JP2017161740A - Light field microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire a three-dimensional image in which a difference between fluorescence intensities at a front and back of a focal plane is reduced.SOLUTION: A light field microscope 1 is provided that comprises: illumination optical systems 2 and 3 that irradiate a sample X with excitation light; an objective lens 7 that converges fluorescence generated in the sample X thanks to the irradiation with the excitation light by the illumination optical systems 2 and 3; and a detection optical system 4 that includes an image pick-up element 16 imaging the fluorescence converged by the objective lens 7, and a microlens array 15 arranged between the image pick-up element 16 and the objective lens 7, in which the illumination optical systems 2 and 3 are configured to irradiate both sides with the excitation light across a focal plane A to be arranged within the sample X of the objective lens 7.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a light field microscope.

  2. Description of the Related Art Conventionally, a light field microscope is known that can construct three-dimensional image data of a specimen by focusing on the deep part of the specimen and performing one-time image acquisition (see, for example, Patent Document 1).

US Pat. No. 7,723,662

However, since conventional light field microscopes detect fluorescence returning to the direction of excitation light irradiation by epi-illumination, the amount of excitation light at a position deeper than the focal position of the excitation light decreases due to the effects of light absorption and scattering of the sample. There is an inconvenience that a three-dimensional image with low fluorescence intensity is acquired in a portion deeper than the focal plane.
The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a light field microscope that can acquire a three-dimensional image with reduced fluorescence intensity difference before and after the focal plane.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention includes an illumination optical system that irradiates a specimen with excitation light, an objective lens that collects fluorescence generated in the specimen when the illumination optical system irradiates excitation light, and the objective lens. A detection optical system including an imaging device for photographing the emitted fluorescence and a microlens array arranged between the imaging device and the objective lens, and the illumination optical system is arranged in the sample Provided is a light field microscope that irradiates the excitation light from both sides with the focal plane of the objective lens interposed therebetween.

According to this aspect, when the sample is irradiated with the excitation light by the illumination optical system, the fluorescence generated in the sample is collected by the objective lens and taken by the image sensor after passing through the microlens array. Thereby, not only the focal plane of the objective lens in the sample but also information on each part of the sample through which the excitation light has passed can be acquired by the imaging device, and a three-dimensional image of the sample can be obtained based on the acquired image information. Can be acquired.
In this case, the illumination optical system irradiates excitation light from both sides across the focal plane of the objective lens arranged in the specimen, so that not only the objective lens side but also the opposite side of the objective lens with respect to the focal plane It is possible to generate fluorescence with sufficient intensity at each of the positions, and to obtain a three-dimensional image with reduced fluorescence intensity difference before and after the focal plane.

In the above aspect, the illumination optical system includes an epi-illumination optical system that irradiates the specimen with the excitation light via the objective lens, and a condenser lens that is disposed on the opposite side of the objective lens across the specimen. And a transmission illumination optical system for irradiating the specimen with the excitation light.
In this way, the sample is irradiated with excitation light from the objective lens side through the objective lens by the epi-illumination optical system, and from the side opposite to the objective lens through the condenser lens by the transmission illumination optical system. Is irradiated with excitation light. Thereby, excitation light can be irradiated from both sides across the focal plane of the objective lens, and a three-dimensional image with reduced fluorescence intensity difference before and after the focal plane can be acquired.

In the above aspect, the transmission illumination optical system includes a slit member that is disposed at a substantially focal position on the opposite side of the condenser lens from the specimen, and that forms zonal light. It is preferable that an exit angle of the excitation light to be emitted is larger than an angle corresponding to the numerical aperture of the objective lens.
By doing so, the excitation light formed in the annular light by passing through the slit member by the transmission illumination optical system is condensed by the condenser lens and irradiated onto the specimen. By making the emission angle of the excitation light emitted from the condenser lens to the specimen larger than the angle corresponding to the numerical aperture of the objective lens, the excitation light from the condenser lens side that has passed through the specimen is collected by the objective lens. It is possible to obtain a good fluorescence image.

