JP2010286566A - Laser scanning-type fluorescence microscope and fluorescence observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To adjust the focal depth of a fluorescence image, which is momentarily obtained, without changing space resolution in the fluorescence image obtained by scanning laser light. <P>SOLUTION: A laser scanning-type fluorescence microscope 1 includes: a laser light source 8; a scanner 9 which scans laser light from the laser light source 8; an objective lens 10 which irradiates a specimen (A) with the laser light scanned by the scanner 9 to converge fluorescence generated in the specimen (A); an area illumination source 18 which conducts area illumination to the specimen (A); a fluorescence-divergent part 11 which makes the fluorescence, which is generated by the irradiation with the laser light, diverge from the optical path of the laser light; a light-detecting device 14 which detects the fluorescence made to diverge by the fluorescence-divergent part 11; an optical-path divergence part 21 which makes the fluorescence diverge between the objective lens 10 and the scanner 9; an imaging element 26 which photographs the fluorescence which is made to diverge by the optical-path divergence part 21; and a variable diaphragm 23 which is arranged at a position which is optically conjugational to the position of the pupil of the objective lens 10 between the imaging element 26 and the optical-path divergence part 21. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a laser scanning fluorescence microscope and a fluorescence observation method.

  Conventionally, there is a confocal laser microscope in which a fluorescent beam collected by an objective lens is branched by a scanner that scans laser light and a beam splitter disposed in an optical path between the objective lenses, and a CCD camera is disposed in the branched optical path. Are known. This confocal laser microscope obtains a confocal image with high spatial resolution near the focal position of the objective lens by passing the fluorescence returning through the scanner through a confocal pinhole, while two-dimensionally using a CCD camera. A non-confocal image is acquired instantaneously (see, for example, Patent Document 1).

JP 2005-148484 A

  Since photographing with a CCD camera generally has a depth of focus corresponding to the magnification (numerical aperture) of the objective lens, it is advantageous for grasping the entire image of the specimen including the thickness direction. However, for example, when observing fluorescence with a CCD using a water immersion objective lens having a magnification of 40 times and a numerical aperture of 1.2 when observing a living cell having a thickness of about 10 μm, the depth of focus is about 0.5 μm. . Therefore, since the depth of focus is too shallow with respect to the thickness of the living cell, it is impossible to observe from the surface of the living cell to the internal structure at a time.

  Conventional confocal laser microscopes can acquire confocal images with high spatial resolution (low depth of field) and non-confocal images with lower spatial resolution (high depth of field). Although it is possible, in order to set the depth of focus of the non-confocal image acquired by the CCD camera deeper, for example, by providing a variable aperture stop in the objective lens and reducing the aperture stop, the beam diameter of the laser beam is also reduced. Therefore, there is a disadvantage that the resolution of the confocal image is lowered.

  The present invention has been made in view of the above-described circumstances, and adjusts the depth of focus of a fluorescent image acquired instantaneously without changing the spatial resolution of the fluorescent image acquired by scanning laser light. An object of the present invention is to provide a laser scanning fluorescence microscope and a fluorescence observation method that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a laser light source, a scanner that scans laser light from the laser light source, an objective lens that irradiates the sample with laser light scanned by the scanner and collects fluorescence generated in the sample, and the sample A surface illumination light source for surface illumination, a photodetector for detecting fluorescence collected by the objective lens, an optical path branching unit for branching fluorescence between the objective lens and the scanner, and an optical path branching unit A laser scanning fluorescence microscope comprising: an image pickup device for photographing the branched fluorescence; and a variable stop disposed at a position optically conjugate with the pupil position of the objective lens between the image pickup device and the optical path branching portion. I will provide a.

  According to the present invention, a sample is irradiated with laser light emitted from a laser light source and scanned by a scanner, whereby a fluorescent substance existing in the sample is excited to generate fluorescence, and the generated fluorescence is generated by an objective lens. It is collected and detected by a photodetector. A fluorescence image is acquired by storing the scanning position by the scanner and the intensity of the fluorescence detected by the photodetector in association with each other. In this case, by ensuring a sufficiently large numerical aperture of the objective lens, a fluorescent image with a small focal depth and a high spatial resolution can be obtained.

