JP2001013135A - Fluorescence observation method and fluorescence observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence observation method capable of high-precision and high-sensitivity observation relative to a sample labeled by a phosphor handled easily, and capable of executing observation easily by using a general optical microscope. SOLUTION: A biological tissue sample 32 labeled by a phosphor is loaded on a slide glass 30. A chemiluminescent reagent 34 emitting excitation light for exciting the phosphor is dropped onto the biological tissue sample 32, and thereafter a cover glass 36 is put thereon to form a preparation. Optical filters 70, 72 for cutting excitation light and transmitting fluorescence are formed on the surfaces facing to the inside of the preparation, of the slide glass 30 and the cover glass 36, namely, the surfaces abutting on the chemiluminescent reagent 34. By this formation, excitation light emitted from the chemiluminescent reagent 34 held between the slide glass 30 and the cover glass 36 does not enter the slide glass 30 and the cover glass 36, to suppress generation of pseudo fluorescence therein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence observation method, that is, a method for observing the presence and occurrence of a specific substance or the like corresponding to a probe using a probe labeled with a fluorescent substance.

[0002]

2. Description of the Related Art Conventionally, immunostaining and ISH (in situ hybridization) methods used for observing biological tissues have been used.
Radioactive isotopes such as ³ H and ³² P (RI: Radioisotop
The treatment is performed with the reagent containing the probe labeled in e). The probe stays where the target substance (for example, protein or gene) exists, and observes the distribution of the radioisotope of the label by autoradiography to detect the protein or gene that specifically occurs in the sample. The existence and distribution of the target substance such as are analyzed.

[0003] When using this RI-labeled probe, if the RI is short-lived, the RI disappears before observation is performed. Therefore, the average life of the RI is long to some extent. For that, and for accurate analysis, R
Since it is necessary to accumulate the number of events of I decay, there is an inconvenience that observation using RI is generally time-consuming. In addition, since the use of the RI substance in a dedicated facility is particularly required, there is an inconvenience that handling is complicated in that point.

[0004] In recent years, non-radioactive methods have attracted attention and have been used. As one of the non-radioactive methods, an enzyme such as HRP (horseradish peroxidase) is used as a label, and the distribution of the target substance is visualized by an enzyme histochemical method, or an antigenic substance such as biotin, DIG is used as a label. There is a method of performing observation by visualizing the distribution of a target substance using an immunohistochemical technique.

[0005] A disadvantage of these methods of labeling with an enzyme or an antigenic substance is that the labeling substance must be treated by an enzyme histochemical technique or an immunohistochemical technique, and the processing steps are complicated. In general, quantitative analysis is difficult with these methods.

As a technique for solving the above problem, another non-radioactive method using a probe labeled with a fluorescent substance is known. In a method called the FISH method, a fluorescent substance-labeled probe such as FITC is used, and fluorescence emitted from the fluorescent substance is observed using a confocal fluorescence microscope or the like. The biological tissue sample is irradiated with an excitation light source such as a laser. The labeling substance is excited by the excitation light source and emits fluorescence (including phosphorescence) when transitioning from the excited state to a lower energy state. By measuring the generation and intensity of this fluorescence, the presence and concentration of the target substance can be observed. In addition, since fluorescence is generated almost simultaneously with irradiation of excitation light and repeatedly generated each time excitation light is applied, there is an advantage that the time and accuracy required for observation are good.

FIG. 5 is a schematic diagram showing a schematic configuration of a laser fluorescence microscope apparatus based on a conventional observation method using a fluorescent substance-labeled probe. A sample 6 such as a biological tissue section is sandwiched between a cover glass 2 and a slide glass 4 together with a filler 8 such as glycerin. The sample 6 is irradiated with laser light from a laser light generator 10. The fluorescent substance added as a label to the probe in sample 6 is as follows:
It is excited by this laser light to generate fluorescence. This fluorescence is observed with the microscope 12. The light generated from the sample 6 is transmitted to the bandpass filter 1 placed in the microscope 12.
In step 4, only the wavelength associated with the labeled phosphor is selected. The amount of fluorescence that has passed through the bandpass filter 14 is measured by the photomultiplier unit 16.

