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WO2004063683A1 - Dispositif d'analyse d'image spectrale en temps reel et procede d'analyse - Google Patents

Dispositif d'analyse d'image spectrale en temps reel et procede d'analyse Download PDF

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Publication number
WO2004063683A1
WO2004063683A1 PCT/JP2003/016891 JP0316891W WO2004063683A1 WO 2004063683 A1 WO2004063683 A1 WO 2004063683A1 JP 0316891 W JP0316891 W JP 0316891W WO 2004063683 A1 WO2004063683 A1 WO 2004063683A1
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WO
WIPO (PCT)
Prior art keywords
sample
wavelength
image
spectral
dimensional position
Prior art date
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Ceased
Application number
PCT/JP2003/016891
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English (en)
Japanese (ja)
Inventor
Takayuki Sota
Katsuo Aizawa
Nakanobu Hayashi
Shinya Ohtsubo
Fumihiko Ichikawa
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Waseda University
JFE Techno Research Corp
Original Assignee
Waseda University
Kawatetsu Techno Research Corp
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Filing date
Publication date
Application filed by Waseda University, Kawatetsu Techno Research Corp filed Critical Waseda University
Priority to AU2003292679A priority Critical patent/AU2003292679A1/en
Publication of WO2004063683A1 publication Critical patent/WO2004063683A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2823—Imaging spectrometer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2889—Rapid scan spectrometers; Time resolved spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/10—Scanning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/10—Scanning
    • G01N2201/103—Scanning by mechanical motion of stage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/10—Scanning
    • G01N2201/11—Monitoring and controlling the scan