  On the other hand, when the specimen is surface illuminated by the surface illumination light source, the fluorescent material is excited at once in a wide range of the specimen, and fluorescence is generated. The generated fluorescence is collected by the objective lens and branched by the optical path branching portion between the objective lens and the scanner. The fluorescence branched to an optical path different from the optical path to the scanner by the optical path branching section is photographed by the image sensor after passing through the variable aperture. Thereby, a fluorescent image with a wide visual field range is acquired instantaneously.

  In this case, according to the present invention, when the depth of focus of the fluorescent image having a wide field of view acquired instantaneously is increased, the variable diaphragm arranged between the optical path branching unit and the image sensor is narrowed down. Thereby, the numerical aperture of the fluorescence incident on the image sensor can be reduced, and the depth of focus of the fluorescence image is deepened. Therefore, in this way, fluorescence generated from a wide range in the thickness direction of the specimen is captured in a fluorescent image with a wide field of view acquired instantaneously, so the location of the fluorescence generation site can be confirmed instantly can do.

  In this case, since the variable diaphragm is disposed between the optical path branching unit and the image sensor, the influence of the variable diaphragm on the fluorescence image acquired from the fluorescence branched to another optical path in the optical path branching unit. High spatial resolution can be maintained.

In the above invention, a fluorescence branching portion that branches fluorescence in an optical path between the laser light source and the scanner, and an optical conjugate with a focal position of the objective lens between the fluorescence branching portion and the photodetector. It is good also as providing the confocal pinhole arrange | positioned in various positions.
By doing in this way, the fluorescence condensed by the objective lens, returning through the scanner, and passing through the confocal pinhole can be detected by the photodetector. By placing the confocal pinhole at a position optically conjugate with the objective lens in the optical path returning through the scanner, a confocal fluorescence image with high spatial resolution can be acquired.

In the invention described above, a fluorescence branching unit that branches fluorescence in the optical path between the objective lens and the optical path branching unit is provided, and the photodetector detects the fluorescence branched by the fluorescence branching unit. It is good.
By doing in this way, the fluorescence condensed by the objective lens and branched in the fluorescence branching section is detected by the photodetector before returning to the scanner. For example, when an extremely short (femtosecond) pulse laser light source is used as the laser light source, a multiphoton fluorescence image with high spatial resolution can be acquired without using a confocal pinhole.

Moreover, in the said invention, you may provide the surface illumination light confluence | merging part which merges the illumination light from the said surface illumination light source in the optical path which goes to the said objective lens between the said objective lens and the said optical path branching part.
By doing in this way, the surface illumination light radiate | emitted from the surface illumination light source is made to merge on the same optical path as a laser beam by the surface illumination confluence | merging part. As a result, it is possible to simultaneously obtain an epi-fluorescence image with a wide field of view obtained instantaneously by irradiating surface illumination light and a fluorescence image with high spatial resolution obtained by scanning with laser light.

In the above invention, a condenser lens that irradiates the specimen with illumination light from the surface illumination light source from the side opposite to the objective lens across the specimen may be provided.
In this way, the surface illumination light from the surface illumination light source is condensed by the condenser lens to illuminate the sample from the side opposite to the objective lens, and the fluorescence emitted in the direction of transmitting the sample is collected by the objective lens. By doing so, a transmission fluorescent image can be obtained.

  In the above invention, the position of the light source is adjusted by the stimulus position adjusting unit, the stimulus position adjusting unit for adjusting the irradiation position of the sample of the laser light for stimulating light from the laser source for stimulating light, and the stimulus position adjusting unit. A stimulating light combining unit that combines the optical stimulation laser light into an optical path between the scanner and the objective lens toward the objective lens.

  By doing in this way, the irradiation position of the light stimulation laser light emitted from the light stimulation laser light source is adjusted by the stimulation position adjustment unit, and the optical path between the scanner and the objective lens is generated by the stimulation light merging unit. To join. As a result, the irradiation position adjusted by the stimulation position adjusting unit is irradiated with the laser light for light stimulation to give the stimulus, and the reaction is performed by the fluorescence image obtained by irradiating the laser light for obtaining the fluorescent image or the surface illumination light. Can be observed.

