JP2013011527A - Fluorescence microscope system and quantitative method of fluorescent substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence microscope system capable of always highly accurately quantifying a fluorescent substance in a measurement sample.SOLUTION: The fluorescence microscope system obtains a fluorescent image of a measurement sample 30 by a fluorescence microscope 10 and quantifies a fluorescent substance from the obtained fluorescent image using calibration curves. The fluorescence microscope system includes: a distance measurement part 110 for measuring distance from a surface of the measurement sample 30 to a part of which fluorescent image is obtained; a storage part 120 for storing multiple calibration curves according to distance from the surface of the measurement sample 30; and a fluorescent substance quantifying part 130 for selecting a calibration curve corresponding to the distance measured at the distance measurement part 110, from the storage part 120 and quantifying the fluorescent substance from the fluorescent image using the selected calibration curve.

Description

  The present invention relates to a fluorescence microscope system for quantifying a fluorescent substance in a measurement specimen and a fluorescent substance quantification method.

  In a fluorescence microscope system that quantifies fluorescent substances in measurement specimens such as tissues and cells, a fluorescence image of the measurement specimen is acquired using a fluorescence microscope, and a calibration indicating the relationship between the fluorescence intensity and the concentration from the acquired fluorescence image The fluorescent substance is quantified using a line (see, for example, Patent Document 1).

JP 2007-155549 A

  However, in the conventional fluorescence microscope system, the fluorescent substance of the measurement specimen is quantified using one calibration curve. Therefore, for example, using a calibration curve created with a fluorescent substance at a short distance (shallow position) from the surface of the standard specimen for the calibration curve, the fluorescent substance at a long distance (deep position) from the surface of the measurement specimen is quantified. There is a concern that the influence of fluorescence scattering cannot be ignored and the quantitative accuracy is lowered.

  Accordingly, an object of the present invention made in view of such a point is to provide a fluorescence microscope system and a fluorescent substance quantification method capable of always quantitatively quantifying a fluorescent substance of a measurement specimen with high accuracy.

The invention of the fluorescence microscope system according to the first aspect for achieving the above object is as follows:
A fluorescence microscope system that acquires a fluorescence image of a measurement specimen with a fluorescence microscope and quantifies a fluorescent substance using a calibration curve from the acquired fluorescence image,
A distance measuring unit for measuring a distance from the surface of the measurement specimen for acquiring the fluorescence image;
A storage unit for storing a plurality of calibration curves according to the distance from the surface of the measurement sample;
Selecting a calibration curve corresponding to the distance measured by the distance measurement unit from the storage unit, and using the selected calibration curve, a fluorescent substance quantification unit that quantifies the fluorescent substance from the fluorescence image;
It is characterized by providing.

The invention according to the second aspect is the fluorescence microscope system according to the first aspect,
The fluorescence microscope has a lens position detection unit that detects a lens position of an objective lens for forming the fluorescence image,
The distance measuring unit measures a distance from the surface of the measurement specimen based on a focus position characteristic indicating a focus position of the objective lens with respect to lens position information from the lens position detecting unit. is there.

The invention according to the third aspect is the fluorescence microscope system according to the first or second aspect,
The fluorescence microscope is a confocal laser microscope.

The invention according to a fourth aspect is the fluorescence microscope system according to the first or second aspect,
The fluorescence microscope comprises a transmission type fluorescence microscope, and acquires the fluorescence image by dark field observation.

The invention according to a fifth aspect is the fluorescence microscope system according to any one of the first to fourth aspects,
The fluorescence microscope is characterized in that a fluorescence image in the near infrared region is acquired from the measurement specimen.

The invention according to a sixth aspect is the fluorescence microscope system according to any one of the first to fourth aspects,
The fluorescence microscope has a multispectral camera that acquires the fluorescence image.

Furthermore, the invention of the fluorescent substance quantification method according to the seventh aspect of achieving the above object,
In obtaining a fluorescence image of a measurement specimen with a fluorescence microscope and quantifying a fluorescent substance using a calibration curve from the acquired fluorescence image,
In the distance measuring unit, measuring the distance from the surface of the measurement specimen to obtain the fluorescence image;
In the fluorescent substance quantifying unit, selecting a calibration curve corresponding to the measured distance from the storage unit, and quantifying the fluorescent substance from the fluorescent image using the selected calibration curve;
It is characterized by including.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescence microscope system which can always quantify the fluorescent substance of a measurement sample with high precision, and the quantitative determination method of a fluorescent substance can be provided.