Definitions

  • the present invention relates to a real-time spectral image analyzer and a method for measuring a spectral spectrum of a biological sample such as cancer as a spectral image in real time.
  • the main method is to visually observe the endoscopy using endoscopic images, collect living tissue suspected of having the disease, and analyze the tissue pathologically. It is a means.
  • collection diagnosis is not preferable because of the possibility of metastasis depending on the procedure.
  • the observation with the naked eye had problems such as oversight of the diseased part and the possibility that the diagnosis result would be affected by the experience and skill of the diagnostician.
  • early detection and appropriate treatment of diseases such as cancer can provide a high cure rate, so reliable early diagnosis and analysis are required.
  • a spectroscopic image measuring method using a spectroscopic image measuring device without collecting living tissue can be cited.
  • the conventional spectral image measurement method for diagnosis has a problem that only one spectral spectrum in a displayed image can be collected, and sufficient information for diagnosis cannot be obtained.
  • an apparatus for measuring the color and density of a printed surface for diagnosis as disclosed in Japanese Patent Application Laid-Open No. 2000-365652.
  • This device irradiates linear light onto the printing surface, and simultaneously separates the reflected light into minute areas divided on one line, and simultaneously obtains the color and density on one line from the obtained spectral data. You can do it.
  • the measurement range of this device was limited to one line, there was a problem that the measurement range was too narrow to be used for diagnosis.
  • An object of the present invention is to solve the above problem, and to provide a two-dimensional position It is an object of the present invention to provide a real-time spectroscopic image analyzer and an analysis method capable of simultaneously measuring a spectrum and wavelength information as a spectral image in real time. Disclosure of the invention
  • the real-time spectroscopic image analyzer according to claim 1, wherein the movable stage on which the sample is placed, a light source that irradiates white light on the surface of the sample, and the white light is reflected on the surface of the sample
  • a spectroscope that splits the area on one line of the reflected light generated at one time, and a spectral spectrum that is split from the reflected light by the spectroscope.
  • a monochrome CCD camera that captures one-dimensional position information and wavelength information in one frame, and stores, analyzes, and processes the one-dimensional position information and the wavelength information captured by the monochrome CCD camera, and a frame of the monochrome CCD camera.
  • a computer for operating the movable stage by one axis in units.
  • the two-dimensional position information and the wavelength information of the sample can be measured simultaneously and in real time as a spectral image, and reliable and quick measurement for diagnosis can be performed.
  • a spectral spectrum obtained by spectrally analyzing a region on one line of the sample surface at a time is taken as one-dimensional position information and wavelength information, and the one-axis position is obtained. It is characterized in that two-dimensional position information and wavelength information are obtained from the moved one-dimensional position information and wavelength information.
  • two-dimensional position information and wavelength information of a sample can be measured simultaneously and in real time as a spectral image, and reliable and quick diagnostic measurement can be performed.
  • FIG. 1 is a schematic diagram showing one embodiment of the real-time spectral image analyzer of the present invention.
  • FIG. 2 is an explanatory view showing the basic configuration of the spectrometer, as in the above.
  • Fig. 3 shows an example of a spectral image of one line.
  • Fig. 4 shows an example of the spectral spectrum.
  • FIG. 5 is an image showing the surface of the sample in the measurement example of mouse cancer tissue.
  • FIG. 6 is a graph showing the spectral spectrum of a plurality of points on the sample.
  • FIG. 7 is a graph in which the data of A in FIG.
  • FIG. 8 is an image showing the surface of the sample at a wavelength of 425 nm as above.
  • FIG. 9 is an image showing the surface of the sample at a wavelength of 505 nm as in the above.
  • FIG. 10 is an image showing the surface of a sample at a wavelength of 535 nm as in the above.
  • FIG. 11 is an image showing the surface of a sample at a wavelength of 600 nm as in the above.
  • FIG. 12 is an image showing the surface of a sample in another measurement example of a cancer tissue as in the above.
  • FIG. 13 is a graph showing a spectral spectrum at a point A in FIG.
  • FIG. 14 is a graph showing the spectral spectrum at point B based on the spectral spectrum at point A in FIG.
  • FIG. 15 is a graph showing the spectral spectrum at point C based on the spectral spectrum at point A in FIG.
  • FIG. 16 is an image showing the surface of the sample at a wavelength of 450 nm as in the above.
  • FIG. 17 is an image showing the surface of a sample at a wavelength of 500 nm as in the above.
  • FIG. 18 is an image showing the surface of the sample at a wavelength of 560 nm as in the above.
  • FIG. 19 is an image showing the surface of the sample at a wavelength of 600 nm as in the above.
  • FIG. 20 is an image showing the surface of the sample at a wavelength of 600 nm as in the above.
  • FIG. 21 is an image showing the surface of a sample at a wavelength of 700 nm as in the above.
  • FIG. 22 is an image showing the surface of the sample in the measurement example of hemoglobin change in the back of the hand, as in the above.
  • FIG. 23 is a graph showing the spectral spectrum of FIG.
  • FIG. 24 is a graph showing the spectral spectrum of a DNA sample labeled with the fluorescent dye FAM, as in the above.
  • FIG. 25 is a graph showing the spectral pattern of a DNA sample labeled with the fluorescent dye HEX, as in the above.
  • FIG. 26 is a graph showing the spectral spectrum of a DNA sample labeled with the fluorescent dye TET, as described above.
  • FIG. 27 is a graph showing the spectral spectrum of a DNA sample labeled with the fluorescent dye NED, as described above.
  • FIG. 28 is a diagram showing spectroscopic spectra of a DNA sample labeled with different fluorescent dyes and a DNA sample mixed with four types of fluorescent dyes, respectively.
  • reference numeral 1 denotes a movable stage on which a sample is placed.
