JPH11271636A - Scanning type laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope capable of obtaining images of optimum confocal effect and brightness for respective fluorescence wavelengths. SOLUTION: This microscope is provided with a laser beam source 10 for emitting plural laser beams with different wavelengths (for instance, 488 nm, 530 nm and 648 nm), a laser beam selection means 30 for selecting the laser beam of a specified wavelength from the laser beams of respective wavelengths emitted from the source 10, a means for irradiating a sample 9 with the laser beam selected by the selection means 30 through a laser scanning means 5, a confocal pinhole 42 installed at a position conjugate to the surface of the sample 9 so as to pass through fluorescence emitted by the sample 9 excited by the laser beam irradiated by the means and switchable to an aperture diameter corresponding to the wavelength of the fluorescence, and a controller 41 for changing over the aperture diameter of the confocal pinhole 42 matched with the wavelength (520 nm, 615 nm and 670 nm) of the fluorescence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for scanning a laser beam emitted from a laser light source to irradiate a sample, and forming an image of fluorescence emitted from the sample excited by the laser beam irradiation through a confocal optical system. The present invention relates to a scanning laser microscope in which this is spectrally detected for each fluorescence wavelength and detected.

[0002]

2. Description of the Related Art FIG. 2 is a diagram showing a configuration of an optical system of a conventional scanning laser microscope. In FIG. 2, reference numeral 1 denotes a laser light source, 2 denotes a beam expander for expanding the beam diameter of laser light, 3 denotes a laser line filter for selecting a laser wavelength, and 4 denotes a dichroic mirror. Also,
5 is an XY laser scanning optical system such as a galvanometer mirror, 6 is a pupil relay lens, 7 is an observation optical system, 8 is an objective lens, 9
Is a specimen.

Reference numeral 11 denotes an image forming lens, 12 denotes a confocal pinhole disposed at an image forming position of the image forming lens 11, and 13 denotes divergent light (light having a spread angle) passing through the confocal pinhole 12. It is a collimating optical system that converts parallel rays,
These 11 to 13 constitute a confocal optical system. 14 and 15 are dichroic mirrors for spectroscopy,
Reference numeral 16 denotes a mirror, and reference numerals 24, 25, and 26 denote photodetectors.

In this scanning laser microscope, when a laser beam is emitted from a laser light source 1, the laser beam is expanded by a beam expander 2 to a light beam diameter corresponding to the NA of an objective lens 8, and the laser beam is filtered by a laser line filter 3. After a desired laser wavelength is selected, it is reflected by the dichroic mirror 4 and deflected by the XY laser scanning optical system 5 in the XY direction, and then the pupil relay lens 6, the observation optical system 7, and the objective lens 8
Irradiates the specimen 9 through the.

The fluorescence of a predetermined wavelength emitted by the specimen 9 excited by the irradiation returns along the path from the objective lens 8 to the dichroic mirror 4 and passes through the dichroic mirror 4. The fluorescence transmitted through the dichroic mirror 4 is collected by the imaging lens 11 and passes through the confocal pinhole 12. Divergent light (a beam having a divergence angle) that has passed through the confocal pinhole 12 is converted into a parallel light by passing through the collimating optical system 13. The parallel light is separated and detected for each fluorescence wavelength range. That is, light having a wavelength equal to or lower than the first level is reflected by the dichroic mirror 14 and detected by the photodetector 24. Light having a wavelength equal to or less than the second level is reflected by the dichroic mirror 15 and
Is detected by Then, light having a wavelength exceeding the second level is reflected by the mirror 16 and detected by the photodetector 26.

Thus, according to this scanning laser microscope, the fluorescent light after passing through the confocal pinhole 12 is separated at predetermined wavelengths, and detected by the photodetectors 24, 25 and 26 as light of a plurality of different wavelengths. Is measured and measured. An image displayed two-dimensionally on a monitor has been obtained by associating the luminance information with each scanning point.