[0008]

Generally, as described above,
When a sample such as a tissue section is observed clearly, a section glass fixed on a slide glass is covered with a cover glass via a filler such as glycerin. The cover glass also has a function of holding a liquid sample such as blood on a slide glass. When observing a sample sandwiched between such a cover glass and a slide glass, in the above-described method using the conventional fluorescent labeling probe, excitation light such as a laser is transmitted to the sample through the cover glass. . The light transmitted through the sample also enters the slide glass. When excitation light is incident on these slide glasses, cover glasses, or the like, light (pseudo fluorescence) in the same wavelength region as the fluorescence emitted by the labeling substance can be generated from those members. This pseudo fluorescence becomes noise with respect to the fluorescence from the sample. Also,
The scattering component of the excitation light in these members and the like also causes a background. Therefore, the conventional method for observing a sample using a fluorescent-labeled probe has a problem in that the S / N ratio is reduced, and it is difficult to detect a target substance with high sensitivity. In addition, there is a problem that an expensive optical system such as a special laser generator or a confocal fluorescence microscope is required for the observation.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to enable high-precision and high-sensitivity observation of a sample labeled with a phosphor which is easy to handle. An object of the present invention is to provide a fluorescence observation method that can easily perform observation using a microscope.

[0010]

According to the fluorescence observation method of the present invention, a sample is prepared by sandwiching a luminescent reagent emitting excitation light between a first plate and a second plate together with a sample labeled with a fluorescent substance. A specimen preparation step; and an observation step of observing fluorescence emitted by the phosphor upon receiving the excitation light from outside the specimen through the first plate.

According to the present invention, the luminescent reagent is used as a luminescent source for exciting the phosphor, and the luminescent source is held between the first plate and the second plate together with the sample labeled with the phosphor. . Therefore, the phosphor placed in the space between the plates is irradiated with the excitation light generated from inside the space. That is, the excitation light from the light emission source is applied to the phosphor without passing through both the first plate and the second plate, and the phosphor emits fluorescence. Here, the first plate is a plate of the two plates on which an observation unit such as a microscope or a light receiving element for observing fluorescence is arranged. The observation means can observe the fluorescence that has passed through the first plate, and analyze the distribution and concentration of the substance labeled with the phosphor in the sample based on the distribution and intensity. Now, when the excitation light is incident on a substance such as the first plate or the second plate, there is a possibility that pseudo fluorescence is generated or scattered. According to the present invention, since the excitation light is generated in the internal space between the plates as described above, the generation of pseudo-fluorescence and the generation of scattered light that are noises for the observation of the fluorescence are suppressed.

In the fluorescence observation method according to the present invention, the first plate has a filter function for blocking the transmission of the excitation light on a surface facing the sample.

According to the present invention, the excitation light generated in the gap between the plates is suppressed from being incident on the first plate. Therefore, the generation of pseudo-fluorescence or scattering due to the excitation light in the first plate, which is transmitted to the outside and observed by the observation means is suppressed.

In the fluorescence observation method according to the present invention, the second plate has a filter function for blocking transmission of the excitation light on a surface facing the sample.

According to the present invention, the excitation light generated in the gap between the plates is suppressed from being incident on the second plate. Pseudo-fluorescence and scattering generated in the second plate by the excitation light return to the first plate side by being reflected at the boundary surface of the second plate, and adversely affect the fluorescence observation. But,
According to the present invention, the adverse effect is suppressed by suppressing the pseudo-fluorescence and scattering in the second plate.

A preferred embodiment of the present invention is a fluorescence observation method in which the first plate is a cover glass and the second plate is a slide glass.

[0017] The fluorescence observation device according to the present invention has a temperature control means for controlling the temperature of the luminescent reagent and the sample held in the gap between the first plate and the second plate.

