WO2017175374A1

WO2017175374A1 - Image acquisition device

Info

Publication number: WO2017175374A1
Application number: PCT/JP2016/061531
Authority: WO
Inventors: 兼太郎井元; 厚志土井
Original assignee: オリンパス株式会社
Priority date: 2016-04-08
Filing date: 2016-04-08
Publication date: 2017-10-12

Abstract

With the objective of accurately acquiring a signal light image regardless of environmental variation without an operator adjusting the intensity ratio of illumination light at every observation, an image acquisition device (1) according to the present invention is provided with: a modulation unit (3) for modulating light from a light source (2), thereby generating an illumination light which switches between two differing states over a time cycle; an illumination light detection unit (6) for detecting the generated illumination light (L1, L2); an illumination optical system (4) for irradiating a sample (X) with the generated illumination light (L1, L2); a signal light detection unit (5) for focusing and detecting the signal light using the illumination light (L1, L2) generated at the illumination position of the sample (X); a demodulation unit (7) for demodulating the modulation of the detected intensity signal of the signal light and the detected intensity signal of the illumination light; and an image generation unit (8) for generating a signal light image on the basis of the demodulated intensity signal of the signal light, the modulation unit (3) correcting the modulation intensity of the illumination light on the basis of the intensity signal of the illumination light demodulated by the demodulation unit (8).

Description

Image acquisition device

The present invention relates to an image acquisition device.

Corresponding to excitation light in each state by irradiating the sample with excitation light in two states generated by modulation and demodulating after detecting fluorescence generated in the sample in an image acquisition device such as a laser scanning microscope A technique for generating an image by acquiring fluorescence is known (for example, see Patent Document 1).

WO2015 / 163261 pamphlet

In the microscope of Patent Document 1, it is necessary that the intensity of the excitation light in the two states is ideally the same. However, since the characteristics of the optical components change due to environmental changes such as temperature, the intensity ratio is kept constant. It is difficult to hold. For this reason, it is necessary for the operator to adjust the intensity ratio of the excitation light to be constant before observation, which is troublesome and troublesome.

The present invention has been made in view of the above-described circumstances, and an operator can accurately acquire a signal light image regardless of environmental changes without adjusting the intensity ratio of illumination light for each observation. An object of the present invention is to provide an image acquisition device capable of performing the above.

According to one embodiment of the present invention, a modulation unit that generates illumination light in which two different states are periodically switched by modulating light from a light source, and illumination that detects the illumination light generated by the modulation unit A light detection unit; an illumination optical system that irradiates the sample with the illumination light generated by the modulation unit; and the signal light generated at the illumination light irradiation position on the sample by the illumination optical system is collected and detected. A signal light detector; a demodulator that demodulates the modulation of the intensity signal of the signal light detected by the signal light detector and the intensity signal of the illumination light detected by the illumination light detector; and An image generation unit that generates a signal light image based on the intensity signal of the signal light demodulated by the demodulation unit, and the modulation unit is based on the intensity signal of the illumination light demodulated by the demodulation unit Serial is an image acquisition device that corrects the modulated intensity of the illumination light.

According to this aspect, when the light emitted from the light source is incident on the modulation unit, illumination light in which two different states are switched periodically is generated, and the generated illumination light is irradiated onto the sample by the illumination optical system. Is done. The signal light generated at the irradiation position of the illumination light on the sample is detected by the signal light detector. The intensity signal of the detected signal light is demodulated by the demodulator, and a signal light image is generated by the image generator based on the demodulated signal light intensity signal.

On the other hand, the illumination light generated by the modulation unit is detected by the illumination light detection unit, and the intensity signal of the detected illumination light is demodulated by the demodulation unit. The intensity signal in each state of illumination light having two states can be compared by the modulation unit, and the intensity signal of the illumination light in two states can be obtained even when the modulation ratio by the modulation unit varies due to environmental changes. Correction can be performed in the modulation section so that the ratio is constant. Thus, the signal light image can be accurately acquired regardless of the environmental change without the operator adjusting the intensity ratio of the illumination light for each observation.

In the above aspect, the two states are states in which time integrated intensity is equal, the demodulator outputs a difference in time integrated intensity of the illumination light demodulated for each state, and the modulator is The modulation intensity may be corrected so that the difference becomes zero.
In this way, the modulation intensity is corrected by the modulation unit until the difference in time integration intensity of the illumination light output from the demodulation unit becomes zero, thereby making the time integration intensity of the illumination light in the two states equal. Can be maintained. The state where the time integrated intensities of the two states are equal includes not only the case where the intensity of the illumination light is equal but also the case where the intensities are different.