It is the schematic which shows the structure of the principal part of the fluorescence microscope system which concerns on 1st Embodiment of this invention. It is an enlarged view of the preparation of FIG. It is a block diagram of the distance measurement part of FIG. It is a figure which shows the calibration curve memorize | stored in the memory | storage part of FIG. It is a figure which shows the correspondence of the calibration curve of FIG. 4, and the distance from the surface of a measurement sample. FIG. 2 is a block diagram of a fluorescent substance quantification unit in FIG. 1. It is a flowchart explaining operation | movement of 1st Embodiment. It is an enlarged view of a preparation in a 2nd embodiment. It is a flowchart explaining operation | movement of 2nd Embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a schematic diagram showing the configuration of the main part of the fluorescence microscope system according to the first embodiment of the present invention. The fluorescence microscope system shown in FIG. 1 includes a fluorescence microscope 10 and an arithmetic processing unit 100.

  The fluorescence microscope 10 includes a known fluorescence microscope such as a transmission fluorescence microscope, an epi-illumination fluorescence microscope, a confocal laser microscope, and the like. FIG. 1 illustrates the case where the fluorescence microscope 10 is an epi-illumination fluorescence microscope, and includes an observation light source 11, a specimen stage 12, a fluorescence excitation light source 13, an objective lens 14, a lens position detection unit 15, and a camera 16.

  On the specimen stage 12, a preparation 31 having a measurement specimen 30 such as a tissue stained (labeled) with a fluorescent substance is placed. As shown in an enlarged view in FIG. 2, the preparation 31 is produced by holding the measurement specimen 30 together with the encapsulant 34 between the slide glass 32 and the cover glass 33. In the present embodiment, as the cover glass 33, the fluorescent dyes 35 and 36 to be measured are respectively applied to the upper surface and the lower surface of the region where the thickness d1 is known, for example, d1 = 50 μm and the measurement specimen 30 is not located. Use the same thing.

  The fluorescence excitation light source 13 includes an ultra-high pressure mercury lamp, a xenon lamp, an ultraviolet LCD, a laser light source, and the like, and is driven when the fluorescent substance in the measurement specimen 30 is quantified. The excitation light emitted from the fluorescence excitation light source 13 is reflected by, for example, the dichroic mirror 18 through the excitation filter 17, and is applied to the measurement sample 30 through the objective lens 14. As a result, the fluorescent material of the measurement specimen 30 is excited and fluorescence is generated from the measurement specimen 30. This fluorescence is incident on the camera 16 through the objective lens 14, the dichroic mirror 18, and the absorption filter 19, and a fluorescence image is formed.

  The focus position of the objective lens 14 is adjusted manually or automatically by a focus adjustment mechanism such as a focus handle or an autofocus mechanism. As the autofocus mechanism, a known method such as a passive optical path difference method, a contrast method, or an active phase difference method using a knife edge can be employed.

  The lens position detection unit 15 detects the lens position of the objective lens 14. The detected lens position information is supplied to the arithmetic processing unit 100. The camera 16 is configured using a CCD camera, a CMOS camera, a photomultiplier, a multispectral camera, or the like according to the fluorescent image to be captured. The image signal of the measurement specimen 30 captured by the camera 16 is supplied to the arithmetic processing unit 100.

  The observation light source 11 is driven when a transmission optical image of the measurement specimen 30 is observed. The illumination light emitted from the observation light source 11 is irradiated to the measurement specimen 30 through an illumination optical system including the reflection mirror 20, and the transmitted light passes through the objective lens 14 to the eyepiece optical system (not shown) or the camera 16. Led. As a result, similarly to the fluorescence image, the transmission optical image of the measurement specimen 30 can be observed through the eyepiece optical system, and the transmission optical image captured by the camera 16 is displayed on a dedicated display unit or an arithmetic processing unit described later. 100 is displayed on the display unit.

  The arithmetic processing unit 100 includes a distance measurement unit 110, a storage unit 120, a fluorescent substance quantification unit 130, a control unit 140, and a display unit 150.

  The distance measurement unit 110 measures the distance from the surface from which the fluorescence image of the measurement specimen 30 is acquired, and includes a storage unit 111 and a distance calculation unit 112, for example, as shown in FIG. The storage unit 111 stores the thickness information of the cover glass 33 used for the preparation 31 of the measurement specimen 30, and the lens position information from the lens position detection unit 15 and the image signal from the camera 16. Remembered. Further, the storage unit 111 stores a focus position characteristic indicating a focus position with respect to lens position information of an objective lens 14 described later. The image signal stored in the storage unit 111 is output to the fluorescent substance quantification unit 130 as appropriate.