[0007]

The above-mentioned conventional scanning laser microscope has the following problems. In the scanning laser microscope having the above configuration, when the fluorescence of the specimen 9 is observed by multiple staining, fluorescence of a plurality of wavelengths passes through one confocal pinhole 12. The fact that a plurality of fluorescent lights having different diffraction diameters pass through one confocal pinhole 12 means that among the diffraction diameters D satisfying the following expression (1), only the diffraction diameter D of one fluorescence wavelength λ is the confocal pin. It only fits the hole diameter. In other words, the pinhole diameter d is not the optimum for other fluorescence wavelengths, so that an image having a weak confocal effect or an image having lost brightness is obtained.

D = 1.22 (λ / NA) (1) where D: diffraction diameter λ: fluorescence wavelength NA: numerical aperture of light incident on confocal pinhole Here, pinhole diameter d and diffraction diameter D When d ≦ D, the confocal effect does not change but the brightness decreases. In the case of d> D, the image becomes brighter but the confocal effect is reduced. Therefore, if d = D at a short wavelength, brightness is lost at a long wavelength. Conversely, if d = D at the long wavelength, the confocal effect is reduced at a short wavelength, but the confocal effect is reduced.

An object of the present invention is to provide a scanning laser microscope capable of obtaining an image having a high confocal effect and a small loss of brightness for all fluorescent wavelengths emitted from a specimen to be observed.

[0010]

In order to solve the above-mentioned problems and achieve the object, a scanning laser microscope according to the present invention is configured as follows. (1) A scanning laser microscope according to the present invention includes a laser light source that emits a plurality of laser lights having different wavelengths, and a laser light selection unit that selects a laser light of a specific wavelength from the laser lights of each wavelength emitted from the laser light source. Means for scanning the sample with the laser light selected by the laser light selecting means, and irradiating the sample with the laser light; and irradiating the sample with fluorescence emitted by the sample excited by the laser light irradiated by the irradiating means. A confocal pinhole that is installed at a position conjugate to the plane and can be switched to an aperture diameter corresponding to the wavelength of the fluorescence, and a control device that controls switching of the aperture diameter of the confocal pinhole in accordance with the wavelength of the fluorescence. It is characterized by having. (2) The scanning laser microscope of the present invention is the microscope according to the above (1), wherein the control device is configured to control the sample based on the wavelength information of the laser light emitted from the laser light source or the fluorescent dye information of the sample. Is characterized by calculating the diffraction diameter of the fluorescence wavelength emitted by the controller, and switching and controlling the aperture diameter of the confocal pinhole based on the calculation result.

[0011]

(First Embodiment) FIG. 1 is a view showing a configuration of an optical system of a scanning laser microscope according to a first embodiment of the present invention. Parts having the same functions as those in FIG. 2 are denoted by the same reference numerals, and detailed description is omitted. One of the features of the optical system shown in FIG. 1 is that a 488 nm laser line filter 31, a 530 nm laser line filter 32, 6 is provided between the beam expander 2 and the dichroic mirror 4.
The point is that a laser beam selecting means 30 capable of selectively inserting and removing the 48 nm laser line filter 33 with respect to the optical path is provided. Another characteristic point is that the control device 41 adjusts the opening diameter of the confocal pinhole 42 arranged at the image forming position of the image forming lens 11 according to the type of the filter of the laser beam selecting means 30 according to the wavelength of the fluorescent light. And a confocal control system 40 that controls opening and closing of shutters 44, 45, and 46 disposed in the respective light receiving units of the photodetectors 24, 25, and 26 in conjunction with the switching control. The confocal pinhole 42 has a single pinhole whose diameter is enlarged or reduced, or a plurality of pinholes having different diameters arranged on a rotating disk and selectively inserted into an optical path. Etc. can be used.

The laser light source 10 according to the present embodiment
Is a Kr-Ar multi-wavelength oscillation laser light source capable of simultaneously oscillating laser beams of each wavelength of, for example, 488 nm, 530 nm, and 648 nm. Specimen 9 is a fluorescent dye FITC, P
It is assumed that the cells are stained with I, CY5.