In the above invention, a light beam diameter adjusting unit for adjusting a light beam diameter of the laser light for photostimulation from the laser light source for photostimulation may be provided.
By doing so, it is possible to adjust the depth of focus by adjusting the NA of the light stimulation laser light irradiated on the specimen by the operation of the light beam diameter adjusting unit. That is, when a fluorescence image with a deep focal depth is acquired, it is impossible to determine from which position in the depth direction the fluorescence is generated, so the focal depth of the fluorescence image is increased by increasing the focal depth of the laser light for light stimulation. Stimulation can be given to the area covering. In addition, when a fluorescence image with a shallow depth of focus is acquired and the position in the depth direction of the part to be stimulated can be specified precisely, the NA of the laser light for light stimulation is increased by the light beam diameter adjusting unit to increase the depth of focus. It is possible to give a stimulus to a narrow range by shallowing.

In the above invention, the display unit for displaying the fluorescence image of the specimen acquired by the imaging device, the region designating unit for designating the region in the fluorescence image displayed on the display unit, and the region designating unit And a control unit that controls the scanner so as to acquire a fluorescent image of a specified region.
In this way, when the fluorescent image of the specimen acquired instantaneously by the image sensor is displayed on the display unit and the region in the fluorescent image displayed by the region specifying unit is specified, the control unit controls the scanner. , It is possible to acquire a high-resolution fluorescent image of the designated area.

In the above invention, a sensitivity adjustment unit may be provided that adjusts the sensitivity of the image sensor based on the aperture amount of the variable aperture.
By doing this, the amount of fluorescence incident on the image sensor is reduced when the variable aperture is reduced to obtain a fluorescence image with a deep focal depth. Therefore, the sensitivity adjustment unit increases the sensitivity of the image sensor and is acquired. It is possible to prevent the fluorescent image from becoming dark. In addition, when the variable aperture is opened and the amount of fluorescence incident on the image sensor is increased, the sensitivity adjustment unit can reduce the sensitivity of the image sensor so that the acquired fluorescence image does not become too bright. .

In the above invention, the objective switching unit that switches the objective lens is provided, and the sensitivity of the imaging element is adjusted based on the type of the objective lens switched by the objective switching unit and / or the aperture amount of the variable aperture. A sensitivity adjustment unit may be provided.
In this way, when the objective lens is switched by the operation of the objective switching unit, the numerical aperture of the objective lens differs depending on the type of the objective lens. Independently, the sensitivity of the image sensor can be adjusted according to the type of the objective lens so that the fluorescent image does not become too bright or too dark.

  Further, the present invention irradiates a specimen with spot-like laser light through an objective lens, scans the irradiation position of the laser light on the specimen, and generates a fluorescence generated in the specimen and collected by the objective lens. An image generation step for generating a fluorescence image based on the detected fluorescence and the scanning position, and surface illumination light is applied to the specimen, and the fluorescence generated in the specimen is collected by the objective lens. A step of taking an image after branching from the optical path of the laser beam, and an aperture amount of the fluorescence condensed by the objective lens after the branching from the optical path of the laser beam. Provided is a fluorescence observation method including an iris adjustment step for adjusting a lens pupil position and an optically conjugate position.

  According to the present invention, in the image generation step, a spot-like laser beam is scanned on the specimen, and the fluorescence generated at each position is collected and detected by the objective lens, so that a fluorescence image with high spatial resolution can be obtained. Can be acquired. In the imaging step, the surface illumination light is irradiated onto the specimen, and the fluorescence generated in a relatively wide range on the specimen is condensed and photographed by the objective lens, thereby making it more space than the fluorescence image acquired in the image generation step. A fluorescence image with low resolution can be acquired instantaneously. In the aperture adjustment step, the depth of focus of the fluorescence image acquired in the imaging step can be adjusted by adjusting the amount of fluorescence aperture. In this case, the spatial resolution of the fluorescence image acquired by the image generation step is affected by adjusting the amount of aperture of the fluorescence at a position optically conjugate with the pupil position of the objective lens after branching from the optical path of the laser beam. In this case, it is possible to adjust the depth of focus of the fluorescent image acquired in the photographing step.