  The distance calculation unit 112 calculates a focus position characteristic based on the thickness information of the cover glass 33 and the lens position information stored in the storage unit 111 and stores them in the storage unit 111. Further, the distance calculation unit 112 is based on the focus position characteristic stored in the storage unit 111 and the lens position information from the lens position detection unit 15, and the distance of the focus position from the lower surface of the cover glass 33, that is, the measurement sample. The distance from the surface from which 30 fluorescent images are acquired is calculated. The distance calculated by the distance calculation unit 112 is output to the fluorescent substance quantification unit 130 as distance information.

  The storage unit 120 stores various data such as a plurality of calibration curves indicating the relationship between the fluorescence intensity and the concentration according to the distance from the surface of the measurement specimen 30 as shown in FIG. FIG. 4 shows five types of calibration curves from the calibration curve 1 to the calibration curve 5, and the correspondence between each calibration curve and the distance from the surface of the measurement sample 30 is as shown in FIG. ing. As apparent from FIGS. 4 and 5, the fluorescence intensity decreases as the distance from the surface of the measurement specimen 30 increases even at the same concentration.

  The fluorescent substance quantification unit 130 includes a calibration curve selection unit 131 and a fluorescent molecule quantification unit 132, for example, as shown in FIG. The calibration curve selection unit 131 selects a corresponding calibration curve from the storage unit 120 based on the distance information from the distance calculation unit 112. The fluorescent molecule quantification unit 132 quantifies the fluorescent substance in the image signal (fluorescence image) from the distance calculation unit 112 using the calibration curve selected by the calibration curve selection unit 131. The quantitative result is output to the control unit 140 as quantitative information.

  The control unit 140 controls the overall operation of the fluorescence microscope 10 and the arithmetic processing unit 100, and is configured by a personal computer, for example. Then, the control unit 140 displays the quantitative information from the fluorescent substance quantitative unit 130 on the display unit 150. Moreover, the control part 140 displays the fluorescence image and transmission optical image of the measurement sample 30 which were acquired with the fluorescence microscope 10 on the display part 150 as needed.

  Hereinafter, a method for quantifying a fluorescent substance using the fluorescence microscope system according to the present embodiment will be described with reference to the flowchart shown in FIG.

  In the present embodiment, the preparation 31 as shown in FIG. 2 is set on the specimen stage 12 of the fluorescence microscope 10 and the fluorescent excitation light source 13 is turned on to quantify the fluorescent substance. Here, the preparation 31 includes, for example, when the measurement specimen 30 is a pathological tissue specimen of a lung adenocarcinoma patient, a fluorescently labeled anti-TFF1 antibody that specifically binds to the nucleus of a human lung cancer cell on the collected human lung cancer pathological specimen. It is made by bonding.

  First, the distance measuring unit 110 acquires lens position information output from the lens position detecting unit 15 when the objective lens 14 is focused on the upper surface of the cover glass 33 (step S11). Here, the focusing operation of the objective lens 14 on the upper surface of the cover glass 33 is performed manually or automatically with reference to the image of the fluorescent dye 35 applied on the upper surface of the cover glass 33. The lens position information on the upper surface of the cover glass 33 is stored in the storage unit 111.

  Next, the distance measuring unit 110 acquires lens position information output from the lens position detecting unit 15 when the objective lens 14 is focused on the lower surface of the cover glass 33 (step S12). Here, the focusing operation of the objective lens 14 on the lower surface of the cover glass 33 is performed manually or automatically with reference to the image of the fluorescent dye 36 applied on the lower surface of the cover glass 33, as in step S11. Is called. The lens position information on the lower surface of the cover glass 33 is stored in the storage unit 111. Note that step S11 and step S12 may be performed in the reverse order.

  Thereafter, the distance measuring unit 110, in the distance calculating unit 112, the thickness information of the cover glass 33 stored in the storage unit 111, and the lens position information corresponding to the acquired focus positions of the upper and lower surfaces of the cover glass 33, Based on the above, the focus position characteristic indicating the focus position with respect to the lens position information of the objective lens 14 is calculated (step S13). This focus position characteristic is stored in the storage unit 111.

  After calculating the focus position characteristic of the objective lens 14 as described above, the distance calculation unit 112 thereafter receives the lens position information and the image signal from the lens position detection unit 15 and uses the calculated focus position characteristic. The distance of the focus position from the surface of the measurement specimen 30 corresponding to the lens position information is calculated (step S14). The calculated distance information is stored in the storage unit 111 and output to the fluorescent substance quantification unit 130. Further, the image signal at that time is also output to the fluorescent substance quantification unit 130.