In the aspect described above, at least one of the epi-illumination optical system and the transmission illumination optical system may include an intensity adjustment unit that adjusts the intensity of the excitation light.
In this way, the intensity adjusting unit adjusts at least one of the intensity of the excitation light applied to the specimen by the epi-illumination optical system and the intensity of the excitation light applied to the specimen by the transmission illumination optical system, and the focus of the objective lens Fine adjustment can be made so as to eliminate the difference in fluorescence intensity between the front and back surfaces.

Moreover, in the said aspect, the said intensity adjustment part may adjust the intensity | strength of the said excitation light based on the position of the focal plane of the said objective lens with respect to the said sample.
By doing so, even when the ratio of the thickness of the specimen on the epi-illumination optical system side and the thickness of the specimen on the transmission illumination optical system side changes, such as when the position of the focal plane of the objective lens changes Fine adjustment can be made so as to eliminate the difference in fluorescence intensity before and after the focal plane of the objective lens.

Further, in the above aspect, the intensity adjusting unit is configured to obtain a luminance of a fluorescent image acquired by the imaging device when the excitation light is applied to the sample via the epi-illumination optical system, and the transmission illumination optical system. The intensity of the excitation light may be adjusted so that the luminance of the fluorescence image acquired by the imaging device is substantially equal when the sample is irradiated with the excitation light via the.
By doing in this way, the fluorescence intensity difference before and behind the focal plane can be reduced without detecting the position of the focal plane of the objective lens.

Further, in the above aspect, a transmitted bright field illumination optical system that causes bright field illumination light to be incident on the sample via the condenser lens, and a bright field imaging device that captures the bright field illumination light transmitted through the sample; May be provided.
By doing so, by transmitting the bright field illumination light to the specimen through the condenser lens by the transmission bright field illumination optical system and photographing the bright field illumination light transmitted through the specimen by the bright field imaging device, The focal plane of the objective lens in the sample can be determined by bright field illumination without damaging the sample by the excitation light.

  According to the present invention, it is possible to obtain a three-dimensional image in which a difference in fluorescence intensity between before and after the focal plane can be acquired.

1 is a schematic overall configuration diagram showing a light field microscope according to a first embodiment of the present invention. It is a typical whole block diagram which shows the light field microscope which concerns on the 2nd Embodiment of this invention. It is a typical whole block diagram which shows the light field microscope which concerns on the 3rd Embodiment of this invention. It is a typical whole block diagram which shows the light field microscope which concerns on the 4th Embodiment of this invention.

The light field microscope 1 according to the first embodiment of the present invention will be described below with reference to FIG.
As shown in FIG. 1, the light field microscope 1 according to this embodiment is an epi-illumination optical system (illumination optical system) that makes excitation light incident on a specimen X placed on a stage from one direction, for example, from above. 2, a transmission illumination optical system (illumination optical system) 3 that makes excitation light incident on the sample X from the other direction, for example, from below, and a detection optical system 4 that detects fluorescence generated in the sample X.

The epi-illumination optical system 2 includes a first light source 5 that generates excitation light, an illumination lens 6 that condenses the excitation light emitted from the first light source 5, and the excitation light collected by the illumination lens 6. While irradiating the specimen X, an objective lens 7 that condenses the fluorescence generated in the specimen X is provided.
The transmission illumination optical system 3 includes a second light source 8 that generates excitation light, an illumination lens 9 that condenses the excitation light emitted from the second light source 8, and the excitation light collected by the illumination lens 9. And a condenser lens 10 for irradiating the specimen X. The epi-illumination optical system 2 and the transmission illumination optical system 3 are general bright-field illumination optical systems such as Koehler illumination.

The epi-illumination optical system 2 and the transmission illumination optical system 3 are configured to irradiate the specimen X with excitation light from the light sources 5 and 8 simultaneously.
In the figure, reference numeral 11 denotes a mirror for forming an optical path of the transmission illumination optical system 3.