  • Reference numeral 2 denotes a light source, which irradiates the sample S mounted on the movable stage 1 with uniform white light. Any light source can be used as the light source 2 as long as it can emit white light.
  • the light source 2 can be constituted by a halogen lamp.
  • excitation light corresponding to the fluorescent label is used, two-dimensional positional information and wavelength information of the fluorescent-labeled biomolecule can be observed.
  • a spectroscope 4 integrally provided with a slit 3 is provided above the movable stage 1.
  • the slit 3 allows the spectroscope 4 to transmit only a minute area on one line of the reflected light generated by the white light of the light source 2 reflected on the surface of the sample S.
  • the spectroscope 4 is an imaging spectroscope equipped with a transmission type grating, and simultaneously disperses while maintaining one-dimensional position information on one line of the reflected light transmitted through the slit 3 to obtain a spectral spectrum. It is configured to obtain a vector. Then, the one-dimensional position information and the wavelength information of the spectral spectrum separated by the spectroscope 4 are configured to be captured in one frame of the monochrome CCD camera 5 as a spectral image. You.
  • the slit 3 includes a slit body 3a and a lens 3b, and the lens 3b extends one line of the sample S. It is configured to collect the reflected light on the straight line A—B on the slit body 3a. Further, the spectroscope 4 includes two lenses 4 a and 4 c and a prism 4 b called PGP. Then, the reflected light on the straight line A—B of the sample S transmitted through the slit body 3a is split by the lenses 4a, 4c and the prism 4b while maintaining the positional relationship on the straight line A—B.
  • Black and white CCD camera 5 In other words, the region on the straight line AB of the sample S is separated at once while maintaining the positional relationship, and is captured in one frame of the monochrome CCD camera 5.
  • the black-and-white CCD camera 5 acquires the one-dimensional position information of the spectral spectrum of the straight line AB of the sample S on the X axis and the wavelength information on the Y axis.
  • the spectroscope 4 ImSpecterJ (spectral range: 380 to 780 nm, wavelength resolution: 2 nm) manufactured by Spectral Imaging Ltd of Finland is suitably used.
  • the black-and-white CCD camera 5 of the present embodiment has a photomultiplier tube 5 a (Image Intensifies) (Luminance gain: 250 000, sensitivity: 20 ⁇ 1 u, wavelength band 380). ⁇ 850 nm, wavelength resolution 2 nm), so that the weak light can be broadly acquired by the monochrome CCD camera 5.
  • a photomultiplier tube 5 a Image Intensifies
  • ⁇ 850 nm, wavelength resolution 2 nm so that the weak light can be broadly acquired by the monochrome CCD camera 5.
  • Reference numeral 6 denotes a computer that stores, analyzes, and processes the one-dimensional position information and wavelength information captured by the monochrome CCD camera 5. Further, the movable stage 1 is configured to operate in one axis, and the movable stage 1 is provided with adjusting means 7 for adjusting the speed of the one-axis operation and the like. The computer 6 is configured to control the adjusting means 7 so that the movable stage 1 moves one axis for a fixed distance for each frame unit of the monochrome CCD camera 5.
  • the two-dimensional position information and the wavelength information of the sample S can be obtained from the individual one-dimensional position information and the wavelength information of the sample S stored in the computer 6. I'm familiar. Based on this information, analysis and image processing are performed by the computer 6 to obtain, for example, the color values R, G, and B from the intensity of the spectral spectrum at each wavelength of 5 nm, and obtain the color value of the surface of the sample S.
  • Appearance As a color RGB image, spectral spectrum at any point on sample S, and simultaneous display of spectral spectrum and difference spectrum at any two points, or raw data, reflection It is configured to display the transmittance (transmittance) and the like, and to display the spectral spectrum intensity of an arbitrary wavelength on the surface of the sample S as a monochrome gradation image (gray scale).
  • the sample S to be measured is placed on the movable stage 1, and the light source 2 irradiates the surface of the sample S with white light. Only the reflected light from a minute area on one line on the surface of sample S passes through slit 3, and the reflected light that has passed through slit 3 Then, the one-dimensional position information and the wavelength information of this spectral spectrum are taken into one frame of the monochrome CCD camera 5 as a spectral image. More specifically, the reflected light on the straight line A—B of the sample S is collected by the lens 3b on the slit body 3a, and is reflected on the straight line A—B of the sample S transmitted through the slit body 3a.
  • the reflected light is split by the lenses 4a, 4c and the prism 4b while maintaining the positional relationship on the straight line AB, amplified by the photomultiplier tube 5a, and taken into the monochrome CCD camera 5. Then, the monochrome CCD camera 5 acquires the one-dimensional position information of the spectral spectrum of the straight line AB of the sample S on the X-axis, the wavelength information on the Y-axis, and obtains the spectral image as a spectral image.
  • Figure 3 shows an example of this spectral image.
  • the vertical axis (X-axis) is position information
  • the horizontal axis (Y-axis) is wavelength information.
  • the one-dimensional position information and the wavelength information acquired by the monochrome CCD camera 5 are stored by the computer 6.
  • the computer 6 controls the adjusting means 7 so that the movable stage 1 moves one axis at a fixed distance for each frame unit of the monochrome CCD camera 5.
  • the computer 6 controls the adjusting means 7 so that the movable stage 1 moves one axis at a fixed distance for each frame unit of the monochrome CCD camera 5.
  • the computer 6 After that, based on these two-dimensional position information and wavelength information, the computer 6 After analyzing and image processing, if necessary, color values R, G, and B are obtained from the intensity of the spectral spectrum at each wavelength of 5 nm, and the state of the surface of sample S is displayed as a color RGB image. Or the spectral spectrum of any point on sample S, or the spectral spectrum of any two points and the difference spectrum simultaneously, or raw data, reflectance (transmittance), etc. Or display the spectral spectrum intensity at an arbitrary wavelength on the surface of sample S as a monochrome gradation image (gray scale). As an example, Fig. 4 shows the spectral spectrum at any point of sample S. In Fig. 4, the horizontal axis represents wavelength, and the vertical axis represents intensity.