  According to the present invention, a laser scanning fluorescence microscope and fluorescence observation capable of adjusting the focal depth of a fluorescent image acquired instantaneously without changing the spatial resolution of the fluorescent image acquired by scanning with laser light. It aims to provide a method.

1 is an overall configuration diagram showing a laser scanning fluorescence microscope according to a first embodiment of the present invention. It is a whole block diagram which shows the 1st modification of the laser scanning fluorescence microscope of FIG. FIG. 6 is an overall configuration diagram showing a second modification of the laser scanning fluorescence microscope of FIG. 1. It is a whole block diagram which shows the laser scanning fluorescence microscope which concerns on the 2nd Embodiment of this invention.

A laser scanning fluorescence microscope 1 and a fluorescence observation method according to a first embodiment of the present invention will be described below with reference to the drawings.
A laser scanning fluorescence microscope 1 according to the present embodiment is an apparatus for observing a specimen A placed on a stage 2 as shown in FIG. 1, and includes an LSM observation optical system 3 and a CCD observation optical system 4. And a control unit 5 for controlling them, a monitor 6 and an input unit 7. The stage 2 is provided so that the placed specimen A can be moved in a three-dimensional direction.

  The LSM observation optical system 3 includes a laser light source 8 that emits laser light, a scanner 9 that two-dimensionally scans the laser light emitted from the laser light source 8, and a laser beam scanned by the scanner 9 as a specimen A. An objective lens 10 that condenses the fluorescence generated in the specimen A, a dichroic mirror 11 that branches the fluorescence collected by the objective lens 10 and returned through the scanner 9 from the optical path of the laser beam, A condensing lens 12 that condenses the fluorescence branched by the dichroic mirror 11, a confocal pinhole 13 disposed at the focal position of the condensing lens 12, and the fluorescence that has passed through the confocal pinhole 13 is detected. And a photodetector (PMT: photomultiplier tube) 14. In the figure, reference numeral 15 is a pupil projection lens, reference numeral 16 is an imaging lens, and reference numeral 17 is a barrier filter.

  The CCD observation optical system 4 includes a surface illumination light source 18 such as a mercury lamp that generates white light, an excitation filter 19 that selects excitation light in a predetermined wavelength range from the white light emitted from the surface illumination light source 18, and A dichroic mirror 20 for merging the excitation light selected by the excitation filter 19 into the optical path between the imaging lens 16 and the objective lens 10, and the excitation light is emitted from the sample A and collected by the objective lens 10. A dichroic mirror 21 that branches the fluorescent light from the optical path to the scanner 9 between the imaging lens 16 and the pupil projection lens 15, and a pupil projection lens 22 that is arranged in the optical path branched by the dichroic mirror 21; A variable aperture 23, a photometric filter 24, an imaging lens 25, and a CCD camera 26 are provided.

In the figure, reference numeral 27 denotes a condenser lens, which converts white light from the surface illumination light source 18 into substantially parallel light.
The excitation filter 19 is, for example, a filter turret capable of selectively arranging a plurality of types of filters having different pass wavelength bands in the optical path.

The variable diaphragm 23 is disposed at a position optically conjugate with the pupil position of the objective lens 10 relayed by the imaging lens 16 and the pupil projection lens 22, and the diameter of the fluorescent light beam passing through the position is stepless. It can be adjusted.
The photometric filter 24 is also a filter turret, for example, and can select a fluorescence wavelength band to be photographed by the CCD camera 26 from the fluorescence condensed by the pupil projection lens 22.

  The control unit 5 controls the scanner 9 to adjust the scanning range, scanning position, and scanning speed of the laser beam. In addition, the control unit 5 controls the aperture diameter of the variable diaphragm 23 to adjust the fluorescent light beam diameter and adjust the incident NA (numerical aperture) of the fluorescent light incident on the CCD camera 26. Further, the control unit 5 controls the excitation filter 19 and the photometric filter 24 to adjust the wavelength range of the surface illumination light applied to the specimen A, and adjust the wavelength range of the fluorescence imaged by the CCD camera 26. ing.