  The detection optical system 4 includes a dichroic mirror 12 that branches the fluorescence collected by the objective lens 7 from the optical path of the excitation light, and an emission filter 13 that transmits the fluorescence branched by the dichroic mirror 12 and blocks the excitation light. The imaging lens 14 that condenses the fluorescence that has passed through the emission filter 13, the microlens array 15 disposed near the focal position of the imaging lens 14, and the fluorescence that has passed through the microlens array 15 are photographed. An image sensor 16.

The operation of the light field microscope 1 according to this embodiment configured as described above will be described below.
In order to acquire a three-dimensional image of the specimen X using the light field microscope 1 according to this embodiment, the first light source 5 of the epi-illumination optical system 2 is operated to emit excitation light, and the transmission illumination optical system 3 The second light source 8 is activated to emit excitation light.

The excitation light emitted from the first light source 5 incidentally illuminates the specimen X by the objective lens 7 via the illumination lens 6 and the dichroic mirror 12.
On the other hand, the excitation light emitted from the second light source 8 illuminates and transmits the sample X through the condenser lens 10 via the illumination lens 9 and the mirror 11.

  By preliminarily fluorescently labeling the specimen X, the fluorescent substance in the specimen X is excited by the irradiated excitation light, and fluorescence is generated. The fluorescence generated in the sample X is collected by the objective lens 7 and then separated from the optical path of the excitation light by the dichroic mirror 12. Then, after passing through the emission filter 13, the excitation light is removed, and then condensed by the imaging lens 14, passes through the microlens array 15, and is photographed by the imaging device 16.

  The fluorescence image acquired by the imaging device 16 includes information on each position in the direction along the optical axis of the objective lens 7 as well as in the focal plane A of the objective lens 7 in the sample X by the microlens array 15. ing. Therefore, by performing deconvolution processing by software on the acquired fluorescence image, it is possible to extract each three-dimensional position in correspondence with the fluorescence intensity emitted from that position. Fluorescent images can be generated.

In this case, according to the light field microscope 1 according to the present embodiment, excitation light is incident from both sides across the focal plane A of the objective lens 7 disposed in the deep portion in the sample X. The difference in the intensity of the fluorescence generated in can be reduced.
That is, when the excitation light is irradiated only by the epi-illumination optical system 2 or only the transmission illumination optical system 3, the intensity of the excitation light decreases as it goes deeper due to scattering and absorption in the specimen X, so that the objective lens 7 There may be a large difference in the fluorescence intensity generated before and after the focal plane A.

  As in the light field microscope 1 according to this embodiment, the sample X is simultaneously irradiated with the excitation light from both the epi-illumination optical system 2 and the transmission illumination optical system 3, whereby the difference in fluorescence intensity before and after the focal plane is obtained. There is an advantage that a three-dimensional image that is reduced and easy to view can be generated.

Next, a light field microscope 17 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the light field 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. 2, the light field microscope 17 according to the present embodiment has a slit member 20 having a ring-shaped slit 19 disposed in front of the condenser lens 10 of the transmission illumination optical system 18. This is different from the light field microscope 1 according to the first embodiment.
The slit member 20 is disposed in the vicinity of the focal position on the opposite side (rear side) of the condenser lens 10 from the sample X. The size of the slit 19 is such that the exit angle α at which the excitation light formed in an annular shape by passing through the slit 19 is condensed by the condenser lens 10 and emitted to the specimen X is the objective lens. It is set to be larger than an angle corresponding to a numerical aperture (NA) of 7.

  According to the light field microscope 17 according to the present embodiment configured as described above, the excitation light applied to the specimen X from the transmission illumination optical system 18 passes through the specimen X as shown in FIG. It does not enter the lens 7. That is, it is possible to prevent the fluorescence condensed by the objective lens 7 from including the direct light of the excitation light incident by the transmission illumination optical system 3.

  Although the dichroic mirror 12 and the emission filter 13 alone may not sufficiently remove the excitation light due to their optical characteristics, according to the present embodiment, the direct light of the excitation light irradiated by the transmission illumination optical system 18 is fluorescent. In addition, there is an advantage that a good three-dimensional image with less noise can be acquired by preventing the inconvenience of being photographed together.