  • the hematoxylin and eosin-stained sample S is placed on the movable stage 1, and the movable stage 1 is driven at 1 mm / min, and synchronized with the monochrome CCD camera 5 to obtain a two-dimensional position on the surface of the sample S. Information and wavelength information were collected. At this time, an objective lens 3b having a magnification of 10 was used.
  • A is a site where cancer tissue is not present
  • B is a site with strong eosin staining in the cytoplasm
  • C is a site with weak eosin staining
  • D is a region around the tissue.
  • E indicates the nucleus area, which indicates hematoxin and eosin staining.
  • the spectral spectra at each of the points A to E shown in FIG. 5 are obtained from the spectral spectral information recorded in the real-time spectral image analyzer of the present invention, that is, from the image spectral information.
  • Figure 6 shows the result of the instant image processing.
  • the spectral spectrum shown in FIG. 6 is raw information because it is a spectral spectrum measured using a halogen light source. Therefore, the intensity is weak near 400 nm. Therefore, the data of A in Fig. 6 was set as transmittance 1 and the data values of B, C, D, and E for A were converted, and the data were corrected.
  • the absorption wavelengths at the eosin-stained sites B and C a spectral spectrum having a peak at 5355 nm and a shoulder at 500 nm was observed.
  • the absorption wavelength of E which is the site of hematoxin and eosin staining, has a peak at 5355 nm and a shoulder at 500 nm, as in B and C stained only with eosin.
  • a spectral spectrum with a shoulder was observed up to 100 nm.
  • Orange site D although weakly absorbed, had a peak at 420-460 nm in addition to the peak at the same wavelength as the eosin-stained site.
  • Fig. 8 is an image of sample S at a wavelength of 425 nm
  • Fig. 9 is an image of wavelength 505 nm
  • Fig. 10 is an image of sample S at a wavelength of 535 nm
  • Fig. 11 is an image of sample S at a wavelength of 600 nm.
  • a dark portion is seen around the cancer tissue. This corresponded to an orange absorption band at 420 to 460 nm in the D site in FIG.
  • the images at the wavelengths of 500 nm and 5355 nm showed the eosin-stained sites in the cytoplasm densely.
  • the nucleus was clearly observed from the cytoplasm, and the image was dependent on hematoxin, which had an absorption band at 560 to 600 nm. was clearly confirmed.
  • a to C shown in Fig. 12 indicate that A is a site where no cancer tissue is present, B is a site where cytochrome has strong eosin staining, and C is a nucleus site where hematoxin and eosin staining are present. is there.
  • the spectral spectrum at each of the points A to C shown in FIG. 12 is determined based on the spectral spectrum of A, which is the background, based on the image spectral information recorded in the real-time spectral image analyzer of the present invention.
  • Figures 13 to 15 show the images processed at the instants of the spectral spectrum at points A, B, and C, respectively.
  • B which is a site of eosin staining
  • a spectral spectrum having a peak at 560 nm and a shoulder at 600 nm was observed.
  • the absorption wavelength of hematoxin and eosin-stained C is the same as that of B stained only with eosin, at 560 nm, and has a shoulder from 600 to 700 nm. Spectral spectrum was observed.
  • FIG. 16 is an image of cancer cell tissue at a wavelength of 460 nm.
  • Fig. 17 shows a wavelength of 550 nm
  • Fig. 18 shows a wavelength of 560 nm
  • Fig. 19 shows a wavelength of 600 nm
  • Fig. 20 shows a wavelength of 600 nm
  • Fig. 21 shows a wavelength of 700 nm.
  • It is an image of cancer cell tissue in nm.
  • FIG. 16 in the image of a single wavelength of 46 Onm, a dark portion can be seen around the cancer tissue.
  • an intensity distribution diagram of a spectral spectrum at each wavelength is created, and a stained sample set is prepared. The distribution state of the disease site in the weave was observed.
  • the excised blood vessel was quickly frozen at 180 ° C, and the aortic arch was cut in a cross section to a thickness of 10 ⁇ m using a cryostat, and this was pasted on a slide glass to obtain Sample S. .
  • the sample S is placed on the movable stage 1, and the movable stage 1 is driven at 1 mm / min.
  • the two-dimensional position information and the wavelength of the surface of the sample S are synchronized with the monochrome CCD camera 5. Collected information.
  • an objective lens 3b having a magnification of 10 was used.
  • Npe 6 has a structure in which one asparaginate is attached to the side chain via an amide bond at carbon position 17 of the phosphorus ring at which one double bond in the D ring of the tetrapyrrole ring has been cleaved. It is a photosensitizer with a molecular weight of 799. It is accumulated in pathological tissues such as tumors and atherosclerotic sites, but is immediately excreted from normal tissues. In addition, NP e6 was observed in the phosphate band (pH 394) and the band (502, 530, 620, 654 nm) in the phosphate buffer at pH 7.4, respectively. It has absorption peaks, and these absorption peaks move to the longer wavelength side by about 10 II m when they are taken into biological components such as tumors and atherosclerotic sites.
  • the computer 6 Based on the collected two-dimensional position information and the spectral spectrum as wavelength information, the computer 6 analyzed and processed the image to calculate RGB, and displayed the surface of Sample S as a single RGB image. Further, by using the real-time spectral image analyzer of the present invention, a spectral spectrum at an arbitrary point on the sample S can be instantaneously displayed using image processing by the computer 6, and these spectral spectra can be displayed. By comparing the absorption wavelength of the vector with NPe6, it was possible to quickly diagnose the site where the arteriosclerosis site exists. In addition, a single wavelength image at the absorption wavelength of NP e 6 is also available. It could be displayed instantly using Pewter6, and the location of the atherosclerotic site could be confirmed at a glance.
  • the sample S was placed on the movable stage 1, and the movable stage was moved by 1 mmZ, and two-dimensional position information and wavelength information on the surface of the sample S were collected in synchronization with the monochrome CCD camera 5. At this time, an objective lens 3b having a magnification of 10 was used.
  • hemoglobin contained in blood has two absorption bands of 577 and 540 nm for oxyhemoglobin bound to oxygen, and deoxyhemoglobin for deoxygenated has 5 absorption bands. It has one absorption band at 55 nm.