  Further, the control unit 5 receives the intensity of the fluorescence detected by the light detector 14 and stores it in association with the irradiation position of the laser beam from the scanner 9, thereby generating an LSM fluorescence image. Further, the control unit 5 generates a CCD fluorescent image based on image information acquired instantaneously by the CCD camera 26.

  The monitor 6 displays the LSM fluorescence image and the CCD fluorescence image generated by the control unit 5 and displays the GUI. The input unit 7 inputs various observation conditions according to the GUI display on the monitor 6, or designates, for example, a range in the CCD fluorescence image displayed on the monitor 6 where the LSM fluorescence image is desired to be acquired with a cursor. Can be done.

A fluorescence observation method using the laser scanning fluorescence microscope 1 according to the present embodiment configured as described above will be described below.
In order to perform fluorescence observation of the specimen A using the laser scanning fluorescence microscope 1 according to the present embodiment, an observer inputs observation conditions from the input unit 7. As the observation conditions, first, the wavelength range of excitation light irradiated as surface illumination light and the wavelength range of fluorescence acquired by the CCD camera 26 can be mentioned.

  When an observation condition is input from the input unit 7, the control unit 5 controls the excitation filter 19 and the photometric filter 24 according to the input observation condition, and sets a pass wavelength band and an imaging wavelength range. In the initial state, the control unit 5 sets the variable diaphragm 23 to a predetermined aperture diameter, for example, a minimum aperture diameter sufficiently smaller than the pupil diameter of the objective lens 10.

  After various settings are made, the control unit 5 activates the surface illumination light source to emit white light. As a result, the excitation light in the wavelength band selected by the excitation filter 19 is reflected by the dichroic mirror 20 and irradiated onto a relatively wide range of the specimen A through the objective lens 10.

  In the specimen A, fluorescence is generated by exciting a fluorescent substance existing in a region irradiated with excitation light. The generated fluorescence is collected by the objective lens 10, collected by the imaging lens 16, and transmitted through the dichroic mirror 21. By being transmitted through the dichroic mirror 21, the light is branched from the optical path of laser light from a laser light source 8 described later, and is condensed by the pupil projection lens 22.

Then, the fluorescence condensed by the pupil projection lens 22 passes through the variable diaphragm 23 arranged at a position optically conjugate with the pupil position of the objective lens 10, and the wavelength range is selected by the photometric filter 24. Later, the light is condensed by the imaging lens 25 and photographed by the CCD camera 26.
Fluorescence image information over a relatively wide visual field range of the specimen A acquired instantaneously by the CCD camera 26 is sent to the control unit 5 to be generated as a CCD fluorescence image (imaging step) and displayed on the monitor 6.

  The observer checks the CCD fluorescence image over a relatively wide range of the specimen A displayed on the monitor 6 and searches for a part to be observed in detail. Since the variable aperture 23 is limited to the minimum aperture diameter, the CCD fluorescent image is dark and the contrast is low. That is, when the variable stop 23 is made sufficiently smaller than the pupil diameter of the objective lens 10, the depth of focus of the CCD fluorescent image is deepened, and from a relatively wide range in the depth direction around the focus position of the objective lens 10. A low-contrast CCD fluorescence image in which the generated fluorescence overlaps is acquired. If necessary, the control unit 5 increases the sensitivity of the CCD camera 26 or increases the gain by which the fluorescence image information is multiplied.

  In this case, the observer operates the input unit 7 as necessary to adjust the aperture diameter of the variable diaphragm 23 within a range smaller than the pupil diameter of the objective lens 10. Increasing the aperture diameter of the variable aperture 23 increases the incident NA to the CCD camera 26, so that the CCD fluorescent image becomes brighter and the contrast increases, but the depth of focus decreases. The observer appropriately adjusts the aperture diameter of the variable aperture 23 so that the position where the fluorescence is generated in the direction intersecting the optical axis of the objective lens 10 can be specified by a trade-off between the contrast and the focal depth. Set (aperture adjustment step).