Next, a light field microscope 21 according to a third embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the light field 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. 3, the light field microscope 21 according to the present embodiment gives the incident illumination optical system 22 and the transmission illumination optical system 23 the intensity of the excitation light emitted from the first light source 5 and the second light source 8. The light field microscope 1 is different from the light field microscope 1 according to the first embodiment in that it includes intensity adjusting units 24 and 25 for adjustment and a control unit 26 for controlling the intensity adjusting units 24 and 25.

  The intensity adjusting units 24 and 25 are, for example, disk-shaped ND filters 27 and 27 arranged on the optical path between the first light source 5 and the illumination lens 6 and on the optical path between the second light source 8 and the illumination lens 9, respectively. 28, and motors 29 and 30 for rotating the ND filters 27 and 28 about an axis substantially parallel to the optical axis. The ND filters 27 and 28 change the transmittance along the circumferential direction, and are rotated by the motors 29 and 30 to continuously change the intensity of the excitation light incident on the illumination lenses 6 and 9. It can be done.

For example, the control unit 26 operates the motors 29 and 30 of the intensity adjustment units 24 and 25 based on the intensity of the fluorescent image acquired by the image sensor 16.
That is, when the observer sets the focal plane A of the objective lens 7 to a desired position in the sample X, the control unit 26 first sets the transmittance of the ND filters 27 and 28 by preliminary processing, and sets the transmittance. The main shooting is now done.

  Specifically, in the preliminary process, the control unit 26 operates the motor 30 of the intensity adjustment unit 25 on the transmission illumination optical system 23 side to place the ND filter 28 at the cutoff position where the transmittance is zero, and the incident illumination optical system. The motor 29 of the intensity adjusting unit 24 on the system 22 side is operated, the transmittance of the ND filter 27 is set to a predetermined value, and a temporary image is acquired. Next, the control unit 26 operates the motor 29 of the intensity adjusting unit 24 on the epi-illumination optical system 22 side to place the ND filter 27 at the cutoff position where the transmittance is zero, and the intensity adjusting unit on the transmission illumination optical system 23 side. The transmittance of the ND filter 28 is adjusted so that a fluorescent image having substantially the same brightness as that of the temporary image is acquired by operating the motor 30.

  This completes the preliminary process. Since the intensity ratio between the excitation light from the epi-illumination optical system 22 and the excitation light from the transmission illumination optical system 23 is obtained by the preliminary process, the control unit 26 determines the intensity ratio obtained by the preliminary process in the actual photographing. The fluorescent image is acquired by adjusting the ND filters 27 and 28 so as not to change. Thereby, there is an advantage that a difference in fluorescence intensity before and after the focal plane can be further reduced with accuracy and an easy-to-see three-dimensional image can be generated.

  In the present embodiment, the intensity adjusting units 24 and 25 are provided in both the epi-illumination optical system 22 and the transmission illumination optical system 23. Alternatively, the intensity adjustment units 24 and 25 may be provided in either one of them. . In addition, the intensity adjustment units 24 and 25 are set based on the intensity of the fluorescence image acquired by the image sensor 16, but instead, the position of the focal plane A of the objective lens 7 is detected, The intensity adjusting units 24 and 25 may be set according to the position of the focal plane A.

  Further, in the present embodiment, the strength adjustment units 24 and 25 are automatically adjusted by the control unit 26, but instead of this, manual adjustment may be made.

Next, a light field microscope 31 according to a fourth embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the light field microscope 21 according to the third embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 4, the light field microscope 31 according to the present embodiment includes a transmitted bright field illumination optical system 32 that irradiates a specimen X with bright field illumination light for bright field transmission observation, and a bright field that transmits the specimen X. The light field microscope 21 according to the third embodiment is different from the light field microscope 21 in that a bright field imaging device 33 for photographing illumination light is provided.