In this embodiment, the confocal pinhole 4 is used.
2, the switching control of the aperture diameter is linked to the laser light selecting means 30. However, the present invention is not limited to this. For example, the confocal pinhole 4 is independent of the laser light selecting means 30.
The switching control of the opening diameter of No. 2 may be performed by the control device 41.

The control device 41 controls the wavelength information (for example, 488 nm, 530 nm) of the laser light output from the laser light source 10.
nm, 648 nm) or the fluorescent dye information (F
ITC, PI, CY5) are stored in, for example, a built-in memory, and the diffraction diameter of the fluorescence wavelength emitted by the specimen 9 is calculated based on the stored information, and the confocal point is determined according to the calculated diffraction diameter. The size of the pinhole 42 is variably controlled.

According to the present embodiment having the above-described configuration, the laser light output from the laser light source 10 is expanded to a light flux diameter corresponding to the NA of the objective lens 8 by passing through the beam expander 2. The laser light selecting means 30 selects only laser light having a predetermined laser wavelength as described below.

First, for example, when the laser line filter 31 is inserted into the optical path, a laser beam having a wavelength of 488 nm is selected. The selected laser beam is reflected by a dichroic mirror 4 and is XY-deflected by an XY laser scanning optical system 5 such as a galvanometer mirror.
The light is irradiated onto the sample 9 via the observation optical system 7 and the objective lens 8. That is, only the laser beam having a wavelength of 488 nm is irradiated on the sample 9 and two-dimensional scanning is performed.

By irradiating a laser beam having a wavelength of 488 nm,
When the fluorescent dye of the specimen 9 is excited, mainly about 520 n
FITC fluorescent dye having a wavelength of m (hereinafter referred to as FITC)
Is issued. The fluorescence of the FITC returns along the path from the objective lens 8 to the dichroic mirror 4 and passes through the dichroic mirror 4. The fluorescent light transmitted through the dichroic mirror 4 is condensed by the imaging lens 11, and the confocal pinhole 4 disposed at the imaging position of the imaging lens.
Pass 2

Based on the fact that the filter of the laser beam selecting means 30 is the laser line filter 31, the control device 41 determines the fluorescence wavelength of FITC (about 52
0 nm), and the size of the confocal pinhole 42 (pinhole diameter d) is variably set according to the diffraction diameter. Therefore, the fluorescent light having a wavelength of about 520 nm that has passed through the confocal pinhole 42 has a small flare, that is, a high confocal effect, and a small light amount loss. Therefore, a good image can be obtained.

The divergent light (a beam having a divergent angle) passing through the confocal pinhole 42 is converted into a parallel light by passing through the collimating optical system 13. This parallel ray is
The light having a wavelength of 575 nm or less is reflected by the dichroic mirror 14, passes through a shutter 44 opened by the control device 41, and is detected by the photodetector 24.
At this time, the specimen 9 also emits fluorescent light having a wavelength exceeding 575 nm, but this fluorescent light passes through the dichroic mirror 14 and is spectrally or reflected by the dichroic mirrors 15 and 16 to enter the photodetectors 25 and 26. I do. However, at this time, since the shutters 45 and 46 are closed by the control device 41, the photodetectors 25 and 2
There is no fear that extra light is incident on 6.

Therefore, only the fluorescence of FITC having a wavelength of about 520 nm that has passed through the confocal pinhole 42 is separated and extracted by the dichroic mirror 14 and the shutter 44 and detected by the corresponding photodetector 24 to obtain luminance information. Will be.

Next, for example, when the laser line filter 32 is inserted into the optical path, a laser beam having a wavelength of 530 nm is selected. The selected laser beam is reflected by the dichroic mirror 4 and XY-deflected by an XY laser scanning optical system 5 such as a galvanomirror, as in the case described above, and the pupil relay lens 6, the observation optical system 7, and the objective lens 8 The sample 9 is radiated on the specimen 9 through the light source.