Moreover, in the said aspect, the state which irradiates the said illumination light to the different position of a sample may be sufficient as said two states.
By doing so, for example, it is applied to a scanning confocal microscope, and the illumination light in one state is condensed at a position optically conjugate with the pinhole in front of the detector, and the illumination light in the other state is condensed. The light is condensed at a position optically unconjugated with the pinhole.

In one state, the focus signal light from the condensing position and the out-of-focus signal light from other than the condensing position pass through the pinhole, and in the other state, only the out-of-focus signal light passes through the pinhole. By acquiring, out-of-focus signal light can be removed.
In this case, the out-of-focus fluorescence can be accurately removed by correcting the intensity of the illumination light applied to the sample in the two states in the modulation unit so as to match with high accuracy.

Further, in the above aspect, the two states may be a state where the signal light generated in the sample is saturated and a state where the signal light is not saturated.
In this way, for example, when applied to a SAX (Saturated Excitation) microscope, the time integrated intensity of the illumination light is the same in the two states, but the signal light generated in the sample is saturated with the illumination light with the higher intensity. When the signal light is not saturated with the illumination light having the lower intensity, high-precision observation can be performed by accurately matching the time integrated intensities of the illumination light in the two states.

Further, in the above aspect, the optical system includes a branching unit that branches the light from the light source into two optical paths, and the modulation unit modulates the light of each optical path branched by the branching unit with a different frequency, respectively. The modulation intensity of the illumination light may be corrected so that the intensity ratio of the two illumination lights demodulated by the demodulator has a preset value.
By doing in this way, the light from the light source branched into the two optical paths by the branching unit is modulated at different frequencies and irradiated to two locations of the sample, and the intensity signal of the detected signal light is demodulated by the demodulation unit Thus, two points of the sample can be observed simultaneously.

When applied to a nonlinear microscope typified by a two-photon excitation microscope, the obtained signal light intensity is proportional to the square of the instantaneous intensity of the illumination light. Even if the intensities are matched, there is a difference in the intensity of the detected signal light. According to this aspect, it is possible to acquire a signal light image without color unevenness by correcting the modulation intensity of the illumination light so as to obtain a preset intensity ratio in consideration of the irradiation pattern.

Moreover, in the said aspect, you may provide the signal correction | amendment part which correct | amends the intensity signal of the said illumination light detected by the said illumination light detection part with the preset correction coefficient.
In this way, when the illumination light detection unit has different characteristics depending on the polarization state and frequency of the illumination light, it is possible to eliminate a detection error due to the characteristic difference by the correction coefficient set in advance in the signal correction unit.

According to the present invention, there is an effect that a signal light image can be obtained with high accuracy regardless of environmental changes without an operator adjusting the intensity ratio of illumination light for each observation.

1 is an overall configuration diagram illustrating an image acquisition device according to a first embodiment of the present invention. It is a figure which shows an example of the illumination light modulated by the modulation part of the image acquisition apparatus of FIG. It is a figure which shows an example of the illumination light used for the modification of the image acquisition apparatus of FIG. It is a whole block diagram which shows the image acquisition apparatus which concerns on the 2nd Embodiment of this invention. It is a figure which shows an example of the illumination light modulated by the 1st modulator of the image acquisition apparatus of FIG. It is a figure which shows an example of the laser beam inject | emitted from the light source of the image acquisition apparatus of FIG. It is a figure which shows an example of the illumination light modulated by the 2nd modulator of the image acquisition apparatus of FIG.

Hereinafter, an image acquisition device 1 according to a first embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the image acquisition apparatus 1 according to the present embodiment includes a laser light source (light source) 2 that emits laser light having a constant intensity, and a modulation unit that modulates laser light from the laser light source 2. 3 and a microscope optical system (illumination optical system) 4 for condensing fluorescence (signal light) generated in the sample X while irradiating the illumination light modulated in the modulation unit 3 to the sample X (not shown), and the microscope A photodetector (signal light detection unit) 5 for detecting fluorescence that has passed through the pinhole 16 of the optical system 4; a detector (illumination light detection unit) 6 for detecting the illumination lights L1 and L2 modulated by the modulation unit 3; Based on the fluorescence intensity signal detected by the photodetector 5 and the intensity signal of the illumination lights L1 and L2 detected by the detector 6, the demodulator 7 and the fluorescence intensity signal demodulated by the demodulator 7 To generate fluorescent images And an image generation unit 8 that.