The transmitted bright field illumination optical system 32 includes a third light source 34 that emits bright field illumination light, an illumination lens 35 that collects the bright field illumination light emitted from the third light source 34, and the transmitted illumination optical system 23. And a mirror 36 detachably provided on the optical path of the excitation light from the second light source 8.
The mirror 36 is inserted on the optical path of the transmissive illumination optical system 23, thereby causing bright field illumination light to enter the optical path of the transmissive illumination optical system 23 and blocking the excitation light from the second light source 8. Yes.

  The bright field imaging device 33 has an imaging surface disposed in the vicinity of the focal position of the imaging lens 14, and a mirror 37 detachably provided on the optical path of the detection optical system 4 is inserted on the optical path. Thus, the bright field illumination light transmitted through the specimen X and condensed by the objective lens 7 and the imaging lens 14 can be photographed.

  According to the light field microscope 31 according to the present embodiment configured as described above, when the operator searches for the observation location of the specimen X, the third mirrors 36 and 37 are inserted into the optical path, thereby providing the third The bright field illumination light from the light source 34 is irradiated onto the specimen X through the illumination lens 9 and the condenser lens 10, and the bright field illumination light transmitted through the specimen X is captured through the objective lens 7 and the imaging lens 14 for bright field imaging. Images can be taken with the element 33.

By doing in this way, as the bright field illumination light, visible light or the like that does not damage the specimen X can be used, and it is possible to prevent the specimen X from being damaged by searching for the observation location. There is.
After the observation place is searched, the two mirrors 36 and 37 are separated from the optical path to constitute the light field microscope 31 according to the third embodiment, and a three-dimensional image of the specimen X is acquired. Can do.

1,17,21,31 Light field microscope 2,22 Epi-illumination optical system (illumination optical system)
3, 18, 23 Transmission illumination optical system (illumination optical system)
DESCRIPTION OF SYMBOLS 4 Detection optical system 7 Objective lens 10 Condenser lens 15 Micro lens array 16 Image sensor 20 Slit member 24, 25 Intensity adjustment part 32 Transmission bright field illumination optical system 33 Bright field image sensor X Sample (alpha) Output angle

Claims (7)

An illumination optical system for irradiating the specimen with excitation light;
An objective lens that condenses the fluorescence generated in the specimen by being irradiated with excitation light by the illumination optical system, an imaging element that photographs the fluorescence condensed by the objective lens, and an imaging element and the objective lens A detection optical system comprising a microlens array disposed between,
A light field microscope in which the illumination optical system irradiates the excitation light from both sides across a focal plane of the objective lens disposed in the specimen. The illumination optical system is an epi-illumination optical system that irradiates the sample with the excitation light through the objective lens;
The light field microscope according to claim 1, further comprising: a transmission illumination optical system that irradiates the excitation light to the specimen via a condenser lens disposed on the opposite side of the objective lens with the specimen interposed therebetween. The transmission illumination optical system includes a slit member that is disposed at a substantially focal position on the opposite side of the condenser lens from the sample, and forms annular light;
The light field microscope according to claim 2, wherein an emission angle of the excitation light emitted from the condenser lens to the specimen is larger than an angle corresponding to a numerical aperture of the objective lens.   4. The light field microscope according to claim 2, wherein at least one of the epi-illumination optical system and the transmission illumination optical system includes an intensity adjusting unit that adjusts the intensity of the excitation light.   The light field microscope according to claim 4, wherein the intensity adjusting unit adjusts the intensity of the excitation light based on a position of a focal plane of the objective lens with respect to the specimen.   The intensity adjusting unit emits the excitation light through the incident illumination optical system, the brightness of the fluorescence image acquired by the imaging device when the sample is irradiated with the excitation light, and the excitation light through the transmission illumination optical system. The light field microscope according to claim 4, wherein the intensity of the excitation light is adjusted so that a luminance of a fluorescent image acquired by the imaging device when the specimen is irradiated is substantially equal. A bright-field illumination optical system that makes bright-field illumination light incident on the sample via the condenser lens, and a bright-field imaging device that photographs the bright-field illumination light transmitted through the sample. Item 7. The light field microscope according to any one of Items 6.

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