  That is, by reducing the aperture diameter of the variable aperture 23 to be smaller than the pupil diameter of the objective lens 10, a CCD fluorescence image having a deep focal depth can be obtained, and fluorescence is emitted at a site different from the focal plane of the objective lens 10 in the depth direction. Can be included in the CCD fluorescence image. Therefore, the observer specifies a location where fluorescence is generated in the CCD fluorescence image displayed on the monitor 6, and sets the acquisition range of the LSM fluorescence image at that position by operating the input unit 7.

  The control unit 5 sets the scanning position and scanning range of the scanner 9 so that the laser beam is scanned within the acquisition range input from the input unit 7, and causes the laser light source 8 to emit the laser beam. The laser light emitted from the laser light source 8 is scanned two-dimensionally by the scanner 9 and condensed on the specimen A via the pupil projection lens 15, the dichroic mirror 21, the imaging lens 16 and the objective lens 10. Thereby, the laser beam is scanned within the acquisition range on the specimen A.

  When fluorescence is generated in the specimen A by being irradiated with the laser light, the fluorescence is collected by the objective lens 10 and is the same as the laser light through the imaging lens 16, the dichroic mirror 21, the pupil projection lens 15, and the scanner 9. It returns along the optical path and is branched by the dichroic mirror 11. Then, after the laser light component is removed by the barrier filter 17, the branched fluorescence is condensed by the condenser lens 12, and only the light that has passed through the confocal pinhole 13 arranged at the focal position is light. It is detected by the detector 14.

  The control unit 5 stores the irradiation position of the laser beam by the scanner 9 and the intensity of the fluorescence detected by the photodetector 14 at each irradiation position in association with each other, and stores the intensity of each fluorescence according to the information on the irradiation position. LSM fluorescence images can be generated by arranging them. Thereby, an LSM fluorescence image having a high spatial resolution in the optical axis direction spreading on the focal plane of the objective lens 10 can be acquired (image generation step).

  If the observer cannot confirm a desired fluorescent region in the LSM fluorescent image displayed on the monitor 6, the observer operates the stage 2 in the vertical direction to move the focal plane of the objective lens 10 in the specimen A in the vertical direction. By doing so, the fluorescent region can be searched.

  As described above, according to the laser scanning fluorescence microscope 1 and the fluorescence observation method according to the present embodiment, the specimen A over the wide visual field range is instantaneously observed by the CCD fluorescence image, and the specimen A is specified by the LSM fluorescence image. It is possible to perform detailed observation of a part with high spatial resolution. In this case, since the variable aperture 23 is arranged in the optical path branched before returning the fluorescence condensed by the objective lens 10 to the scanner 9, the CCD fluorescence image can be reproduced without reducing the spatial resolution of the LSM fluorescence image. The depth of focus can be adjusted.

  In particular, by increasing the focal depth of the CCD fluorescence image, the fluorescence generated at any position in the depth direction can be included in the CCD fluorescence image. Therefore, the entire observation can be performed at once without moving the stage 2 in the vertical direction and searching for a region with high fluorescence intensity. For example, when the specimen A is a living cell, it is possible to observe the entire area of the living cell as a whole at a time by increasing the depth of focus. As a result, the progress of the cell cycle is observed, and when a cell change occurs, a plurality of slice images spread on the focal plane of the objective lens 10 and a plurality of focal positions in the optical axis direction are shifted by a high NA LSM fluorescence image. Detailed observation can be performed by obtaining a laminated image obtained by photographing one slice image.

  In the present embodiment, the LSM observation optical system 3 is exemplified by the confocal observation optical system that disscans the fluorescence condensed by the objective lens 10, but the present invention is not limited to this. For example, as shown in FIG. 2, a pulse laser light source is used as the laser light source 8, and the fluorescence condensed by the objective lens 10 is immediately branched off from the optical path by the dichroic mirror 28 at the subsequent stage of the objective lens 10. Alternatively, a multi-photon excitation type LSM observation optical system 3 ′ that is detected by 14 may be employed. Also in this case, the focal depth of the CCD fluorescent image can be adjusted without impairing the spatial resolution of the LSM fluorescent image, as described above.