The modulation unit 3 is arranged on each optical path and a first beam splitter (branching unit) 9 that branches the laser light from the laser light source 2 into two optical paths, and turns on and off the laser light that passes through each optical path at alternate timings.

Modulators

10a and 10b such as an acousto-optic modulator (AOM) that modulates in this way, a modulation control unit 11 that controls these

modulators

10a and 10b, and illumination light L1 output from the

modulators

10a and 10b And a second beam splitter 12 for multiplexing L2. The second beam splitter 12 is arranged at an angle slightly different from 45 ° with respect to the optical axis of one optical path, and the two illumination lights L1 and L2 after being combined are emitted at different angles. It is like that.

The microscope optical system 4 is an optical system of a laser scanning fluorescence microscope, and includes a scanner 13 that scans two illumination lights L1 and L2 emitted from the modulation unit 3, and an illumination light L1 that is scanned by the scanner 13. , L2 on the sample X, a dichroic mirror 15 for branching the fluorescence condensed by the objective lens 14 from the optical path of the illumination light L1, L2, and a pinhole 16.
In the figure, reference numeral 17 denotes an optical path forming mirror.

The two illumination lights L1 and L2 having different emission angles by the second beam splitter 12 of the modulation unit 3 are incident on the pupil of the objective lens 14 from different angles, thereby being condensed at different positions in the sample X. The fluorescent material existing in the sample X is excited to generate fluorescence.
That is, the illumination light L1 that has passed through one optical path is condensed at a position optically conjugate with the pinhole 16 disposed in the front stage of the photodetector 5, while the illumination light L2 that has passed through the other optical path. Is condensed at a position optically unconjugated with the pinhole 16.

Since the illumination light L1 and L2 passing through the two optical paths are alternately emitted by the operation of the modulation control unit 11, as shown in FIG. 2, the illumination light L1 that is condensed at the conjugate position and the non-conjugated light Illumination light is generated in which the illumination light L1, L2 in two states of the illumination light L2 condensed at the position is switched periodically. The modulation control unit 11 outputs a modulation signal to the demodulation unit 7.

The photodetector 5 is, for example, a photomultiplier tube, and detects the intensity of the fluorescence condensed by the objective lens 14.
The demodulating unit 7 modulates the intensity signals of the fluorescence generated by the illumination lights L1 and L2 in the two states detected by the photodetector 5 by the

modulators

10a and 10b sent from the modulation control unit 11, respectively. The signal is demodulated in synchronization with the timing, and the difference between the fluorescence intensity signals is output.

The image generator 8 generates a fluorescent image by arranging the difference value of the fluorescence intensity signal output from the demodulator 7 in association with the information of the scanning position by the scanner 13. When the illumination light L1 is condensed at a position conjugate with the pinhole 16, the fluorescence detected by the photodetector 5 includes the focus fluorescence generated at the focus position and the path to the focus in the sample X. Part of the generated out-of-focus fluorescence is included.

On the other hand, the fluorescence detected by the photodetector 5 when the illumination light L2 is condensed at a position unconjugated with the pinhole 16 includes the focus fluorescence generated at the focal position because it cannot pass through the pinhole 16. Only the out-of-focus fluorescence is included. Therefore, the difference between these fluorescence intensity signals output from the demodulator 7 includes only the focus fluorescence from which the out-of-focus fluorescence is removed, and a clear fluorescence image with less noise is generated. Will be able to.

The detector 6 is disposed, for example, at the subsequent stage of the modulation unit 3, and includes a beam sampler 18 including a low-reflectance half mirror that separates part of the illumination lights L 1 and L 2 output from the modulation unit 3, and the beam sampler 18. And a single point detector 19 disposed at a position where the illumination lights L1 and L2 in the two states separated by (2) can be detected.

The single point detector 19 may be arranged at a position conjugate with the pupil plane of the objective lens 14 by a relay lens (not shown), may be arranged at the focal position of a condenser lens (not shown), or two illuminations You may employ | adopt the thing of the large diameter which can detect light L1, L2 in a wide range.
In the present embodiment, the output of the single point detector 19 is input to the demodulator 7, and the output from the demodulator 7 is input to the modulation controller 11.