The irradiation of the laser light having a wavelength of 530 nm excites the fluorescent dye of the specimen 9 and mainly emits the fluorescent dye of about 6 nm.
The fluorescence of PI having a wavelength of 15 nm is emitted. The PI fluorescence returns along the path from the objective lens 8 to the dichroic mirror 4 and passes through the dichroic mirror 4. The fluorescent light transmitted through the dichroic mirror 4 is condensed by the imaging lens 11 and passes through a confocal pinhole 42 arranged at an imaging position of the imaging lens.

The control device 41 determines the fluorescence wavelength of the PI (approximately 615n) emitted from the sample 9 based on the fact that the filter of the laser beam selecting means 30 is the laser line filter 32.
The diffraction diameter of m) is calculated, and the size of the confocal pinhole 42 is variably set according to the diffraction diameter. Therefore,
The fluorescent light having a wavelength of about 615 nm that has passed through the confocal pinhole 42 also has a small flare, that is, a high confocal effect, and a small light amount loss, and a good image is obtained.

As in the case described above, the fluorescence of PI having a wavelength of 615 nm having passed through the confocal pinhole 42 is converted into a parallel light by the collimating optical system 13.
After passing through the dichroic mirror 14, the parallel light is reflected by the dichroic mirror 15 that reflects light having a wavelength of 640 nm or less. In this case, the parallel light passes through the shutter 45 that is open and is detected by the photodetector 25. . At this time, similarly to the above, fluorescence having a wavelength of 575 nm or less is reflected by the dichroic mirror 14, and fluorescence having a wavelength exceeding 640 nm is transmitted by the dichroic mirror 15 and then reflected by the mirror 16. Attempts to enter the photodetectors 24 and 26. However, at this time, since both the shutters 44 and 46 are closed, there is no possibility that unnecessary light is incident on the photodetectors 24 and 26.

Next, for example, when the laser line filter 33 is inserted into the optical path, a laser beam having a wavelength of 648 nm is selected. The selected laser beam is reflected by the dichroic mirror 4 and XY-deflected by an XY laser scanning optical system 5 such as a galvanomirror, as in the case described above, and the pupil relay lens 6, the observation optical system 7, and the objective lens 8 The sample 9 is radiated on the specimen 9 through the light source.

The irradiation of the laser light having a wavelength of 648 nm excites the fluorescent dye of the specimen 9 and mainly emits the fluorescent dye of about 6 nm.
CY5 fluorescence having a wavelength of 70 nm is emitted. This fluorescence returns along the path from the objective lens 8 to the dichroic mirror 4 and passes through the dichroic mirror 4.
The fluorescent light transmitted through the dichroic mirror 4 is condensed by the imaging lens 11 and passes through a confocal pinhole 42 arranged at an imaging position of the imaging lens.

Based on the fact that the filter of the laser beam selecting means 30 is the laser line filter 33, the control device 41 determines the fluorescent wavelength of CY 5 (about 67
0 nm), and the size of the confocal pinhole 42 is variably set according to the diffraction diameter. Therefore, the wavelength passing through this confocal pinhole 42 is about 67
The fluorescent light of 0 nm also has a small flare, that is, a high confocal effect, and a small loss of light amount, resulting in a good image.

As in the case described above, the CY having a wavelength of about 670 nm passing through the confocal pinhole 42 is used.
The fluorescence of No. 5 is converted into a parallel light by the collimating optical system 13.

This parallel light is transmitted to the dichroic mirror 1
4, the light passes through the dichroic mirror 15, and then is reflected by the mirror 16. In this case, the light passes through the shutter 46 which is in the open state and is detected by the photodetector 26. In this case, the wavelength is 575 in the same manner as described above.
Fluorescence having a wavelength of less than nm is reflected by the dichroic mirror, and fluorescence having a wavelength of less than 640 nm is reflected by the dichroic mirror 15 and is going to enter the photodetectors 24 and 25, respectively. However, at this time, since both the shutters 44 and 45 are closed, there is no possibility that extra light is incident on the photodetectors 24 and 25.