  When receiving the distance information from the distance measuring unit 110, the fluorescent substance quantifying unit 130 selects a calibration curve corresponding to the received distance information from the storage unit 120 in the calibration curve selecting unit 131 (step S15). The selected calibration curve information is supplied to the fluorescent molecule quantification unit 132.

  Thereafter, the fluorescent molecule quantification unit 132 quantifies the fluorescent substance in the received image signal (fluorescence image) using the selected calibration curve information (step S16). The quantitative result is displayed on the display unit 150 via the control unit 140 (step S17).

  As described above, according to the fluorescence microscope system according to the present embodiment, the fluorescence image acquired at the focus position by selecting the calibration curve corresponding to the distance from the surface of the measurement specimen 30 on which the objective lens 14 is focused. Since the fluorescent substance is quantified from the above, the influence of fluorescence scattering can be reduced, and the fluorescent substance can be quantified with high accuracy. In particular, when the fluorescence wavelength region of the fluorescent material is a wavelength in the near infrared region, by selecting an appropriate calibration curve according to the distance from the sample surface, even when the distance from the sample surface is longer, the tissue Accurate quantification is possible without being affected by the scattering of the light.

  In the present embodiment, since the distance from the surface of the measurement specimen 30 is measured based on the focus position characteristic indicating the focus position with respect to the lens position information of the objective lens 14, it is possible to measure the distance quickly. Measurement time can be shortened.

  Further, when the measurement specimen 30 is stained with a plurality of types of fluorescent substances, it is possible to automatically and accurately quantify the plurality of types of fluorescent molecules by using a known multispectral camera as the camera 16.

  Further, when a confocal laser microscope is used as the fluorescence microscope 10, the fluorescent excitation range can be accurately limited, and thus the fluorescent substance can be quantified with higher accuracy.

(Second Embodiment)
In the second embodiment of the present invention, in the fluorescence microscope system described in the first embodiment, a slide glass 32 is used instead of the cover glass 33 of the preparation 31, and the focus position with respect to the lens position information of the objective lens 14 is used. The focus position characteristic indicating is calculated. Therefore, as shown in an enlarged view of the preparation 31 in FIG. 8, as the slide glass 32, the thickness d2 is known, for example, d2 = 1000 μm, and the upper surface and the lower surface of the region where the measurement specimen 30 is not located, respectively. The one to which the fluorescent dyes 37 and 38 to be measured are applied is used. Further, the measurement specimen 30 placed on the slide glass 32 has a thickness d3 of, for example, d3≈5 μm.

  In addition, the storage unit 111 of the distance measurement unit 110 stores the thickness information of the measurement specimen 30 in addition to the thickness information of the slide glass 32 to be used. Other configurations are the same as those of the first embodiment.

  Hereinafter, the fluorescent substance quantification method using the fluorescence microscope system according to the present embodiment will be described according to the flowchart of FIG. 9 with reference to the configuration of the first embodiment and FIG.

  First, in the distance measuring unit 110, when the objective lens 14 is focused on the fluorescent dye 37 applied on the upper surface of the slide glass 32, lens position information output from the lens position detecting unit 15 is acquired (step S21). . Next, in the distance measurement unit 110, when the objective lens 14 is focused on the fluorescent dye 38 applied to the lower surface of the slide glass 32, the lens position information output from the lens position detection unit 15 is used as the distance measurement unit 110. (Step S22).

  Thereafter, in the distance calculation unit 112, based on the thickness information of the slide glass 32 stored in the storage unit 111 and the obtained lens position information corresponding to the focus positions of the upper and lower surfaces of the slide glass 32, the objective lens The focus position characteristic indicating the focus position with respect to the lens position information of 14 is calculated (step S23).

  After calculating the focus position characteristic of the objective lens 14 as described above, the distance calculation unit 112 receives the lens position information and the image signal from the lens position detection unit 15 and then calculates the calculated focus position characteristic and measurement sample. The distance of the focus position from the surface of the measurement specimen 30 corresponding to the lens position information is calculated using the thickness information 30 (step S24).

  Here, the thickness d3 of the measurement sample 30 is defined as d3≈5 μm. Therefore, based on the received lens position information, for example, the distance of the focus position from the upper surface of the slide glass 32 is calculated, and if the distance is subtracted from 5 μm which is the thickness of the measurement specimen 30, the distance from the surface of the measurement specimen 30 The distance of the focus position is calculated. The calculated distance information is stored in the storage unit 111 and output to the fluorescent substance quantification unit 130. Further, the image signal at that time is also output to the fluorescent substance quantification unit 130.