  • the computer 6 Based on the collected two-dimensional position information and the spectral spectrum as wavelength information, the computer 6 analyzes and processes the image to calculate RGB, and displays the surface of sample S as a single RGB image. See Figure 22.
  • Each of points A and B shown in FIG. 22 shows A when the artery is compressed with a large amount of oxyhemoglobin, and B shows when the artery is released with a large amount of oxyhemoglobin.
  • FIG. 23 shows an image obtained by instantaneously processing the spectral spectrum at each of points A and B shown in FIG. 22 from the image spectral information recorded in the real-time spectroscopic image analyzer of the present invention.
  • the absorption wavelength of A at the time of arterial compression a spectroscopic spectrum having a peak at 5.55 nm was observed.
  • the absorption wavelength of B at the time of releasing the arterial compression a spectral spectrum having peaks at 540 and 5777 nm was observed.
  • the spectral spectrum of FIG. It was confirmed that the absorption bands were consistent with those of hemoglobin and oxyhemoglobin.
  • the real-time spectroscopic image analyzer of the present invention to compare the absorption wavelengths of oxyhemoglobin and deoxyhemoglobin, it is possible to determine a change in the oxygen content in blood, and to determine the presence of red blood cells Diagnosis of the affected part was performed quickly.
  • a single-wavelength image of the absorption wavelength of oxyhemoglobin and dexhemoglobin can be displayed instantaneously using the computer 6, and the site where erythrocytes are present can be checked at a glance. Was completed.
  • Example 5 DNA was analyzed using the real-time spectroscopic imager of the present invention.
  • the fluorescent dye can be used as a primer for each base of the DNA (adenine, thymine, cytosine, guanine).
  • Fluorescent dyes include FAM (5'-carboxyfluorescein), HEX (5'-hexaclofluorescein), TET (5'-tetraclorofluorescein), NED (4—dichloro-1-6-carboxy) Fluorescein) was used.
  • FAM 5'-carboxyfluorescein
  • HEX 5'-hexaclofluorescein
  • TET 5'-tetraclorofluorescein
  • NED 4—dichloro-1-6-carboxy Fluorescein
  • a DNA sample S labeled with a different fluorescent dye for each base is placed on the movable stage 1 and the movable stage 1 is driven at 1 mm / min. Two-dimensional position information and wavelength information of the surface were collected.
  • FAM of the fluorescent dye has an absorption peak at 525 nm
  • HEX has an absorption peak at 556 nm
  • TET has an absorption peak at 536 nm
  • NED has an absorption peak at 560 nm.
  • the spectral spectrum on the sample S could be instantaneously displayed using the image processing by the computer 6.
  • FIGS. 24 to 28 show image-processed spectral spectra of the respective DN samples S labeled with the NED fluorescent dye.
  • Figure 24 shows the spectral spectrum of the DNA sample labeled with the fluorescent dye FAM
  • Figure 25 shows the spectral spectrum of the DNA sample labeled with the fluorescent dye HEX
  • Figure 26 shows the fluorescent dye TET.
  • Sign at Figure 2.7 shows the spectral spectrum of the DNA sample labeled with the fluorescent dye NED.
  • FIG. 28 is a diagram showing the spectral spectrum of each DNA sample labeled with different fluorescent dyes and the spectral spectrum of a DNA sample mixed with four types of fluorescent dyes. It was confirmed that these spectral spectra completely matched the absorption wavelength of each fluorescent dye. As described above, by using the real-time spectroscopic image analyzer of the present invention, a plurality of types of fluorescently labeled DNA could be observed.
  • the present invention can be applied not only to the analysis of fluorescently labeled DNA but also to the analysis of proteins.
  • the real-time spectroscopic image analyzer of the present invention includes a movable stage 1 on which a sample S is mounted, a light source 2 for irradiating the surface of the sample S with white light, and the white light of the sample S
  • a spectroscope 4 for separating the area on one line of the reflected light generated by reflection on the surface at one time, and a spectroscope 4 for separating the spectral spectrum separated from the reflected light by the spectroscope 4 into one-dimensional position information.
  • a monochrome CCD camera 5 that captures wavelength information in one frame, and stores, analyzes, and processes the one-dimensional position information and the wavelength information captured by the monochrome CCD camera 5, as well as a frame of the monochrome CCD camera 5.
  • a computer 6 for operating the movable stage 1 by one axis.
  • the two-dimensional position information and the wavelength information of the sample S can be measured simultaneously and in real time as a spectral image, and reliable and quick measurement for diagnosis can be performed. Also, by using it for analysis and observation of disease sites such as cancer tissue sites and arteriosclerosis sites, it is possible to measure the overall distribution of disease sites in real time. In addition, since spectral distribution data can be obtained instantaneously at a raw wavelength without using an RGB filter, accurate spectral information can be obtained unlike conventional color sensors.
  • the real-time spectroscopic image analysis method of the present invention captures, as one-dimensional position information and wavelength information, spectral spectra obtained by spectrally analyzing a plurality of regions on one line of the surface of the sample S at one time. Two-dimensional position information and wavelength information are obtained from the position information and wavelength information. According to the above configuration, the two-dimensional position information and the wavelength information of the sample S can be measured simultaneously and in real time as a spectral image, and reliable and quick measurement for diagnosis can be performed.
  • the sample S is a DNA or a protein which is a biomolecule whose fluorescence has been recognized.
  • two-dimensional position information and wavelength information of fluorescently labeled DNA and protein can be measured simultaneously and in real time as a spectral image, and reliable and quick measurement for diagnosis can be performed.
  • a clear spectral image can be obtained even for a sample with low illuminance of light.
  • the present invention is not limited to the above embodiment, and various modifications can be made within the scope of the present invention.
  • it can be used for various purposes other than diagnosis, such as image recognition related to color and chemical composition.
  • the real-time spectroscopic image analyzer of the present invention may be incorporated in an endoscope for diagnosis.