When the images based on the three wavelengths of fluorescent light are superimposed on each other, an image having a high confocal effect and a small brightness loss can be obtained from fluorescent light of a short wavelength to fluorescent light of a long wavelength.
As a result, favorable multi-staining observation can be performed.

In obtaining a multi-stained image by such a method, since only light having a fluorescence wavelength corresponding to one excitation light is taken into each detector, the fluorescence on the short wavelength side is changed to the fluorescence on the long wavelength side. Can be avoided, and an image with good S / N without so-called crossover can be obtained.

(Second Embodiment) The amount of fluorescence is reduced and darkened until the pinhole diameter d of the confocal pinhole 42 is reduced to a point where the in-plane resolution of the diffraction diameter D is saturated, for example, to 1/3. It has been found that the in-plane resolution can be improved. And depending on the specimen to be observed, the pinhole diameter d
Under sufficiently bright conditions even if the aperture diameter is reduced to the diffraction diameter D, there are cases where it is desired to further narrow the pinhole diameter d to increase the in-plane resolution. In some cases, the pinhole diameter d may be increased to give priority to brightness even at the expense of the confocal effect.

Therefore, in the second embodiment, when the fluorescent light is sufficiently bright, the magnitude relationship between the pinhole diameter d of the confocal pinhole 42 and the diffraction diameter D of each fluorescent light can be varied according to the conditions of the observation sample. It is characterized by adding means for setting.

That is, the diffraction diameter D is obtained by using the following equation (2) modified so as to multiply the above equation (1) by an appropriate coefficient K. D = 1.22 (λ / NA) K (2) where K is a coefficient set in accordance with the condition By setting the coefficient K to be the same regardless of the fluorescent wavelength, the confocal point of each fluorescent wavelength is obtained. The balance between the effect and the brightness can be made constant.

(Features of Embodiment) [1] The scanning laser microscope shown in the embodiment has different wavelengths (for example, 488 nm, 530 nm, and 648 nm).
A laser light source (10) that emits a plurality of laser lights, and a laser light selection unit (30) that selects a laser light of a specific wavelength from the laser light of each wavelength emitted from the laser light source (10).
Means for irradiating the sample (9) with the laser light selected by the laser light selecting means (30) via the laser scanning means (5); and the sample excited by the laser light irradiated by this means. (9) a confocal pinhole (42) which is installed at a position conjugate to the surface of the specimen (9) so as to pass the fluorescence emitted therefrom and which can be switched to an aperture diameter corresponding to the wavelength of the fluorescence, and And a control device (41) for switching and controlling the opening diameter of the pinhole (42) in accordance with the wavelength of the fluorescence (for example, 520 nm, 615 nm, 670 nm).

In the above-mentioned scanning laser microscope, the control device (41) emits each fluorescence wavelength (FITC fluorescence of about 520 nm, P
The fluorescence of I is about 615 nm, and the fluorescence of CY5 is about 670 n
The diffraction diameter D of m) is calculated, and the size of the confocal pinhole (42) is variably set according to the diffraction diameter D. Therefore, the pinhole diameter (d) matches the diffraction diameter (D),
The fluorescent light of a specific wavelength that has passed through the confocal pinhole (42) has a small flare, that is, a high confocal effect, and a small amount of light loss, resulting in a good image.

When the images based on the three wavelengths of fluorescent light are superimposed, an image having a high confocal effect from the short wavelength fluorescent light to the long wavelength fluorescent light and having no brightness loss can be obtained. As a result, good multi-staining observation is possible. [2] The scanning laser microscope shown in the embodiment is a microscope similar to the above [1], and the control device (41) is configured to control the wavelength information of the laser light emitted from the laser light source (10) or the sample ( Based on the fluorescent dye information from (9), the diffraction diameter (D) of the fluorescence wavelength emitted by the specimen (9) is calculated, and the aperture of the confocal pinhole (42) is switched and controlled based on the calculation result. It is characterized by being.