Generally, the reaction rate of a luminescent reagent is affected by temperature. According to the present invention, by controlling the temperature of the luminescent reagent itself and the temperature of the sample in contact with the luminescent reagent, it is possible to control the generation rate of the excitation light and adjust the observation time.

The fluorescence observation apparatus according to the present invention is an apparatus for observing a specimen prepared by sandwiching a luminescent reagent emitting excitation light between a first plate and a second plate together with a sample labeled with a fluorescent substance. And an observation step of observing fluorescence emitted by the phosphor upon receiving the excitation light from outside the specimen through the first plate.

According to the present invention, the phosphor placed in the space between the plates is irradiated with the excitation light generated from inside the space. That is, since the excitation light can excite the phosphor without passing through the first and second plates, the generation of pseudo-fluorescence and scattered light in the first and second plates is suppressed, and noise for observation of the fluorescence is reduced. Is reduced.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below. FIG. 1 shows a configuration of an observation device using the biological tissue observation method according to the present invention. The biological tissue sample 32 affixed to the slide glass 30 is ISH
Fluorescent labeling by the method or immunohistochemistry.

The biological tissue sample 32 is a slide glass 3
0 and the chemiluminescent reagent 34 is
Several hundred μl are dropped and covered with a cover glass 36 thereon.

The biological tissue sample 32 is covered with a cover glass 36.
Is conventionally performed in order to obtain a clear microscope image. A preparation formed by sandwiching the biological tissue sample 32 between the cover glass 36 and the slide glass 30 is mounted on the stage of the microscope.

The chemiluminescent reagent 34 is used for the biological tissue sample 3
2 emits light of a wavelength that excites the fluorescent substance labeling 2.
FIG. 2 is an example of the emission and absorption characteristics of the phosphor. In the figure, the horizontal axis corresponds to the wavelength of light, and the vertical axis represents the relative intensity with the peak of each characteristic curve taken as 100%. Also,
The solid characteristic curve is the absorption characteristic, and the dotted characteristic curve is the emission characteristic. In the example shown in the figure, the maximum absorption wavelength is 780 n.
m, and the maximum fluorescence wavelength is 820 nm. The phosphor is excited by absorbing light having a wavelength shorter than the fluorescence wavelength, that is, light having a high energy, and emits light having a wavelength corresponding to the energy difference between the two states when returning from the excited state to the ground state. This is the fluorescence. Phosphorescence is basically generated by a similar mechanism. In general, the excited state at the time of emitting fluorescence is a state in which excitation energy has been partially lost through various processes from a state excited by absorption, and thus the maximum fluorescence wavelength is larger than the maximum absorption wavelength.

The chemiluminescent reagent 34 is selected so as to emit light having a wavelength shorter than the maximum fluorescence wavelength of the phosphor, generally preferably having a maximum absorption wavelength. As described above, various chemiluminescent reagents are used depending on the fluorescent substance. In order to observe a biological tissue as in the present apparatus, for example, a substance that produces peroxalate chemiluminescence, luminol luminescence, lucigenin luminescence, and gallic acid luminescence can be used.

FIG. 3 is a schematic chemiluminescence spectrum of an example of a chemiluminescent substance suitable for exciting the phosphor exemplified in FIG. The example shown in this figure is for gallic acid / OH ⁻ / O ₂
It is a light emitting system. This light emitting system has an intensity peak at an energy position (approximately 600 and several tens nm in wavelength) larger than the fluorescence energy that gives the maximum intensity of the phosphor, and can excite the phosphor efficiently.

One of the major features of the present invention is that the chemiluminescent reagent 34 is placed between the slide glass 30 and the cover glass 36.
Is sandwiched together with the biological tissue sample 32. The excitation light emitted from the chemiluminescent reagent 34 is absorbed by the phosphor in the biological tissue sample 32, and the energy is emitted as fluorescence.