The modulation control unit 11 corrects the modulation intensity of the illumination lights L1 and L2 modulated by one of the

modulators

10a and 10b in accordance with the difference value input from the demodulation unit 7. That is, based on the intensity of the illumination lights L1 and L2 in two states separated by the beam sampler 18, the modulation control unit 11 sets the intensity of any illumination light so that the output from the demodulation unit 7 becomes zero. Therefore, the intensities of the illumination lights L1 and L2 in the two states are adjusted with high accuracy.

According to the image acquisition apparatus 1 according to the present embodiment configured as described above, the laser light emitted from the laser light source 2 is branched into two optical paths by the first beam splitter 9 in the modulation unit 3 and passes through the optical paths. During this time, the light is modulated by the

modulators

10a and 10b arranged in the respective optical paths, combined by the second beam splitter 12, and emitted at different angles.

Illumination lights L1 and L2 output from the modulation unit 3 and that change in two states in terms of time are made incident on the microscope optical system 4, and a part of the illumination lights L1 and L2 are separated by the beam sampler 18 and single point detector 19 is used. Is detected. The illumination lights L1 and L2 in two states scanned by the scanner 13 in the microscope optical system 4 and condensed by the objective lens 14 are condensed at two different positions in the sample X, and fluorescent at each condensing position. Is generated.

Fluorescence generated in the sample X by irradiation with the illumination lights L1 and L2 in two states is detected by the photodetector 5 and input to the demodulator 7. In the demodulator 7, by receiving demodulation synchronized with the modulation in the modulator 3, the difference between the fluorescence intensity signals generated by the illumination lights L 1 and L 2 in each state is output from the demodulator 7.
As described above, the difference in the intensity signal of the fluorescence output from the demodulator 7 is an intensity signal of only the fluorescence from the focal position of the illumination light from which the out-of-focus fluorescence is removed. By arranging the scanning positions by the scanner 13 in association with each other in the image generation unit 8, a clear fluorescent image without noise can be acquired.

In this case, in order to accurately remove the out-of-focus fluorescence in the demodulator 7, the intensities of the illumination lights L1 and L2 in the two states are required to be the same. Prior to observation, the modulation control unit 11 is set so that the intensity of the illumination lights L1 and L2 in the two states output from the modulation unit 3 are the same, but the use environment such as temperature fluctuates, There may be a difference in the intensity of the illumination lights L1 and L2 output from the

modulators

10a and 10b.

According to the image acquisition device 1 according to the present embodiment, part of the illumination lights L1 and L2 output from the modulation unit 3 are separated by the beam sampler 18, detected by the single point detector 19, and detected illumination light L1. , L2 intensity signals are demodulated by the demodulator 7 so that the difference between the signal intensities of the two illumination lights L1 and L2 is output from the demodulator 7. Then, the difference output from the demodulator 7 is input to the modulation control unit 11, so that the modulation intensity of the illumination lights L1 and L2 modulated by the

modulators

10a and 10b according to the difference is modulated by the modulation control unit 11. It is corrected.

That is, if the difference between the intensity signals of the illumination lights L1 and L2 in the two states is zero, the modulation intensity is not corrected, so that the intensities of the illumination lights L1 and L2 in the two states are matched with high accuracy. As a result, out-of-focus fluorescence can be accurately removed in the demodulator 7, and even if the usage environment fluctuates, the operator does not adjust the intensity ratio of the illumination lights L1 and L2 for each observation. There is an advantage that a clear fluorescent image can be obtained.

In particular, in the present embodiment, the demodulator 7 that outputs the difference between the fluorescence intensity signals is used to obtain the difference between the intensity signals of the illumination lights L1 and L2, so that the beam sampler 18 and the single point detector 19 There is an advantage that it is only necessary to newly install and can be realized at a low cost with a simple configuration.
A demodulator for illumination light may be prepared separately from the demodulator 7 for fluorescence.

Further, in the present embodiment, the image acquisition device 1 that uses modulation / demodulation to remove out-of-focus fluorescence has been described as an example, but instead, this is applied to a SAX microscope as shown in FIG. You may decide.
That is, as shown in FIG. 3, the modulation unit 3 generates illumination lights L1 and L2 having two different peak values and equal time integration intensities, unlike the above embodiment. Yes.
The

modulators

10a and 10b may employ electro-optic modulators instead of the acousto-optic modulators.