The above-mentioned scanning laser microscope has the same operation and effect as the above [1], and furthermore, the means for variably controlling the size of the confocal pinhole is clearly specified, so that the above-mentioned scanning laser microscope is implemented. There are easy advantages. [3] The scanning laser microscope shown in the embodiment is a microscope similar to the above [1], wherein shutters (44, 45, 46) are respectively provided on the respective light receiving portions of the photodetectors (24, 25, 26). ), And opening and closing these shutters (44, 45, 46) in conjunction with switching control of the aperture diameter of the confocal pinhole (42),
Predetermined photodetector (one of 24, 25, 26) for only the fluorescence to be detected
It is characterized by being made to enter.

In the above scanning laser microscope, the same operation and effect as in the above [1] are obtained, and when obtaining a multi-stained image, the fluorescence other than the fluorescence wavelength corresponding to one excitation light is shut out and should be detected. Only fluorescence is the detector (24,
25,26). Thus, it is possible to avoid the fluorescence on the short wavelength side from being overlapped with the fluorescence on the long wavelength side, and it is possible to obtain an image with good S / N without so-called crossover. [4] The scanning laser microscope shown in the embodiment is a microscope similar to the above [1], and includes a confocal pinhole (42).
And a means for variably setting the magnitude relationship between the pinhole diameter (d) and the diffraction diameter (D) of each fluorescence according to the conditions of the observation sample.

In the above scanning laser microscope, the same operation and effect as in the above [1] can be obtained, and the confocal pinhole can be used.
The pinhole diameter (d) of (42) is variably controlled under the condition suitable for the condition of the observation sample.

[0041]

According to the present invention, the aperture diameter of the confocal pinhole is variably controlled by the control device in accordance with the wavelength of the fluorescent light emitted from the specimen, so that the confocal effect and the confocal effect can be controlled for each fluorescent wavelength. A scanning laser microscope capable of obtaining an image with optimal brightness can be provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of an optical system of a scanning laser microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a configuration of an optical system of a scanning laser microscope according to a conventional example.

[Explanation of symbols]

 Reference Signs List 1 laser light source 2 beam expander 3 laser line filter 4 dichroic mirror 5 XY laser scanning optical system 6 pupil relay lens 7 observation optical system 8 objective lens 9 sample 10 multi-wavelength oscillation laser light source 11 ... Imaging lens 12 ... Confocal pinhole 13 ... Collimator optical system 14,15 ... Dichroic mirror 16 ... Mirror 24,25,26 ... Photodetector 30 ... Laser light selecting means 31 ... 488 nm laser line filter 32 ... Laser line filter for 530 nm 40 ... Confocal control system 41 ... Control device 42 ... Confocal pinhole 44-46 ... Shutter

Claims (2)

[Claims] 1. A laser light source for emitting a plurality of laser lights having different wavelengths; a laser light selecting means for selecting a laser light of a specific wavelength from the laser lights of each wavelength emitted from the laser light source; Means for scanning the sample by scanning the laser light selected by the method, and setting at a position conjugate with the surface of the sample so as to pass the fluorescence emitted by the sample excited by the laser light irradiated by the irradiation means. A confocal pinhole that can be switched to an aperture diameter corresponding to the wavelength of the fluorescence, and a control device that controls switching of the aperture diameter of the confocal pinhole in accordance with the wavelength of the fluorescence. Scanning laser microscope. 2. The control device calculates a diffraction diameter of a fluorescence wavelength emitted from a sample based on wavelength information of a laser beam emitted from a laser light source or information on a fluorescent dye of the sample, and confocals based on the calculation result. 2. The scanning laser microscope according to claim 1, wherein the opening diameter of the pinhole is switched and controlled.