  Thereafter, similarly to steps S15 to S17 in FIG. 7, the calibration curve selection unit 131 of the fluorescent substance quantification unit 130 selects a calibration curve corresponding to the received distance information from the storage unit 120 (step S25). Using the selected calibration curve information, the fluorescent molecule quantification unit 132 quantifies the fluorescent substance of the received image signal (fluorescence image) (step S26), and the quantification result is displayed on the display unit 150 via the control unit 140. It is displayed (step S27).

  Therefore, also in this embodiment, the same effect as that of the first embodiment can be obtained.

  In addition, this invention is not limited only to the said embodiment, Many deformation | transformation or a change is possible. For example, it is possible to use a transmission fluorescence microscope as a fluorescence microscope and acquire a fluorescence image by dark field observation to quantify the fluorescent substance. In this case, the background is completely dark, and a high contrast image can be obtained from weak fluorescence. Therefore, coupled with the selection of a calibration curve corresponding to the distance (depth) from the surface of the measurement specimen, the fluorescent substance can be quantified with high accuracy and high sensitivity.

  Further, the focus position characteristic of the fluorescence microscope is determined by a microscope optical system including an objective lens. Therefore, by using the focus position characteristic of the fluorescence microscope, the focus position characteristic is previously associated with the lens position of the top surface or back surface of the cover glass with a known thickness or the top surface or back surface of the slide with a known thickness. It is also possible to calculate the distance of the focus position from the surface of the measurement specimen with respect to the lens position information.

  In the above embodiment, the fluorescent dye is applied to the upper and lower surfaces of the objective lens on the cover glass or the slide glass. However, depending on the fluorescent excitation light source used, objectives other than the fluorescent dye are used. The lens can be any marker that can be focused.

DESCRIPTION OF SYMBOLS 10 Fluorescence microscope 11 Observation light source 12 Specimen stage 13 Fluorescence excitation light source 14 Objective lens 15 Lens position detection part 16 Camera 30 Measurement sample 31 Preparation 32 Slide glass 33 Cover glass 34 Encapsulant 35, 36, 37, 38 Fluorescent dye 100 Calculation Processing unit 110 Distance measurement unit 111 Storage unit 112 Distance calculation unit 120 Storage unit 130 Fluorescent substance quantification unit 131 Calibration curve selection unit 132 Fluorescent molecule quantification unit 140 Control unit 150 Display unit

Claims (7)

A fluorescence microscope system that acquires a fluorescence image of a measurement specimen with a fluorescence microscope and quantifies a fluorescent substance using a calibration curve from the acquired fluorescence image,
A distance measuring unit for measuring a distance from the surface of the measurement specimen for acquiring the fluorescence image;
A storage unit for storing a plurality of calibration curves according to the distance from the surface of the measurement sample;
Selecting a calibration curve corresponding to the distance measured by the distance measurement unit from the storage unit, and using the selected calibration curve, a fluorescent substance quantification unit that quantifies the fluorescent substance from the fluorescence image;
A fluorescence microscope system comprising: The fluorescence microscope has a lens position detection unit that detects a lens position of an objective lens for forming the fluorescence image,
The distance measurement unit measures a distance from the surface of the measurement specimen based on a focus position characteristic indicating a focus position of the objective lens with respect to lens position information from the lens position detection unit. The fluorescence microscope system according to 1.   The fluorescence microscope system according to claim 1, wherein the fluorescence microscope is a confocal laser microscope.   The fluorescence microscope system according to claim 1, wherein the fluorescence microscope includes a transmission fluorescence microscope, and acquires the fluorescence image by dark field observation.   The fluorescence microscope system according to any one of claims 1 to 4, wherein the fluorescence microscope acquires a fluorescence image in a near infrared region from the measurement specimen.   The fluorescence microscope system according to claim 1, wherein the fluorescence microscope includes a multispectral camera that acquires the fluorescence image. In obtaining a fluorescence image of a measurement specimen with a fluorescence microscope and quantifying a fluorescent substance using a calibration curve from the acquired fluorescence image,
In the distance measuring unit, measuring the distance from the surface of the measurement specimen to obtain the fluorescence image;
In the fluorescent substance quantifying unit, selecting a calibration curve corresponding to the measured distance from the storage unit, and quantifying the fluorescent substance from the fluorescent image using the selected calibration curve;
A method for quantifying a fluorescent substance, comprising: