JP2000314839A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser scanning microscope in which an optical filter and a mechanical driving part requiring high position reproducing accuracy are not necessitated and the change of a device constitution is not necessitated for the various kinds of the combinations of exciting wavelength and a fluorescent pigment, by which the multiexcited fluorescent image of a sample multi-colored can be obtained by one photodetector by having a high S/N and whose constitution is simple. SOLUTION: This microscope is provided with laser light sources 14 and 15 by which the fluorescence of a different wavelength area is generated from the sample 8, the photodetector 26 detecting the fluorescence emitted from the sample 8, a prism 22 arranged between the sample 8 and the photodetector 26 and spectrally resolving light from the sample 8, a mirror array 24 arranged between the prism 22 and the photodetector 26 and consisting of plural fine mirror elements 25 respectively receiving the various kinds of light beams spectrally resolved from the sample and reflecting only the fluorescence to the photodetector 26 and an output signal processing device 31 sampling an output from the photodetector 26.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a laser scanning microscope.

[0002]

2. Description of the Related Art In general, a fluorescence detecting apparatus is widely used in medicine and biology in other fields for the purpose of detecting proteins, genes, and the like, which are fluorescently labeled on biological tissues and cells. In particular, in recent years, multi-fluorescence detection, in which a specimen stained with a plurality of fluorescent dyes is observed at a time, has been very effective in analyzing genes and elucidating intracellular structures. A laser scanning microscope is known as an effective means for detecting such fluorescence.

FIG. 9 shows an optical system as a typical example of these laser scanning microscopes for fluorescence detection. In the figure, three laser oscillators 1a, 1b, 1b having different oscillation wavelengths are shown.
The three types of laser beams emitted from 1c are coupled on a common optical axis by coupling dichroic mirrors 2a and 2b, then passed through a beam expander 3, expanded to an appropriate beam diameter, and reflected by a dichroic mirror 4. The sample 8 is deflected by an XY scanning optical system 5 such as a galvanometer mirror, condensed via a pupil relay lens 6 and an objective lens 7, and irradiated onto the sample 8, thereby scanning the sample 8 with a laser spot. Is done. The fluorescence from the sample 8 excited by the laser beam irradiation returns to the path from the objective lens 7 to the dichroic mirror 4, passes through the dichroic mirror 4, and passes through the spectroscopic dichroic mirror 9a.
Spectroscopy. One fluorescent light reflected by the dichroic mirror 9a is condensed by the imaging lens 10a, passes through the confocal stop 11a, and absorbs or reflects fluorescent light having a wavelength other than the target first fluorescent light by the absorption filter 12a. After that, the intensity is detected by the photodetector 13a.

The confocal stop 11a is disposed at a position optically conjugate with the focal position of the objective lens 7, and blocks light other than the fluorescence excited by the laser spot, so that the obtained image has a very high contrast. High, and a three-dimensional image can be obtained by relatively changing the distance between the specimen 8 and the objective lens 7 in the optical axis direction. On the other hand, the fluorescent light transmitted through the dichroic mirror 9a is further separated by the dichroic mirror 9b, and the fluorescent light reflected by the dichroic mirror 9b is collected by the imaging lens 10b, passes through the confocal stop 11b, and passes through The light intensity is detected by the photodetector 13b after passing through the absorption filter 12b that transmits only the fluorescent light. The fluorescent light transmitted through the dichroic mirror 9b is condensed by the imaging lens 10c, passes through the confocal stop 11c, passes through the absorption filter 12c that transmits only the target third fluorescent light, and is detected by the photodetector 13c. Is done.

In this optical system, each laser oscillator 1a, 1
Simultaneous detection of triple excitation fluorescence by three wavelength laser beams emitted from b and 1c is possible, and the dichroic is changed each time the state of multiple excitation changes, such as change of laser wavelength, type of fluorescent dye and number of excitation laser wavelengths. Mirrors 2a, 2b,
4. The spectral dichroic mirrors 9a and 9b and the absorption filters 12a, 12b and 12c are changed to those having optimal spectral characteristics.

[0006] However, the above-mentioned conventional laser scanning microscope for fluorescence using an optical filter has the following problems. That is, 1) Since the spectral characteristics of the optical filter cannot be freely determined due to manufacturing restrictions, the amount of fluorescent light and the S / N are limited. In particular, it is necessary to completely block excitation light with an absorption filter, but it is impossible to design and manufacture so that fluorescence in the wavelength region with the strongest fluorescence intensity near the excitation wavelength is transmitted without loss of light quantity. (2) Exclusive expensive optical filters must be prepared for each excitation wavelength and fluorescent dye. When various multiplex excitations are assumed, the number of filters increases, and the configuration and size of the apparatus become inevitable. 3) As is clear from FIG.
In the laser scanning microscope for fluorescence, since the multiplexed fluorescence is separated through a plurality of optical filters, there is a considerable loss of light amount before the fluorescence reaches the photodetector.

Hitherto, means for selecting and detecting a plurality of fluorescent wavelengths without using an optical filter have been proposed in order to solve these problems. That is, the technique disclosed in Japanese Patent Application Laid-Open No. 9-502269 discloses that a light flux spectrally decomposed by a prism or the like is divided into a first wavelength region transmitted by a slit-shaped mirror and a second wavelength region reflected by the slit mirror. Further, the present invention relates to a spectroscope and a confocal fluorescence microscope capable of selecting and detecting any two wavelength regions by controlling a slit mirror and a second slit position and a slit width for limiting the second wavelength region. Things. Also, Japanese Patent Application Laid-Open No. H8-4373
In the technique disclosed in Japanese Patent Application Publication No. 9-204, a light beam after passing through a confocal stop is split by a grating, a wavelength region and a wavelength width are selected by at least one slit, and the light amount in each wavelength region is detected by a photodetector. The present invention relates to a scanning optical microscope as described above. These two techniques are both scanning optics capable of detecting the fluorescence with good S / N by reliably blocking the excitation wavelength without using an optical filter and securing a sufficient amount of fluorescence in the multiple excitation fluorescence detection. We offer microscopes.

Japanese Patent Application Laid-Open No. 6-207853 discloses a spectroscopic device which can select an arbitrary wavelength without using an optical filter.
Is disclosed in Japanese Patent Application Laid-Open Publication No. HEI 9-203 (1995). This means that the detected light beam is spatially spectrally decomposed by a light dispersion element, and at least a part of the dispersion spectrum is received by a spatial light modulator represented by a deformable mirror device, and only a desired spectral region is reflected or transmitted. Then, the energy intensity is detected. Once the relationship between the light dispersion element, the spatial light modulator, and the energy detector is fixed, all mechanical movements other than the spatial light modulator are not required, so errors that occur there and high-precision mechanical control Can be eliminated. Here, the deformable mirror device is described in detail in U.S. Pat. No. 5,610,149, which comprises a spatial array of micromirrors, each micromirror being selectively preselected only by controlling the applied voltage. Is an element that can be deflected to a specified angle.

[0009]

However, in the method disclosed in Japanese Patent Application Laid-Open No. H8-43939, the slit for determining the wavelength selection involves a mechanical movement, and a very sophisticated control structure is required in its operating part. Not only is it necessary, but the vibration and wear of the mechanical drive cause loss of reproducibility at the time of measurement and cause calibration problems. In particular, since the slit-shaped mirror and the second slit need to move in conjunction with each other at the time of spectroscopy, it is extremely difficult to improve the control accuracy. Further, when the types of fluorescence to be simultaneously detected increase, the number of the above-mentioned spectroscopic means is not enough, and it is necessary to use a plurality of similar wavelength division means, which causes a loss of light quantity and a complicated device. There is a point.

The method disclosed in Japanese Patent Application Laid-Open No. 9-502269 discloses a method in which one photodetector is used in addition to the problem of calibration that guarantees the accuracy and reproducibility of a mechanical drive unit. The wavelength band that can be detected by the method is determined by the spectral dispersing means such as the grating and the initial arrangement of the photodetector. There is a problem that it cannot cope with diversity.

The spectrometer disclosed in Japanese Patent Application Laid-Open No. 6-207853 is capable of detecting the amount of light in an arbitrary wavelength range. Therefore, it is necessary to obtain one fluorescence for one excitation wavelength. Although it can be applied to laser scanning microscopes for fluorescence, it is easy to use a laser for fluorescence as a method for simultaneously detecting the multiplexed fluorescence intensities at the same time according to all combinations of excitation wavelengths and fluorescent dyes. It cannot be incorporated into a scanning microscope.

The present invention has been made in view of the above-mentioned problems of the prior art, and has as its object to eliminate the need for an optical filter and a mechanical drive unit that requires a high degree of position reproduction accuracy. It is not necessary to change the apparatus configuration for various combinations of excitation wavelengths and fluorescent dyes. Multiple excitation fluorescence images of multiple-stained specimens can be obtained with high S / N without using photodetectors by the number of fluorescence. Can be obtained,
An object of the present invention is to provide a laser scanning microscope having a simple configuration.

[0013]

In order to achieve the above object, a laser scanning microscope according to the present invention emits a laser beam to be applied to a specimen to generate fluorescence in a different wavelength range from the specimen. A laser light source, a light detection device that detects fluorescence from the sample, a spectrum decomposition member disposed between the sample and the light detection device, and spatially spectrally decomposing light from the sample, A minute light deflecting element array comprising a plurality of minute light deflecting elements each arranged between a member and the photodetector and receiving light spectrally decomposed by the spectrum resolving member and capable of changing a deflection angle, An output signal processing device capable of sampling an output from the light detection device, wherein the light is deflected by at least two minute light deflecting elements of the minute light deflecting element array. Is incident with a predetermined time difference to the light detecting device, the output from the optical detector by the output signal processing device is characterized in that so as to sample with a said time difference.

According to the present invention, the sample is irradiated with at least two laser beams having different wavelengths with a predetermined time difference.

Further, according to the present invention, at least one of the detection wavelength ranges of the fluorescence generated from the sample includes the wavelength of the laser light emitted from the laser light source, and the fluorescent light of the plurality of minute light deflecting elements includes the fluorescent light and the fluorescent light. The micro-light deflecting element that receives light having a wavelength common to the laser light deflects the light received by the micro-light deflecting element in a direction deviating from the photodetector while the laser light is hitting the sample. During the time when the light beam does not hit the sample, the light received by the minute light deflecting element is made to enter the photodetector.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Before describing the embodiments of the present invention, the operation and effects of the laser scanning microscope according to the present invention will be described. First, according to the laser scanning microscope according to the present invention as set forth in claim 1, after spatially spectrally decomposing the fluorescence generated from the sample and the laser light from the laser light source reflected on the sample surface, only the fluorescence is reduced to a minute. By guiding the photodetectors with a time difference for each wavelength width to the photodetector, and gradually changing the sampling time according to the fluorescence wavelength range of the multi-stained sample, there is no need for a photodetector for each fluorescence to be detected, and many types Can be detected by spectroscopy. That is, the spectrum-resolved light is composed of fluorescence in a wavelength range different from that of the laser light reflected by the specimen. These fluorescences are spatially spectrally resolved and projected onto a micro-optical deflecting element array composed of micro-optical deflecting elements arranged in that direction. First, the micro-light deflecting element causes the first fluorescence in a specific wavelength range to enter the photodetector,
The second fluorescence in the wavelength range different from this and the laser light are deflected in a direction that does not enter the photodetector, and then, after a time lag, the minute light deflector enters the second fluorescence into the photodetector. It is made to be in a state to be made. The micro-light deflecting element can form fluorescence images in different wavelength ranges with one light detection device by repeating the above-described operation and alternately causing the fluorescence in different wavelength ranges to enter the photodetector. In this case, a mechanical driving unit is not required except for changing the light deflection angle of the minute light deflection element in a stepwise manner, and it is not necessary to change the apparatus or replace parts even if the fluorescence wavelength to be detected changes.
Further, since the light deflection angle of the minute light deflection element can be controlled by an electric digital signal, the device itself does not require a high precision or a complicated configuration. The laser light source used may be a continuous wave light source or a pulse light source.

According to the laser scanning microscope of the present invention, since the laser beams of different wavelengths are irradiated on the sample with a time difference, the fluorescence generated from the sample is also synchronized with the laser light emission time. Occur. For this reason, the fluorescence in the different wavelength bands to be detected is already time-resolved at this stage. After spatially spectrally decomposing the fluorescence generated from these specimens and the laser light reflected on the specimen surface, the light is guided to the photodetector for each fluorescence wavelength by the minute light deflecting element, and synchronized with the irradiation time of the laser light source. , The fluorescence in different wavelength ranges can be spectrally detected by one photodetector. Further, when the wavelength of the laser light and the wavelength of the fluorescence do not match,
The light deflection angle of the minute light deflecting element is to deflect light from the minute light deflecting element corresponding to a plurality of fluorescence wavelengths to be detected or from a group of continuous minute light deflecting elements corresponding to different fluorescence wavelength ranges to the photodetector. If the minute light deflecting element corresponding to the wavelength of the laser light source is set in advance so as to guide the light in a direction different from that of the photodetector, there is no need to drive each minute light deflecting element thereafter. . Even in the case of detecting fluorescence generated from a sample by a laser light source that emits laser light of another wavelength and its irradiation,
If the minute light deflecting elements respectively corresponding to the wavelength region of the fluorescence and the wavelength of the laser beam are set as described above, there is no need to change the configuration of the device. Thus, according to the present invention,
Laser light reflected from the specimen, which causes a reduction in image contrast, can be completely eliminated. Separating multiplexed fluorescence without preparing multiple photodetectors,
Fluorescence in different wavelength ranges can be detected. Furthermore,
If the time difference is made sufficiently small, the fluorescence in each wavelength region can be detected almost simultaneously.

According to the third aspect of the present invention, at least one of the detection wavelength ranges of the fluorescent light generated from the sample includes the wavelength of the laser light emitted from the laser light source. The minute light deflecting element that receives light of a common wavelength cannot always be placed in a state where light is incident on the photodetector. Therefore, the minute light deflecting element is deflected in a direction in which no light is incident on the photodetector during the time when the laser light is hitting the sample, and is deflected in a direction in which light is incident on the photodetector during the time when the laser light is not hitting the sample. Need to be controlled as follows. On the other hand, the minute light deflecting element corresponding to the wavelength range where the wavelength of the laser light and the wavelength of the fluorescent light do not overlap, that is, the minute light deflecting element that receives the fluorescent light but does not receive the laser light transmits the received light to the photodetector. The micro-light deflecting element that receives the laser light with a wavelength that overlaps with the wavelength of the fluorescent light is placed so that the received light does not enter the photodetector. There is no need to drive any of them. In this case, it goes without saying that the minute light deflecting element that receives neither laser light nor fluorescence may be placed in any state.

Hereinafter, embodiments of the present invention will be described in detail with reference to various examples shown in the drawings. In the drawing, substantially the same members as those used in the conventional example are denoted by the same reference numerals, and detailed description thereof is omitted. Embodiment 1 FIG. 1 is a view showing a first embodiment of a laser scanning microscope according to the present invention. In the figure, 14 is a light source composed of a multi-line Kr-Ar laser that oscillates light of wavelengths 488 nm, 568 nm and 647 nm simultaneously, 15 is a light source composed of an Ar laser oscillating light of 351 nm wavelength, 16 is a laser line filter, and 17 is a laser line filter. Fiber coupling lens, 18 is a single mode fiber, 19 is a beam collimating lens, 2
0 is a dichroic mirror, 21 is a collimating lens,
22 is a prism which is a spectrum resolving member, 23 is a condensing lens, 24 is a mirror array (micro light deflecting element array) composed of a number of micro mirrors 25, 26 is a photodetector, 27 is a light trap, 28 is a controller , 29 are a memory unit, 30 is an input unit, and 31 is an output signal processing device. The sample 8, the lasers 14 and 15, the fiber coupling lens 17, the fiber 18, the controller 2
The components except for the memory section 8, the memory section 29, the input section 30, and the output signal processing device 31 are housed in the laser scanning microscope main body.

In the first embodiment, each of the lasers 14,
The laser light emitted from 15 passes through a single mode fiber 18 via a fiber coupling lens 17 and is introduced into the laser scanning microscope main body. In this case, the excitation wavelength of the laser light emitted from the laser 14 is selected by the laser line filter 16. The laser light introduced into the laser scanning microscope main body is converted into a parallel light beam having an appropriate beam diameter by a lens 19 and mixed by a dichroic mirror 20. The mixed laser light is reflected by an excitation dichroic mirror 4 and deflected by an XY scanning optical system 5 such as a galvanometer mirror, reaches a sample 8 via a pupil relay lens 6 and an objective lens 7, and Is performed.

Thus, the fluorescence from the specimen 8 excited by the irradiation of the laser beam returns along the path from the objective lens 7 to the dichroic mirror 4 and
And condensed by the imaging lens 10 through the
Pass through. Collimating lens 2 passing through confocal stop 11
The fluorescent light flux converted into parallel light by 1 converts its wavelength information into a difference in emission angle by a prism 22, and further forms an image on a mirror array 24 via a condenser lens 23. At this time, the wavelength information converted into the difference in the emission angle from the prism 22 is replaced with position information on the mirror array 24,
The position of the minute mirror element 25 can be made to correspond to the wavelength as it is. In this case, instead of the prism 22, another spectrum resolving element such as a grating, an acousto-optic element, or a holographic element may be used.
Further, the condenser lens 23 may be replaced by any optical system having power in the spectral resolution direction such as a cylindrical lens.

The micromirror element 25 can select a deflection angle at which the light beam incident thereon is reflected toward the detection device 26 and a deflection angle at which the light beam is reflected toward the optical trap 27. An electric signal from the controller 28 via the input unit 30 via the output signal processing device 31 can be used for each element. When any input is made to the laser or the fluorescent dye via the input unit 30, the controller 28 calls up the angle state of each micro mirror element 25 stored in the memory unit 29 and reproduces the optimum measurement state at any time. You can do it. Conversely, the angle state of the micromirror element 25 can be stored in the memory unit 29.

Hereinafter, the spectral action of the multiplexed fluorescence will be described. First, the micro mirror elements 25 corresponding to all wavelengths are set so as to reflect the input light toward the optical trap 27. Then, the micromirror element corresponding to the first fluorescence wavelength is controlled so as to reflect the fluorescence toward the detection device 26. As a result, the output signal processing device 31 samples the signal from the light detection device 26, detects the intensity of the first fluorescence, receives the detected signal, and the controller 28 causes the micromirror element to convert the incident light into light. The light is reflected toward the trap 27 or the memory 2
The micromirror element reflects the incident light toward the light detecting device 26 for a certain time recorded in 9, and then reflects the incident light again toward the optical trap 27. Thereafter, the micro mirror element corresponding to the second fluorescence wavelength performs the same operation, and the intensity of the second fluorescence is sampled. By repeating this, the intensities of all observed fluorescence wavelengths are detected.

Therefore, the observed fluorescence wavelength range is three fields.
For example, fluorescent dyes that emit fluorescence in three different wavelength ranges
When used, the signal output from the photodetector 26 is:
As shown in FIG. In FIG. 2, the vertical axis indicates detection.
The horizontal axis represents the intensity of the obtained fluorescence. Each fluorescence
Are sequentially incident on the photodetector 26 with a time lag.
At different times on the time axis,
A peak occurs. And the first, second and third respectively
It corresponds to the signal representing the fluorescence. t₁, T_TwoAnd t _ThreeThe
First, second and third fluorescence sampling times
As a result, the intensity of each fluorescence is detected. t₁, T_TwoAnd t
_ThreeT to completely contain_TwoAnd this T _TwoIs scanning means
Scans each pixel if is less than the time to scan one pixel
Sampling of the three fluorescent signals during
And run the sample once for all output pixels.
Inspection alone gives fluorescent images in three wavelength ranges. This place
Light deflecting element regardless of the number of multiplexed fluorescent dyes
Since the light is spectrally reflected by one reflection by 25,
The volume loss is very low.

Here, a case where the excitation wavelength and the fluorescent dye are changed will be considered. Now, 351 nm, 488 nm, 568 nm and 6
For a quadruple excitation wavelength of 47 nm, it is assumed that the specimen 8 is stained with four types of fluorescent dyes excited at each wavelength.
The fluorescent dye excited by the excitation light having a wavelength of 351 nm does not emit fluorescence having a wavelength longer than 488 nm. Similarly, the fluorescent dye excited by the excitation light having the wavelength of 488 nm does not emit fluorescence having a wavelength longer than 568 nm, and the fluorescent dye excited by the excitation light having the wavelength of 568 nm is 6.
It does not emit fluorescence with a wavelength longer than 47 nm.

FIG. 3 shows the arrangement of the micro mirror elements 25 in the mirror array 24. In FIG. 3A,
The micro mirror elements 25e, 25f, 25g, and 25h correspond to the laser wavelengths of 351 nm, 488 nm, 568 nm, and 647 nm, respectively, and are arranged so as to deflect incident light to the optical trap 27. Micro mirror elements 25a, 25
b, 25c, and 25d represent wavelengths of 351 nm, 488 nm, and 5 respectively.
Corresponding to the fluorescence wavelength range excited at 68 nm and 647 nm, the direction can be switched between a direction in which incident light is deflected so as to be incident on the photodetector 26 and a direction in which it is deflected so as to deviate from the photodetector 26. I have. Specimen 8 emits fluorescence in four different wavelength ranges when excited simultaneously with light of four different wavelengths. Now, the mirror array 24 detects only light incident on the area of the minute mirror element 25a by the photodetector 26.
Assuming that it is in a state where
Only the fluorescence of the dye excited by the 1 nm excitation light is detected by the detector 2
6 is detected. After a certain period of time, the mirror array 24
Of the micromirror element 25
When only the light incident on the region b is directed to the photodetector 26, only the fluorescence of the dye excited by the excitation light having the wavelength of 488 nm is detected by the photodetector 26. Similarly, the fluorescence of the dye excited by the excitation light having the wavelengths of 568 nm and 647 nm is sequentially detected by the photodetector 26. Therefore, if this series of operations is set to be completed within the time required to scan one pixel, it is possible to obtain a quadruple-stained fluorescent image by scanning the specimen 8 with laser beams of a plurality of wavelengths. I can do it.

Next, a case where the specimen 8 is stained with two kinds of fluorescent dyes excited by excitation light having wavelengths of 351 nm and 568 nm will be described. The fluorescent dye in this case is
Unlike the dye in the case of quadruple excitation as described above, the fluorescent wavelength range is wide, and the fluorescent dye excited by excitation light having a wavelength of 351 nm emits fluorescence with excitation light having a wavelength shorter than 568 nm, and the wavelength of 5
It is assumed that the fluorescent dye excited by the excitation light of 68 nm emits fluorescence even with the excitation light having a wavelength exceeding 700 nm. When lasers of two wavelengths are applied to the sample 8, the sample emits fluorescence of two different wavelength ranges, but as shown in FIG. 3B, a micro mirror element corresponding to the excitation wavelength. 25
e, 25 g use the light trap 27
And the fluorescence excited by the excitation light having the wavelength of 351 nm is reflected by the micromirror elements 25a, 25a corresponding to the wavelength.
5f, 25b reflects toward the photodetector 26,
After the fluorescence is detected, the light is directed to the optical trap 27. During this time, the fluorescence intensity signal directed to the photodetector 26 is sampled. At this time, the minute mirror elements 25c, 25h,
Reference numeral 25d denotes a state where incident fluorescence is directed to the optical trap 27. Next, the minute mirror elements 25c and 25c corresponding to the fluorescence excited by the excitation light having the wavelength of 568 nm.
h, 25d change the direction of the incident light and detect the fluorescence
6 and set such that this series of operations for sampling and detecting the fluorescence intensity is completed within the time for scanning one pixel, and scanning the sample 8 with a laser beam containing two wavelengths. A double-stained fluorescent image can be obtained.

Similarly, even when the laser light source itself is changed, a micromirror element for reflecting the incident light beam toward the photodetector 26 or for reflecting the light beam toward the optical trap 27 is attached to any mirror element. Or by selecting appropriately. As described above, the optimum spectroscopy can always be performed for various combinations of the excitation wavelength and the fluorescent dye. The reason is that the direction of each micromirror element is arbitrarily controlled and the fluorescence is incident on the photodetector 26 for each fluorescence. This is because sampling is always performed. As described above, according to the present embodiment, a simple configuration that does not use an optical filter and does not require a mechanical driving unit that requires high position reproducibility, furthermore, various combinations of excitation wavelengths and fluorescent dyes However, multiple excitation fluorescence images of multiple stained specimens can be obtained with high S / N without changing the device configuration. Further, according to this embodiment, after spatially spectrally decomposing the fluorescence generated from the specimen and the light from the laser light source reflected on the specimen surface, the spectrum-resolved light is detected with a time difference for each minute wavelength width. By guiding the sample to the device and changing the sampling time little by little according to the fluorescence wavelength range of the multi-stained specimen, it is possible to use a single photo-detector without providing a photo-detector for each fluorescence to be detected. Excitation fluorescence can be spectrally detected. In addition, a mechanical drive unit is not required except for changing the light deflection angle of the micromirror element in a step-like manner, so that even if the fluorescence wavelength to be detected changes, there is no need to change the apparatus or replace parts. Further, since the light deflection angle of the micromirror element can be controlled by an electric digital signal, the device itself does not require a high precision or a complicated configuration. The laser light source may be either a continuous wave or a pulsed wave. Further, by making the time difference sufficiently small, it is possible to detect each fluorescence almost simultaneously.

Embodiment 2 FIG. 4 is a view showing a laser scanning microscope according to a second embodiment of the present invention. In the drawing, substantially the same members as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted. This embodiment is different from the first embodiment in that a shutter 32 for blocking a beam from each of the lasers 14 and 15 is provided in an optical path between each of the lasers 14 and 15 and the dichroic mirror 20.

Hereinafter, the case of detecting fluorescence in two different wavelength ranges will be described, taking as an example the case of double excitation using excitation light having wavelengths of 351 nm and 568 nm. First, the micromirror elements 25 corresponding to all the wavelengths are set so that the incident light is reflected toward the photodetector 26. FIG.
When the micromirror element of (b) is used, the micromirror elements 25e and 25g corresponding to the laser wavelength of the excitation light are oriented so as to reflect incident light toward the optical trap 27. There is no need to control 25. In FIG. 5, t ₁₁ and t ₁₂ represent the time during which the laser beam is irradiated from the lasers 14 and 15 onto the sample 8 with the time difference T ₃ due to the opening and closing of the shutter 32. Fluorescence generated from the sample 8 by this laser irradiation is converted into minute mirror elements 25a, 25
5f, 25b and 25c, 25h, 25d are reflected toward the photodetector 26, but each of the laser beams is alternately irradiated on the specimen 8 by opening or closing the shutter 32. The two types of fluorescence do not enter the photodetector 26 at the same time. The change in the intensity of the generated fluorescence with respect to time due to the incidence of the laser beam on the specimen 8 is as shown in FIG. In this case, by setting the sampling time of each fluorescent light to ts ₁ and ts ₂ , each fluorescent light can be separated and its intensity can be detected.

In this embodiment, the shutter 32 is not limited to a mechanical shutter, but may be any other such as a car cell or a micro-mirror element as long as it can control transmission and blocking of light. Furthermore, any method can be used as long as the lasers 14 and 15 can be turned on and off alternately without using a shutter. The relationship between the time and time T ₂ the scanning means scans one pixel is omitted because it is similar to that described in the first embodiment.

Embodiment 3 As a third embodiment, referring again to FIG.
The case of double excitation using excitation light of 488 nm and 488 nm will be described, and the case of detecting fluorescence in two different wavelength ranges will be described. First, the micromirror elements 25 corresponding to all the wavelengths are set so that the incident light is reflected toward the photodetector 26. It is assumed that 25e (see FIG. 3C) of the micromirror element 25 corresponding to the excitation light having the wavelength of 351 nm is oriented so as to reflect the incident light toward the optical trap 27. First, the fluorescence generated from the sample 8 during the time tl ₁ (see FIG. 5) during which the sample 8 is irradiated with the laser beam having a wavelength of 351 nm is reflected by the micro mirror elements 25a, 25f, and 25b corresponding to the fluorescence wavelength range. Photodetector 2
Go to 6. Next, a laser beam having a wavelength of 488 nm
The fluorescent light generated from the specimen 8 during the time tl ₂ (see FIG. 5) is reflected by the minute mirror elements 25b, 25g, and 25c (see FIG. 3 (c)) corresponding to the fluorescent wavelengths. It goes to the detecting device 26. At this time, the wavelength 488
The micromirror element 25f is controlled such that the laser light incident on the micromirror element 25f corresponding to the laser beam of nm is reflected toward the optical trap 27. Since the shutter 16 does not irradiate the sample 8 with two types of laser beams at the same time, the two types of fluorescence do not enter the photodetector 26 at the same time. In this embodiment, the change of the intensity of the two types of fluorescence with respect to time is shown in FIG. The sampling time of each fluorescence is ts ₁ , t
With s _2, each fluorescence intensity can be separated are detected.

In FIG. 6, ta indicates the sampling end time of the first fluorescent light, and tb indicates the start time of the second fluorescent light entering the photodetector 26. As shown in this figure, when the time ta is earlier than tb, the signal intensity obtained by this sampling is only due to the first fluorescence, and the signal intensity due to the second fluorescence is not mixed. . By doing so, sampling can be performed without mixing the intensities of the respective fluorescent lights, so that a decrease in the contrast of the obtained image can be reliably prevented. Further, in FIG. 6, T ₄ is the irradiation time of the laser beam, T ₅ is the fluorescence lifetime generated by irradiation of the laser beam, T ₆ is the fluorescence of the sampling time.
As is clear from this figure, since the total time of the times T ₄ and T ₅ is shorter than the time T 6 and is included in the time zone, the generated fluorescence can be sampled without waste. I can do it. Further, as shown in FIG. 7, the sum of the irradiation time tl ₁ (tl ₂ ) of each laser beam and the lifetime tf ₁ (tf ₂ ) of each fluorescent light becomes equal to the sampling time ts ₁ (ts ₂ ) of each fluorescent light. In this way, if the irradiation time tl ₁ (tl ₂ ) is further shortened, it becomes easy to obtain a multicolor fluorescent image in all pixels.

Embodiment 4 FIG. 8 is a view showing a laser scanning microscope according to a fourth embodiment of the present invention. In the figure, substantially the same members as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted. This embodiment differs from the first embodiment in that two photodetectors are provided. That is, 26a and 26b are two photodetectors, and in the case of quadruple excitation,
The first and second fluorescences are detected by the light detection device 26a, and the third and fourth fluorescences are detected by the light detection device 26b. In the case of triple excitation, the first and second fluorescences are detected by the photodetector 26a and the third fluorescence is detected by the photodetector 26b. In this case, the light detection device 26b
Does not need to be time-resolved since it represents one fluorescence intensity. Other functions and effects of this embodiment are the same as those described in the first embodiment, and thus description thereof will be omitted. Note that the number of photodetectors is not limited to two.

As described above, the laser scanning microscope of the present invention has the following features in addition to the features described in the claims. (1) The laser light source is configured to be capable of simultaneously generating fluorescence in different wavelength ranges.
2. A laser scanning microscope according to claim 1.

(2) When the predetermined time difference is T ₁ , and when the scanning means of the laser scanning microscope scans one pixel is T ₂ , the condition that T ₂ > T ₁ is satisfied. The laser scanning microscope according to claim 1 or (1), wherein: Thus, it is possible to sample all the intensities of fluorescence in different wavelength ranges while the scanning unit scans one pixel. If this is done for all the pixels, it is possible to obtain a fluorescent image of each wavelength region to be detected on the output screen by scanning the detection range of the sample only once.

(3) The scanning provided in the laser scanning microscope
The time for the inspection means to scan one pixel is T _Two, Different wavelengths
Predetermined time during which at least two laser beams are irradiated on the specimen
T difference_ThreeAnd T_Two> T_ThreeTo meet certain conditions
4. The method according to claim 2, wherein
Laser scanning microscope. This allows one scanning unit
All the fluorescence intensities in different wavelength ranges while scanning
Can be sampled. For all pixels
If this is done, only one scan of the detection range of the specimen is required
For each pixel that you want to detect at every pixel of the output screen.
A long fluorescent image can be obtained.

(4) With respect to the two wavelength ranges of the fluorescence which are successively incident on the photodetector in time, the sampling end time of the intensity of the previously incident fluorescence is determined by the time when the later incident fluorescence is transmitted to the photodetector. The laser scanning microscope according to claim 2, wherein the laser scanning microscope is configured to be earlier than the incident time. As a result, the fluorescence incident on the photodetector does not overlap with time. By resetting the sampling of the fluorescent light during the time when the fluorescent light is not incident on the photodetector, the sampling can be performed without mixing the intensity of each fluorescent light, so that a decrease in the contrast of the fluorescent image can be prevented.

(5) When the irradiation time of the laser light is T ₄ , the lifetime of the fluorescence generated by the irradiation of the laser light is T ₅ , and the sampling time of the fluorescence is T ₆ , T ₆ ≧ T
The laser scanning microscope according to the above (4), characterized by being configured so as to satisfy the ₄ + T ₅ becomes conditions. As a result, the entire amount of generated fluorescence can be sampled, so that fluorescence can be measured even when the fluorescence intensity per unit time is small.

(6) The start of the time T ₆ starts at the time T ₄
And the end of the time T ₆ is before the time T
The laser scanning microscope according to the above (5) than ₅ end, characterized by being configured such that later. Thereby, the time including the time T ₄ is completely included in the time T ₆ , which is more preferable.

(7) The laser scanning microscope according to any one of (1) to (3) or (3) to (5), wherein a pulse laser is used as the laser light source. Thus, the sample can be irradiated with the laser light with a time difference.

(8) A shutter which can block laser light is disposed between the laser light source and the sample, according to any one of claims 1 to 3, or (3) to (5).
The laser scanning microscope according to any one of the above. This makes it possible to open and close the shutter and irradiate the sample with laser light having different wavelengths with a time difference.

(9) The laser scanning microscope according to (8), wherein a car cell is used as the shutter. This makes it easy to switch between the transmitting state and the shielding state at a high speed, and it is possible to efficiently irradiate the sample surface with the laser light with a time difference.

(10) The laser scanning microscope according to the above (8), wherein a micro light deflecting element arranged between the laser light source and the sample is used as the shutter. This makes it possible to quickly switch between a state in which the laser light is deflected in a specific direction and a state in which the laser light is deflected in a direction different from the specific direction, thereby irradiating the sample surface with the laser light with a time difference. I can do it.

[0045]

As described above, according to the present invention, there is no need for an interference filter or a mechanical drive unit requiring a high degree of positional reproducibility, and it is necessary to change the device configuration for various combinations of excitation wavelengths and fluorescent dyes. Instead, it is possible to provide a laser scanning microscope that can detect fluorescence in different fluorescence wavelength ranges with a single light detection device and has high fluorescence detection sensitivity.

[Brief description of the drawings]

FIG. 1 is a view showing a first embodiment of a laser scanning microscope according to the present invention.

FIG. 2 is a graph showing the output signal intensity of the photodetector in the first embodiment.

FIGS. 3A and 3B are enlarged views of a micromirror element used in the laser scanning microscope according to the present invention, wherein FIG. 3A is a state in the first embodiment, and FIG. 3B is a modified example and a second embodiment of the first embodiment; The state in the example and (c) show the state in the third embodiment described later.

FIG. 4 is a view showing second and third embodiments of the laser scanning microscope according to the present invention.

FIG. 5 is a graph showing the output signal strength of the photodetector in the second and third embodiments.

FIG. 6 is a graph showing the output signal strength of a photodetector according to a modification of the third embodiment.

FIG. 7 is a graph showing an output signal intensity of a photodetector in another modification of the third embodiment.

FIG. 8 is a diagram showing a fourth embodiment of the laser scanning microscope according to the present invention.

FIG. 9 is a diagram showing a conventional laser scanning microscope for fluorescence.

[Explanation of symbols]

1a, 1b, 1c Laser oscillator 2a, 2b Dichroic mirror for laser coupling 3 Beam expander 4, 20 Dichroic mirror 5 XY scanning optical system 6 Pupil relay lens 7 Objective lens 8 Sample 9a, 9b Dichroic mirror for spectrum 10, 10a , 10b, 10c Imaging lens 11, 11a, 11b, 11c Confocal stop 12a, 12b, 12c Absorption filter 13a, 13b, 13c Photodetector 14 Multiline Kr-Ar laser 15 Ar laser 16 Laser line filter 17 Fiber coupling Lens 18 Single-mode fiber 19 Beam collimating lens 21 Collimating lens 22 Prism (spectral resolution member) 23 Condensing lens 24 Mirror array (Micro-optical deflection element array) 25, 25a to 25h Micro-mirror Over device (micro optical deflector) 26, 26a, 26b photodetector 27 a light trap 28 controller 29 memory unit 30 input unit 31 outputs the signal processor 32 shutter t _1, t _2, t ₃ fluorescence sampling time tl _1, tl ₂ Laser irradiation time ts ₁ , ts ₂ Fluorescence sampling time tf ₁ , tf ₂ Fluorescence life time ta First fluorescence sampling end time tb Second fluorescence injection start time T ₂ time T ₃ 2 single time difference laser light is irradiated onto the sample surface T ₄ laser light irradiation time T ₅ laser light fluorescence lifetime T ₆ fluorescent sampling time generated by irradiation necessary to sample fluorescence

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G043 AA01 AA04 BA16 CA04 EA01 FA02 GA01 GB01 HA01 HA02 HA05 HA09 HA11 HA15 JA02 JA05 KA02 KA03 KA05 KA09 LA01 MA04 2H052 AA08 AA09 AB24 AB25 AC12 AC15 AC26 AC27 AC34 AF07

Claims (3)

[Claims] In a laser scanning microscope, a laser light source for emitting laser light to be irradiated on a specimen to generate fluorescence in a different wavelength range from the specimen, and a light detecting device for detecting fluorescence from the specimen A spectral decomposition member disposed between the sample and the light detection device and spatially spectrally decomposing light from the sample, and the spectral decomposition member is disposed between the spectrum decomposition member and the light detection device. A minute light deflecting element array including a plurality of minute light deflecting elements each of which can receive light spectrally decomposed by the spectrum resolving member and can change a deflection angle, and an output signal processing device capable of sampling an output from the photodetector Comprising, the light deflected by at least two minute light deflecting elements of the minute light deflecting element array is incident on the photodetector with a predetermined time difference, A laser scanning microscope characterized in that an output from the photodetector is sampled by the output signal processor with the time difference. 2. The laser scanning microscope according to claim 1, wherein the sample is irradiated with at least two laser beams having different wavelengths with a predetermined time difference. 3. At least one of the detection wavelength ranges of fluorescence generated from the sample includes a wavelength of laser light emitted from the laser light source, and is common to fluorescence and laser light among the plurality of minute light deflection elements. The micro-light deflecting element that receives light of a given wavelength deflects the light received by the micro-light deflecting element in a direction deviating from the photodetector during the time when the laser light hits the sample, and during the time when the laser light does not hit the sample. 3. The laser scanning microscope according to claim 2, wherein light received by said minute light deflecting element is incident on said photodetector.