light, wherein the calculating means approximates the intensity of the reflected reference light obtained by the reference light detecting means in advance. A fluorescence yield measuring apparatus for calculating an approximate intensity of the received light intensity of the excitation light by substituting into an arithmetic expression, and calculating the approximated intensity as the received light intensity of the excitation light. 4. The fluorescence yield measuring device includes a jig for determining the approximate operation expression used by being attached to the fluorescence yield measuring device, and the jig irradiates the excitation light and the reference light. 4. The fluorescence yield measuring device according to claim 3, wherein the standard sample can be mounted at a position for receiving the fluorescence. 5. The fluorescence yield measuring apparatus according to claim 4, wherein the jig is capable of applying a part of a human body such as a palm as the standard sample. 6. The two or more types of reference light are at least two or more of light in a blue wavelength region, light in a green wavelength region, light in a red wavelength region, and light in a near-infrared wavelength region. The fluorescence yield measuring apparatus according to any one of claims 3 to 5, comprising: 7. The method according to claim 3, wherein the approximation expression is created for each pixel of the image.
The fluorescence yield measuring device according to any one of the preceding claims. 8. The fluorescence yield measuring device according to claim 3, wherein the approximate calculation expression represents the received light intensity of the excitation light by one expression. 9. The approximation equation is represented by an equation in which r is a variable in an r-θ coordinate having an origin at a rotation center of an intensity distribution of the excitation light or the reference light applied to the standard sample. The fluorescence yield measuring device according to any one of claims 3 to 8, wherein: