JP2013160893A - Microscope and aberration correction method for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To correct aberration at the position of a specimen and perform accurate observation while suppressing damage applied to the specimen.SOLUTION: A microscope 1 comprises: a spatial optical modulation element 13 for modulating the wave front of excitation light from a light source 2; a scanner 10 for two-dimensionally scanning the excitation light on a specimen A; an imaging part 20 for photographing fluorescent light generated from a scanning position on the specimen A of the excitation light by the scanner 10 and acquiring a fluorescent image; and a control part 5 for radiating spot-like excitation light to the position of at least one place on the specimen A by the scanner, causing the imaging part 20 to photograph the fluorescent light generated in the specimen A, determining aberration at the position of the specimen A on the basis of an acquired fluorescent light spot image by software, and controlling the spatial optical modulation element 13 on the basis of the determined aberration.

Description

  The present invention relates to a microscope and an aberration correction method thereof.

  Conventionally, a technique for acquiring a plurality of images while changing a correction amount by a wavefront correction apparatus and comparing the acquired images to determine a correction amount that can correct aberrations best is known (for example, (See Patent Document 1).

“Adaptive harmonic generation microscopy of mammalian embryos”, OPTICS LETTERS, Vol.34, No.20, October 15, 2009

However, in the technique of this non-patent document 1, it is necessary to photograph the sample many times before the correction amount by the wavefront correction device is determined. There is a disadvantage that damage such as photo bleaching is given.
The present invention has been made in view of the above-described circumstances, and provides a microscope and an aberration correction method capable of correcting an aberration at the position of the sample and accurately observing while suppressing damage to the sample. The purpose is that.

In order to achieve the above object, the present invention provides the following means.
One embodiment of the present invention includes a spatial light modulation element that modulates the wavefront of excitation light from a light source, a scanner that two-dimensionally scans excitation light on a sample, and a scanning position of the excitation light on the sample by the scanner. An imaging unit that captures the generated fluorescence and obtains a fluorescence image, and the scanner emits the spot-like excitation light to at least one position on the sample, and the fluorescence generated in the sample by irradiation of the excitation light A microscope including: a control unit that controls the spatial light modulation element based on the determined aberration based on the acquired fluorescence spot image. I will provide a.

  According to this aspect, when the excitation light emitted from the light source is scanned on the specimen by the scanner, the fluorescent substance contained in the specimen is excited to generate fluorescence. Then, the generated fluorescence is photographed by the imaging unit, whereby a fluorescence image is acquired.

  The control unit controls the scanner to irradiate at least one spot on the specimen with spot-like excitation light, and the fluorescence generated at that time is captured by the imaging unit, whereby a fluorescent spot image is acquired. Since this fluorescent spot image is deformed when aberrations based on the refractive index non-uniformity of the optical member existing in the optical path to the specimen are included, the control unit corrects the aberration based on the shape of the optical spot image. It can be determined by software.

  The control unit can modulate the wavefront of the excitation light so as to eliminate the aberration by controlling the spatial light modulation element based on the determined aberration. That is, by scanning excitation light having a wavefront modulated as described above on a specimen with a scanner, a precise fluorescent image can be acquired and observed with high accuracy. In this case, the phase pattern generated by the spatial light modulation element in order to remove the aberration can be determined only by photographing the fluorescent spot image once, and the aberration at the position of the specimen can be reduced while suppressing damage to the specimen. Correction can be performed with high accuracy.

In the above aspect, the control unit compares a plurality of reference spot images generated by software with a spot image including various aberrations at the position of the sample and the fluorescent spot image acquired in the imaging unit, The aberration included in the reference spot image having high similarity may be determined as the aberration at the position of the sample.
In this way, a plurality of reference spot images including various aberrations can be easily generated by software, and the shape of each reference spot image is compared with the shape of the captured fluorescent spot image. The aberration included in the most similar reference spot image can be determined as the aberration at the sample position.

In the above aspect, the reference spot image may be generated by software using a Zernike coefficient.
By doing in this way, the reference spot image containing various aberrations can be easily generated by software.

Moreover, in the said aspect, the said control part may determine the similarity of the said reference spot image and the said fluorescence spot image by a cross correlation.
By doing in this way, the similarity of two spot images can be determined easily and the reference spot image most similar to the acquired fluorescent spot image can be easily determined.

In the above aspect, the imaging unit may acquire a plurality of fluorescent spot images by photographing fluorescence generated in the specimen by irradiation with spot-like excitation light at two or more positions on the specimen. .
By doing in this way, the similarity with a reference spot image can be determined about each of several fluorescent spot images, and the precision of the determined aberration can be improved.

Moreover, in the said aspect, the irradiation pattern of the spot-like excitation light with respect to two or more positions on the said sample may be produced | generated by the said spatial light modulation element.
By doing so, the wavefront of the excitation light can be modulated by controlling the spatial light modulation element, and a plurality of positions can be irradiated with spot-like excitation light simultaneously.

  Another aspect of the present invention is a microscope aberration correction method for irradiating a specimen with excitation light from a light source, photographing fluorescence generated in the specimen, and acquiring a fluorescent image, and spotting the specimen with respect to the spot And determining the aberration at the position of the sample by software based on the fluorescent spot image obtained by irradiating the excitation light in the shape, and modulating the wavefront of the excitation light so as to reduce the determined aberration And a microscope aberration correction method.

  According to this aspect, the modulation to be given to the wavefront of the excitation light in order to remove the aberration can be determined only by photographing the fluorescent spot image once, and the aberration at the position of the specimen can be reduced while suppressing damage to the specimen. Correction can be performed with high accuracy.

  In the above aspect, the step of determining the aberration includes a step of generating a plurality of reference spot images composed of spot images including various aberrations at the position of the specimen in software, the generated reference spot image and the fluorescent spot. An aberration included in a reference spot image having high similarity may be determined as an aberration at the position of the sample.

In the above aspect, the reference spot image may be generated by software using a Zernike coefficient.
Moreover, in the said aspect, the step which determines an aberration may determine the similarity of the said reference spot image and the said fluorescence spot image by a cross correlation.
Further, in the above aspect, the step of determining aberration is based on a plurality of fluorescent spot images acquired by photographing fluorescence generated in the specimen by irradiation of spot-like excitation light at two or more positions on the specimen. May be determined.

  According to the present invention, it is possible to perform observation with high accuracy by correcting aberrations at the position of the specimen while suppressing damage to the specimen.

It is a whole lineblock diagram showing the microscope concerning one embodiment of the present invention. It is a flowchart which shows an example of the phase correction method which concerns on this embodiment by the microscope of FIG. It is a figure which respectively shows an example of (a) fluorescence image acquired by the microscope of FIG. 1, (b) selected spot position, and (c) fluorescence spot image. (A)-(c) is a figure which shows three examples of the reference spot image memorize | stored in the memory | storage part of the microscope with a different aberration vector. It is a whole block diagram which shows the modification of the microscope of FIG.

A microscope 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope 1 according to the present embodiment includes an illumination optical system 3 that irradiates the specimen A with excitation light from the light source 2, a photographing optical system 4 that photographs fluorescence generated in the specimen A, And a control unit 5 for controlling them.

  The illumination optical system 3 includes a collimating lens 6 that collimates the excitation light from the light source 2, a wavefront modulation unit 7 that modulates the wavefront of the excitation light, and a relay optical system 8 that relays the wavefront modulated in the wavefront modulation unit 7. 9, a first scanner 10 that scans the excitation light two-dimensionally, a dichroic mirror 11 that reflects the excitation light and transmits the fluorescence, and an objective lens 12 that irradiates the specimen A with the excitation light.

The wavefront modulation unit 7 includes a reflection type wavefront modulation element (spatial light modulation element) 13, a folding mirror 14 for bringing the incident angle of the excitation light to the wavefront modulation element 13 close to the normal direction of the wavefront modulation element 13, and It has. The wavefront modulation element 13 generates a hologram phase pattern capable of achieving an irradiation pattern according to a command from the control unit 5 on the reflection surface, and modulates the wavefront of the excitation light reflected on the reflection surface into a form according to the phase pattern. It has become. Further, the wavefront modulation element 13 can reflect the incident excitation light without modulating it by making its reflection surface into a mirror surface.
The first scanner 10 is configured by a so-called proximity galvanometer mirror in which two galvanometer mirrors 10a and 10b that are swung around non-parallel axes are arranged close to each other.

The photographing optical system 4 has a relay optical system 15 that relays fluorescence from the specimen A collected by the objective lens 12 and transmitted through the dichroic mirror 11; 2 includes a scanner 16, condenser lenses 17 and 18, a confocal pinhole 19, and a photomultiplier tube 20.
The second scanner 16 is also constituted by a so-called proximity galvanometer mirror in which two galvanometer mirrors 16a and 16b that are swung around a non-parallel axis line are arranged close to each other.

The control unit 5 generates a fluorescence image based on the fluorescence intensity information acquired by the photomultiplier tube 20 and the scanning position information of the first scanner 10 and the second scanner 16.
Further, the control unit 5 stops the second scanner 16 and activates the first scanner 10 to set the position for irradiating the spot-like excitation light based on the acquired fluorescent image and to set the position. The second scanner 16 is operated to acquire a fluorescent spot image in a state where the first scanner 10 is stopped so that the spot-like excitation light is irradiated to the formed position.

In addition, the control unit 5 includes a storage unit 5a that stores a plurality of reference spot images, which are generated in software from spot images including various aberrations, in association with the aberrations.
The reference spot image is generated using, for example, a Zernike coefficient.

That is, the aberration is given based on the pupil position of the objective lens 12 and ZernikeZ (ρ, θ), and the aberration vector is ΔW = (Z ₁ , Z ₂ ,..., Z _n ). In the case of no aberration, ΔW = (0, 0,..., 0).

For example, when Z ₅ includes an aberration of 0.1λ, the aberration vector ΔW = (0, 0, 0, 0, 0.1, 0,..., 0) as shown in FIG. A reference spot image is generated.
Next, as shown in FIG. 4 (b), the aberration vector ΔW = (0,0,0,0,0,0,0,0.1,0 containing aberration of 0.1λ to _{Z 8,} ... , 0) is generated.
Next, as shown in FIG. 4 (c), the aberration vector ΔW = (0,0,0,0,0.1,0 containing the _{Z 5} 0.1 [lambda], the aberrations of 0.1 [lambda] to _{Z 8,} 0, 0.1, 0,..., 0) reference spot images are generated.

In this manner, this process is repeated (aberration range / aberration interval) ^ (number of Zernike coefficients) times at a predetermined aberration interval over a predetermined aberration range for a predetermined number of Zernike coefficients.
For example, when an aberration range of ± 5λ is performed at intervals of 0.1λ for _five Zernike coefficients Z ₅ , Z ₆ , Z ₇ , Z ₈ , Z ₉ , 100 ⁵ times are repeated.

Then, the control unit 5 obtains a cross-correlation between the plurality of reference spot images generated in this way and the fluorescent spot image acquired by photographing, and the aberration vector ΔW = when the evaluation function value becomes the largest. (0, 0, 0, 0, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ ,..., X _n ) is determined as the aberration at the position of the sample A.
Further, the control unit 5 instructs the wavefront modulation element 13 to generate a phase pattern that can correct the obtained aberration vector ΔW on the reflection surface of the wavefront modulation element 13. Thereby, an aberration-free spot can be formed on the specimen A, and a fluorescent image with high intensity can be obtained.

The aberration correction method of the microscope 1 according to this embodiment configured as described above will be described below.
In the aberration correction method of the microscope 1 according to the present embodiment, as shown in FIG. 2, first, the wavefront is not corrected by the wavefront modulation element 13, that is, the reflection surface of the wavefront modulation element 13 is set to a mirror surface. (Step S1).

  Next, the second scanner 16 is stopped (step S2), and the first scanner 10 is operated (step S3). As a result, the excitation light from the light source 2 is reflected unmodulated by the wavefront modulation element 13, relayed by the relay optical system 8, and scanned two-dimensionally by the first scanner 10. The excitation light scanned two-dimensionally by the first scanner 10 is relayed by the relay optical system 9, reflected by the dichroic mirror 11, collected on the sample A by the objective lens 12, and present in the sample A. Excites the fluorescent material.

  The fluorescence generated by exciting the fluorescent substance in the specimen A is collected by the objective lens 12, transmitted through the dichroic mirror 11, relayed by the relay optical system 15, and then stopped. Only the fluorescent light that has passed through the condensing lens 17 and is generated at the focal plane of the objective lens 12 passes through the confocal pinhole 19 and is condensed by the condensing lens 18 to be a photomultiplier tube. 20 is detected. By storing the fluorescence intensity detected by the photomultiplier tube 20 and the scanning position of the first scanner 10 at the time of detecting each fluorescence intensity, the fluorescence image shown in FIG. Obtained (step S4).

Next, the control unit 5 detects a region having a high fluorescence intensity in a predetermined area in the acquired fluorescence image, and sets the position as a spot position as shown in FIG. S5), and sent to the first scanner 10. FIG. 3B is an enlarged view of a region surrounded by a frame in FIG. 3A, and a broken line indicates a boundary of a portion where a fluorescent substance that emits fluorescence exists in the fluorescence image of FIG. The region where the spot image is present is the region where the fluorescent material is present.
Then, the first scanner 10 is stopped so that the set spot position is irradiated with the excitation light (step S6), and the second scanner 16 is operated (step S7).

  Thereby, since the spot-like excitation light is irradiated to the set spot position on the specimen A, the fluorescence image around the spot position is obtained by the operation of the second scanner 16 as shown in FIG. A fluorescent spot image is acquired (step S8).

  Next, the control unit 5 reads the reference spot image stored in the storage unit 5a (step S9), and all the references from which the similarity between the acquired fluorescent spot image and the read reference spot image is read. It calculates with respect to a spot image (step S10). As described above, the similarity is calculated by calculating an evaluation function value of a cross-correlation between each reference spot image generated so as to have a different aberration vector ΔW and a fluorescent spot image obtained by photographing. Is called.

Then, by determining the reference spot image having the largest evaluation function value, the aberration vector ΔW of the reference spot image most similar to the fluorescent spot image is determined (step S11).
The control unit 5 instructs the wavefront modulation element 13 to generate a phase pattern on the reflection surface of the wavefront modulation element 13 that can correct the aberration vector ΔW determined as described above (step S12).

With the wavefront modulation element set in this manner, the second scanner 16 is stopped (step S13), and the first scanner 10 is operated (step S14), thereby forming an aberration spot on the specimen A. The spot is scanned two-dimensionally by the first scanner 10. Thereby, fluorescence with high intensity can be generated at each scanning position.
Then, by detecting the high-intensity fluorescence generated at each scanning position in the photomultiplier tube 20, a clear fluorescence image of the specimen can be acquired (step S15).

  That is, according to the microscope 1 and the phase correction method thereof according to the present embodiment, acquisition of information for determining the phase pattern generated in the wavefront modulation element 13 is attempted by irradiating the specimen A with excitation light a plurality of times. It is not performed by mistake, but by comparing a fluorescent spot image acquired by one photographing with a plurality of reference spot images generated by software. For this reason, it is not necessary to irradiate the specimen A with excessive excitation light, and there is an advantage that the aberration at the position of the specimen A can be corrected and observation can be performed with high accuracy while suppressing damage to the specimen A.

In this embodiment, the generation of the reference spot image is calculated for all the combinations. However, since the calculation amount is large when calculating for all the combinations, the calculation may be performed as follows instead. Good.
That is, first, a reference spot image in the case of an aberration vector ΔW = (0, 0, 0, 0, 0.1, 0,..., 0) including an aberration of 0.1λ in Z ₅ is generated.
Then, the cross-correlation between the photographed fluorescent spot image and the generated reference spot image is taken, and the value is set to M1.

Next, a reference spot image in the case of an aberration vector ΔW = (0, 0, 0, 0, 0.2, 0,..., 0) including an aberration of 0.2λ in the Zernike coefficient Z ₅ is generated, and the fluorescent spot The cross-correlation with the image is taken and the value is M2. This process is repeated over a predetermined aberration range at a predetermined aberration interval, and the aberration vector ΔW = (0, 0, 0, 0, X ₁ , 0,..., 0) having the largest cross-correlation is expressed as a Zernike coefficient. It is determined as the value of Z _5. This process is repeated with Z ₆ , Z ₇ ,... To sequentially determine the value of the Zernike coefficient.

According to this method, the number of repetitions is 100 × 5, and the amount of calculation can be greatly reduced.
Alternatively, the calculation may be terminated when the cross-correlation value exceeds a predetermined value. This also can greatly reduce the amount of calculation.

  In the present embodiment, since the photomultiplier tube 20 is used for the photographing optical system 4 for detecting fluorescence, the second scanner 16 is operated to acquire a fluorescent spot image. Instead, as shown in FIG. 5, an image sensor such as a CCD or a CMOS image sensor may be employed, and the second scanner 16 may be omitted.

  In the present embodiment, one fluorescence spot image in the specimen A is acquired and the similarity to the reference spot image is determined. Instead, a plurality of fluorescence spot images are acquired. The similarity with the reference spot image may be calculated, and the similarity may be determined based on the average of these. In this way, the aberration vector ΔW can be determined with high accuracy.

  In this case, the positions of the plurality of spots formed on the specimen A may be sequentially changed by the operation of the first scanner 10, or the plurality of spots may be simultaneously sampled by the wavefront modulation element 13. A phase pattern as generated in A may be given to the excitation light. When a plurality of spots are simultaneously generated on the specimen A by applying a phase pattern to the excitation light, an imaging element such as a CCD or a CMOS image sensor is arranged in the photographing optical system 4 so that fluorescent spot images at a plurality of points can be simultaneously obtained. Can be acquired.

Further, in the present embodiment, the reflection type wavefront modulation element is illustrated, but instead of this, a transmission type wavefront modulation element may be adopted.
In the present embodiment, the microscope using the confocal pinhole 19 is illustrated in FIG. 1, but instead of this, fluorescence observation by multiphoton excitation is performed using an ultrashort pulse laser as the light source 2. The confocal pinhole may be omitted.

DESCRIPTION OF SYMBOLS 1 Microscope 2 Light source 5 Control part 10 1st scanner (scanner)
13 Wavefront modulator (spatial light modulator)
20 Photomultiplier tube (imaging part)

Claims (11)

A spatial light modulator that modulates the wavefront of the excitation light from the light source;
A scanner for two-dimensionally scanning excitation light on the specimen;
An imaging unit that captures fluorescence generated from a scanning position on the sample of excitation light by the scanner and acquires a fluorescence image;
Based on the acquired fluorescent spot image by irradiating the spot-shaped excitation light to at least one position on the specimen by the scanner, causing the imaging unit to photograph fluorescence generated in the specimen by the irradiation of excitation light And a control unit that determines the aberration at the position of the sample by software and controls the spatial light modulator based on the determined aberration.   The control unit compares a plurality of reference spot images generated by software with a spot image including various aberrations at the sample position and a fluorescent spot image acquired in the imaging unit, and has a high similarity reference. The microscope according to claim 1, wherein an aberration included in the spot image is determined as an aberration at the position of the specimen.   The microscope according to claim 2, wherein the reference spot image is generated by software using a Zernike coefficient.   The microscope according to claim 2 or 3, wherein the control unit determines the similarity between the reference spot image and the fluorescent spot image by cross-correlation.   5. The image capturing unit according to claim 1, wherein the imaging unit captures fluorescence generated in the specimen by irradiation of spot-like excitation light at two or more positions on the specimen to acquire a plurality of fluorescent spot images. Microscope.   The microscope according to claim 5, wherein an irradiation pattern of spot-like excitation light with respect to two or more positions on the specimen is generated by the spatial light modulation element. A microscope aberration correction method for irradiating a sample with excitation light from a light source and photographing fluorescence generated in the sample to obtain a fluorescence image,
Based on a fluorescent spot image obtained by irradiating the specimen with spot-like excitation light, determining the aberration at the specimen position by software;
A method for correcting aberrations of the microscope, comprising: modulating a wavefront of the excitation light so as to reduce the determined aberration.   The step of determining an aberration compares the generated reference spot image and the fluorescent spot image with a step of generating a plurality of reference spot images composed of spot images including various aberrations at the sample position. The method according to claim 7, further comprising: determining an aberration included in a reference spot image having high similarity as an aberration at the position of the sample.   The microscope aberration correction method according to claim 8, wherein the reference spot image is generated by software using a Zernike coefficient.   The method for correcting aberrations of a microscope according to claim 8 or 9, wherein the step of determining an aberration determines the similarity between the reference spot image and the fluorescent spot image by cross-correlation.   The step of determining the aberration is determined based on a plurality of fluorescent spot images acquired by photographing fluorescence generated in the sample by irradiation of spot-like excitation light at two or more positions on the sample. The microscope aberration correction method according to claim 10.

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