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  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un dispositif d'analyse d'image spectrale en temps réel ainsi qu'un procédé d'analyse permettant de mesurer des informations de position bidimensionnelles et des informations de longueur d'onde relatives à un échantillon sous forme d'image spectrale de manière simultanée et en temps réel. Le dispositif comprend un étage mobile (1) sur lequel un échantillon S est placé, une source de lumière (2) pouvant émettre une lumière blanche sur une surface de l'échantillon S, un spectroscope (4) destiné à l'analyse spectrale à un degré de la zone sur une ligne de la lumière réfléchie produite par réflexion de la lumière blanche à partir de la surface de l'échantillon S, une caméra CCD noir-blanc (5) permettant d'acquérir le spectre de la lumière de réflexion obtenu par le spectroscope (4) sous forme d'informations de position unidimensionnelles et d'informations de longueur d'onde dans une trame, ainsi qu'un ordinateur (6) servant à stocker, analyser et soumettre à un traitement d'image les informations de position unidimensionnelles et les informations de longueur d'onde acquises au moyen de la caméra CCD noir-blanc (5), l'étage mobile (1) étant amené à réaliser une opération monoaxiale sur la base de la trame de la caméra CCD noir-blanc (5).
PCT/JP2003/016891 2003-01-09 2003-12-26 Dispositif d'analyse d'image spectrale en temps reel et procede d'analyse Ceased WO2004063683A1 (fr)