Further, in the present embodiment, the so-called epi-illumination type laser scanning fluorescence microscope 1 that irradiates the sample A with the excitation light from the surface illumination light source 18 from one direction and observes the fluorescence returning to the irradiation direction is exemplified. However, instead of this, as shown in FIG. 3, a laser scanning fluorescent microscope 1 ′ of a transmission illumination system may be adopted.
That is, the surface illumination light source 18 is arranged on the opposite side of the objective lens 10 across the specimen A, and on the lower side in FIG. 3, and the specimen A is irradiated with excitation light from the lower side. The fluorescent light emitted upward is condensed and detected by the objective lens 10.

  By doing in this way, the dichroic mirror 20 in the case of an epi-illumination system can be eliminated, and it can be comprised more simply. The mirror 29 shown in FIG. 3 does not need to have special wavelength characteristics, and can be configured at low cost.

  Further, a sensitivity adjustment unit (not shown) that adjusts the sensitivity of the CCD camera 26 based on the aperture amount of the variable aperture 23 may be provided. By doing so, the amount of fluorescence incident on the CCD camera 26 is reduced when the variable aperture 23 is reduced to obtain a fluorescent image with a deep focal depth. Therefore, the sensitivity of the CCD camera 26 is increased by the sensitivity adjustment unit. The acquired fluorescence image can be prevented from becoming dark. Further, when the variable diaphragm 23 is opened and the amount of fluorescence incident on the CCD camera 26 is increased, the sensitivity of the CCD camera 26 is decreased by the sensitivity adjustment unit so that the acquired fluorescence image does not become too bright. be able to.

  In addition, an objective switching unit (not shown) for switching the objective lens 10 is provided, and the sensitivity of the CCD camera 26 is adjusted based on the type of the objective lens 10 switched by the objective switching unit and / or the aperture amount of the variable aperture 23. A sensitivity adjustment unit may be provided.

Next, a laser scanning fluorescence microscope 30 and a fluorescence observation method according to the second embodiment of the present invention will be described with reference to the drawings.
In the description of the present embodiment, portions having the same configuration as those of the laser scanning fluorescence microscope 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 4, the laser scanning fluorescence microscope 30 according to the present embodiment includes a photostimulation optical system 31. The photostimulation optical system 31 is adjusted in position by a laser source 32 for photostimulation, a scanner 33 that adjusts the position of the laser light emitted from the laser light source 32 in a direction orthogonal to the optical axis direction, and the scanner 33. And a dichroic mirror 34 that joins the laser light to the optical path of the LSM observation optical system 3. In the figure, reference numeral 35 denotes a light beam diameter adjusting unit for adjusting the light beam diameter of the laser beam for light stimulation, and reference numeral 36 denotes a mirror.

  The light beam diameter adjusting unit 35 includes, for example, a plurality of lenses arranged in the optical axis direction, and is configured by a beam expander that changes the light beam diameter by moving at least one of the lenses in the optical axis direction. Yes. By adjusting the beam diameter of the laser beam for photostimulation, the incident NA of the laser beam for photostimulation incident on the specimen A through the objective lens 10 can be adjusted.

  According to the laser scanning fluorescence microscope 30 according to the present embodiment configured as described above, an area where high-intensity fluorescence is generated by a CCD fluorescence image having a deep focal depth obtained by narrowing the variable diaphragm 23 is used. When the light source can be identified, fluorescence is generated by causing the light beam diameter adjusting unit 35 to enter the laser light for light stimulation whose depth of focus is deepened by the light beam diameter adjusting unit 35. A light stimulus can be reliably applied to the site. That is, there is an advantage that light stimulation can be given to a desired region without specifying the irradiation position in the depth direction only by specifying the irradiation position of the laser light for light stimulation on the CCD fluorescent image. The reaction before and after applying the light stimulus can be observed in both the CCD fluorescence image and the LSM fluorescence image.