The fluorescence emitted from the biological tissue sample 32 in the preparation passes through the cover glass 36 and leaks out of the preparation. The preparation was an optical microscope 4
The biological tissue sample 32 is arranged so as to enter the zero field of view. In the optical microscope 40, a band pass filter 42 is arranged. The band-pass filter 42 transmits light in a wavelength band corresponding to the wavelength of the fluorescence emitted from the phosphor. By this bandpass filter 42, light having the wavelength of the fluorescence emitted exclusively from the biological tissue sample 32 is incident on the optical microscope 40, and the S / N ratio of the fluorescence observation is improved.

In this apparatus, the light incident from the objective lens 44 is split into two by the prism 46, and one is guided to the eyepiece 48 side. The observer can directly observe the generation of fluorescence in the biological tissue sample 32 from the eyepiece 48 with the naked eye.

On the other hand, a CCD camera 50 is mounted on a destination of the other light emitted from the prism 46. C
Biological tissue sample 3 that emits fluorescence by CD camera 50
2 can be converted into an electric signal. Further, the distribution of fluorescence can be observed from an image obtained by the CCD camera 50. By using the CCD camera 50 in this manner, the distribution and concentration of the target substance labeled with the fluorescent substance in the biological tissue sample 32 can be observed, and the biological tissue sample 32 can be analyzed based on the results. .

The stage 52 on which the slide is placed at the time of observation incorporates a temperature adjusting means such as an electric heater or a Peltier element, and a control circuit 54 is connected to the temperature adjusting means. The slide glass 30 of the preparation is placed on the stage 52 with its lower surface in contact with the slide glass. For example, a temperature sensor 56 is attached to the upper surface of the slide glass 30. The control circuit 54 controls the biological tissue sample 3 placed on the upper surface of the slide glass 30 based on the output of the temperature sensor 56.
2 and the temperature of the chemiluminescent reagent 34 are controlled to desired temperatures. By this temperature control, the generation rate of the excitation light of the chemiluminescent reagent 34 can be adjusted. For example, in the case of a chemiluminescent reagent 34 in which the generation of excitation light is promoted by a rise in temperature, the temperature is kept low before observation to suppress the luminescence reaction, while the temperature is raised during observation to promote the luminescence reaction. Can be. Thereby, the luminescence ability of the chemiluminescent reagent 34 can be effectively used for observation.

FIG. 4 is a schematic view showing a sectional structure of the preparation. This figure shows other features of the present invention that could not be represented due to the small scale in FIG. The features are an optical filter 70 provided on one main surface of the slide glass 30 and an optical filter 72 provided on one main surface of the cover glass 36. These optical filters 70 and 72 have a wavelength characteristic of transmitting the fluorescence from the fluorescent material and cutting off the excitation light emitted by the chemiluminescent reagent 34. For example, according to the examples of the phosphor and the chemiluminescent reagent shown in FIGS. 2 and 3, the optical filters 70 and 72 transmit light in a wavelength region of 750 to 850 nm in which a peak of fluorescence from the phosphor exists, while The wavelength region where the excitation light spectrum emitted by the chemiluminescent reagent 34 has a large intensity is cut. Therefore, the optical filters 70 and 72 have a cutoff wavelength near 750 nm, and transmit light having a longer wavelength than that.
Conversely, it is configured not to transmit light of a shorter wavelength. For example, the optical filters 70 and 72 can be formed by coating that is often performed on optical devices such as lenses, and desired wavelength characteristics can be obtained by selecting the material and the film thickness.

In the preparation of the slide, the slide glass 30 is used with the side provided with the optical filter 70 facing the biological tissue sample 32 and the chemiluminescent reagent 34 side. In the preparation of the preparation, the cover glass 36 is used with the side on which the optical filter 72 is provided facing the biological tissue sample 32 and the chemiluminescent reagent 34 side.