Priority Applications (1)

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AU2003292679A AU2003292679A1 (en) 2003-01-09 2003-12-26 Real time spectral image analysis device and analysis method

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JP2003-003266 2003-01-09
JP2003003266A JP2004219092A (ja) 2003-01-09 2003-01-09 リアルタイム分光画像分析装置及び分析方法

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WO2004063683A1 true WO2004063683A1 (fr) 2004-07-29

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JP (1) JP2004219092A (fr)
AU (1) AU2003292679A1 (fr)
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US9183427B2 (en) 2011-09-29 2015-11-10 Hoya Corporation Diagnostic system

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JP2005114530A (ja) 2003-10-07 2005-04-28 Olympus Corp 画像表示装置及び画像表示方法
JP4112469B2 (ja) 2003-10-07 2008-07-02 オリンパス株式会社 マルチバンドカメラの制御装置及び制御方法
JP4072108B2 (ja) * 2003-10-07 2008-04-09 オリンパス株式会社 画像表示装置及び画像表示方法
JP2008070389A (ja) * 2007-12-03 2008-03-27 Olympus Corp 画像表示装置及び画像表示方法
JP5147468B2 (ja) * 2008-03-11 2013-02-20 キヤノン株式会社 計測装置および露光装置
JP6244945B2 (ja) 2014-01-29 2017-12-13 セイコーエプソン株式会社 電子機器
JP2016011844A (ja) * 2014-06-27 2016-01-21 セイコーエプソン株式会社 分光画像撮像システム、及び分光画像撮像システムの制御方法
DE102021126145A1 (de) * 2021-10-08 2023-04-13 Heidelberg Engineering Gmbh Anordnung mit einem Detektor

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Publication number Priority date Publication date Assignee Title
JPH02226027A (ja) * 1989-02-27 1990-09-07 Hamamatsu Photonics Kk 分光解析装置
JPH08128899A (ja) * 1994-10-31 1996-05-21 Satoshi Kawada 分光画像収集装置
WO2000006980A1 (fr) * 1998-07-27 2000-02-10 Cedars-Sinai Medical Center Topographie spectrale de matiere d'origine mammalienne
JP2000356552A (ja) * 1999-06-11 2000-12-26 Kawatetsu Techno Res Corp 印刷面の色彩・濃度測定方法および装置
JP2002048719A (ja) * 2000-08-07 2002-02-15 Glory Ltd 光学鑑定装置
JP2003214951A (ja) * 2002-01-28 2003-07-30 Matsushita Electric Works Ltd 分光計測装置及び分光計測方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02226027A (ja) * 1989-02-27 1990-09-07 Hamamatsu Photonics Kk 分光解析装置
JPH08128899A (ja) * 1994-10-31 1996-05-21 Satoshi Kawada 分光画像収集装置
WO2000006980A1 (fr) * 1998-07-27 2000-02-10 Cedars-Sinai Medical Center Topographie spectrale de matiere d'origine mammalienne
JP2000356552A (ja) * 1999-06-11 2000-12-26 Kawatetsu Techno Res Corp 印刷面の色彩・濃度測定方法および装置
JP2002048719A (ja) * 2000-08-07 2002-02-15 Glory Ltd 光学鑑定装置
JP2003214951A (ja) * 2002-01-28 2003-07-30 Matsushita Electric Works Ltd 分光計測装置及び分光計測方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9183427B2 (en) 2011-09-29 2015-11-10 Hoya Corporation Diagnostic system

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JP2004219092A (ja) 2004-08-05
AU2003292679A8 (en) 2004-08-10

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