A Specimen 1,30 Laser scanning fluorescence microscope 5 Control unit 6 Monitor (display unit)
7 Input part (area designation part)
8 Laser light source 9 Scanner 10 Objective lens 11 Dichroic mirror (fluorescence branch)
DESCRIPTION OF SYMBOLS 13 Confocal pinhole 14 Optical detector 15 Optical path branch part 18 Surface illumination light source 20 Surface illumination light confluence part 21 Dichroic mirror (optical path branch part)
23 Variable aperture 26 CCD camera (imaging device)
27 Condenser Lens 32 Laser Light Source for Light Stimulation 33 Scanner (Stimulation Position Adjustment Unit)
34 Dichroic mirror (stimulating light junction)
35 Light beam diameter adjustment part

Claims (11)

A laser light source;
A scanner that scans laser light from the laser light source;
An objective lens that irradiates the sample with laser light scanned by the scanner and collects fluorescence generated in the sample;
A surface illumination light source for surface illumination of the specimen;
A fluorescence branching portion for branching the fluorescence generated by the laser light irradiation from the optical path of the laser light;
A photodetector for detecting fluorescence branched by the fluorescence branching unit;
An optical path branching section for branching fluorescence in an optical path between the objective lens and the scanner;
An image sensor for photographing fluorescence branched at the optical path branching section;
A laser scanning fluorescence microscope comprising: a variable stop disposed at a position optically conjugate with a pupil position of the objective lens between the imaging element and the optical path branching unit. The fluorescence branching unit branches the fluorescence in an optical path between the laser light source and the scanner,
The laser scanning fluorescence microscope according to claim 1, further comprising a confocal pinhole disposed at a position optically conjugate with a focal position of the objective lens between the fluorescence branching section and the photodetector. A fluorescence branching portion for branching fluorescence in an optical path between the objective lens and the optical path branching portion;
The laser scanning fluorescence microscope according to claim 1, wherein the photodetector detects fluorescence branched by the fluorescence branching unit.   2. The laser scanning fluorescence microscope according to claim 1, further comprising: a surface illumination light merging unit that merges illumination light from the surface illumination light source in an optical path toward the objective lens between the objective lens and the optical path branching unit.   The laser scanning fluorescent microscope according to claim 1, further comprising a condenser lens that irradiates the specimen with illumination light from the surface illumination light source from the opposite side of the objective lens across the specimen.   A laser source for photostimulation, a stimulation position adjusting unit for adjusting an irradiation position of a sample of the laser beam for photostimulation from the laser source for photostimulation, and a laser beam for photostimulation adjusted by the stimulation position adjusting unit. 2. The laser scanning fluorescence microscope according to claim 1, further comprising a stimulation light merging portion that merges an optical path between the scanner and the objective lens toward the objective lens.   The laser scanning fluorescence microscope according to claim 6, further comprising a light beam diameter adjusting unit that adjusts a light beam diameter of the laser light for light stimulation from the laser light source for light stimulation. A display unit for displaying a fluorescence image of the specimen acquired by the imaging device;
An area designating unit for designating an area in the fluorescent image displayed on the display unit;
The laser scanning fluorescence microscope according to claim 1, further comprising: a control unit that controls the scanner so as to acquire a fluorescence image of a region designated by the region designation unit.   The laser scanning fluorescence microscope according to claim 1, further comprising a sensitivity adjustment unit that adjusts the sensitivity of the imaging element based on a diaphragm amount of the variable diaphragm.   An objective switching unit that switches the objective lens, and a sensitivity adjustment unit that adjusts the sensitivity of the imaging device based on the type of the objective lens switched by the objective switching unit and / or the aperture amount of the variable diaphragm. 2. A laser scanning fluorescence microscope according to 1. The specimen is irradiated with a spot-like laser beam through the objective lens, the irradiation position of the laser light on the specimen is scanned, and the fluorescence generated in the specimen and collected by the objective lens is detected and detected. An image generation step for generating a fluorescence image based on the fluorescence and the scanning position;
An imaging step of irradiating the specimen with surface illumination light, condensing the fluorescence generated in the specimen by the objective lens, and photographing the condensed fluorescence after branching from the optical path of the laser light,
A diaphragm observation method including a diaphragm adjustment step of adjusting a diaphragm amount of the fluorescence condensed by the objective lens at a position optically conjugate with a pupil position of the objective lens after branching from the optical path of the laser light.

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