As a result, the excitation light emitted from the chemiluminescent reagent 34 sandwiched between the slide glass 30 and the cover glass 36 is applied to the slide glass 30 and the cover glass 3.
6 is suppressed. That is, the slide glass 30 and the cover glass 36 are irradiated with the excitation light,
The generation of light (pseudo fluorescence) or scattered light having a wavelength similar to the fluorescence emitted from the phosphor is prevented.

Such pseudo fluorescence or scattered light can enter the optical microscope 40 directly from the cover glass 36 or indirectly via reflection at the boundary surface of the slide glass 30 or the like. Then, they are erroneously observed as fluorescence from the phosphor, which causes a reduction in observation accuracy. The optical filters 70 and 72 of the present apparatus prevent the occurrence of such pseudo-fluorescence and the like that cause a decrease in observation accuracy, and transmit the fluorescence from the fluorescent material well, and perform observation with an excellent S / N ratio. Is made possible.

The optical filters 70 and 72 may be provided not only on one surface of the slide glass 30 and the cover glass 36 but also on both surfaces.

[0037]

According to the present invention, pseudo-fluorescence and scattered light when excitation light is transmitted through a slide glass or a cover glass can be reduced by providing a light-emitting source for exciting a phosphor inside a slide. Thereby, the effect of improving the fluorescence observation accuracy can be obtained.

According to the present invention, a filter that does not transmit the excitation light from the luminescent reagent is formed on the surface of the slide glass in contact with the luminescent reagent or the surface of the cover glass in contact with the luminescent reagent. This also prevents excitation light from entering the slide glass or cover glass from inside the preparation,
The above-described pseudo-fluorescence and scattered light can be further reduced, and the effect of improving the fluorescence observation accuracy can be obtained.

Further, according to the present invention, by adjusting the reaction rate of the luminescent reagent by the temperature control means, the luminescent ability can be effectively used for observation, and the effect of shortening the observation time and improving the sensitivity can be obtained. Achieved.

[Brief description of the drawings]

FIG. 1 shows a configuration of an observation device using a biological tissue observation method according to the present invention.

FIG. 2 is a spectrum showing an example of emission and absorption characteristics of a phosphor.

FIG. 3 is a schematic chemiluminescence spectrum of an example of a chemiluminescent substance.

FIG. 4 is a schematic diagram showing a cross-sectional structure of a preparation.

FIG. 5 is a schematic diagram showing a schematic configuration of a laser fluorescence microscope apparatus based on a conventional observation method using a phosphor-labeled probe.

[Explanation of symbols]

30 slide glasses, 32 biological tissue samples, 34
Chemiluminescent reagent, 36 cover glass, 40 microscope, 4
2 bandpass filter, 50 CCD camera, 52
Stage, 54 control circuit, 56 temperature sensor, 70,
72 Optical filter.

Claims (6)

[Claims] A sample preparation step of sandwiching a luminescent reagent emitting excitation light together with a sample labeled with a fluorescent substance between a first plate and a second plate to form a sample; Receiving the emitted fluorescent light in the first
An observation step of observing the specimen from outside through a plate; 2. The fluorescence observation method according to claim 1, wherein the first plate has a filter function for blocking transmission of the excitation light on a surface facing the sample. 3. The fluorescence observation method according to claim 1, wherein the second plate has a filter function for blocking transmission of the excitation light on a surface facing the sample. Observation method. 4. The fluorescence observation method according to claim 1, wherein the first plate is a cover glass, and the second plate is a slide glass. Fluorescence observation method. 5. The fluorescence observation device used in the fluorescence observation method according to claim 1, wherein the luminescent reagent is held in a gap between the first plate and the second plate. And a temperature control means for controlling the temperature of the sample. 6. An apparatus for observing a specimen created by sandwiching a luminescent reagent emitting excitation light between a first plate and a second plate together with a sample labeled with a fluorescent substance, wherein the fluorescent substance is The fluorescence emitted by receiving the excitation light
A fluorescence observation apparatus comprising an observation step of observing the specimen from outside through a plate.