When the illumination light L1 having a higher peak intensity saturates the fluorescence generated in the sample X and the illumination light L2 having a lower peak intensity does not saturate the fluorescence, even if the usage environment changes, two In this state, the time integrated intensities of the illumination lights L1 and L2 can be maintained in a highly accurate state, and high-precision observation can be performed.

Next, an image acquisition apparatus 20 according to a second embodiment of the present invention will be described below with reference to the drawings.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as that of the image acquisition device 1 according to the first embodiment described above, and description thereof is omitted.

As shown in FIG. 4, the image acquisition device 20 according to the present embodiment is applied to a two-photon excitation microscope, and divides illumination light composed of ultrashort pulse laser light into two optical paths, The light is modulated at different frequencies by the arranged

modulators

10a and 10b, condensed at two different points of the sample X, and the fluorescence emitted from each focal point is detected and demodulated to detect the fluorescence from the two points. Are supposed to be acquired separately.

Specifically, as shown in FIG. 5A, the first modulator 10a in one optical path cuts every other pulse of the ultrashort pulse laser light from the laser light source 2 to become 50% duty. So that it can be modulated.
The other second modulator 10b reduces the peak intensity of all pulses of the ultrashort pulse laser beam from the laser light source 2 shown in FIG. 5B to 70% as shown in FIG. 5C. Yes.

For example, when the light emitted from the laser light source 2 is 80 MHz, the first modulator 10a modulates the ultrashort pulse laser light from the laser light source 2 to generate the 40 MHz illumination light L2. Yes. Thereby, the illumination light L2 has an alternating current pattern whose intensity changes at 40 MHz. The second modulator 10b modulates the peak intensity of the ultrashort pulse laser light from the laser light source 2 to 70% to generate 80 MHz illumination light L1.

Then, the fluorescence intensity signal from the sample X detected by the photodetector 5 is input to the two

demodulation units

21a and 21b. Since the input signal is the sum of the fluorescence signals coming from the two focal points, and the fluorescence signal is proportional to the square of the peak intensity of the illumination light L1, it can be expressed as a time function for each arrival pulse, F _all (t) = [10 ² +7 ² , 7 ² , 10 ² +7 ² , 7 ² ].

On the other hand, in the first demodulator 21a, as a demodulated signal synchronized with the modulated signal of the first modulator 10a, the fluorescence signal integrated by multiplying code1 (t) = (1, -1,1, -1) is integrated. Is output.
Specifically, F1 = F _all (t) · code1 (t) = 10 ² +7 ² −7 ² +10 ² +7 ² −7 ² = 200 corresponding to L1 is output.

The other second demodulator 21b multiplies and integrates code1 = (1,1,1,1) as a demodulated signal synchronized with the modulated signal of the second modulator 10b, and then fluorescence corresponding to L1. The signals are subtracted and a fluorescent signal corresponding to L2 is output. Specifically, since the fluorescence signal is proportional to the square of the peak intensity of the illumination light L1, F2 = F _all (t) · code 2 (t) −F1 = 10 ² +7 ² +7 ² +10 ² +7 ² +7 ² − 200≈200 is output.

As a result, the fluorescence intensity signals output from the two

demodulation units

21a and 21b are substantially equal, and the occurrence of uneven brightness in the fluorescence image generated using these fluorescence intensities can be prevented.
In this case, the intensity signals of the illumination lights L1 and L2 in the two states detected by the single point detector 19 and demodulated by the

demodulation units

21a and 21b are output as a difference, unlike the first embodiment. And output as separate intensity signals.

Therefore, when the intensity signals of the illumination lights L1 and L2 in the respective states output from the

demodulating units

21a and 21b are L1: L2 = 7: 10 in the above example, the modulation intensity in each of the

modulators

10a and 10b. The modulation control unit 11 controls so that correction is not performed. That is, the modulation control unit 11 multiplies the intensity signal of the illumination light L1 output from the first demodulation unit 21a by a preset ratio, 10/7 in the above example, and the second value. The two

modulators

10a and 10b are controlled so that the difference from the intensity signal of the illumination light L2 output from the demodulator 21b becomes zero.

As described above, according to the image acquisition device 20 according to the present embodiment, even if the usage environment changes, two-point simultaneous detection is performed without the operator adjusting the intensity ratio of the illumination lights L1 and L2 for each observation. There is an advantage that a fluorescent image with little luminance unevenness can be acquired while improving the frame rate by the above.

In the present embodiment, the case where the illumination lights L1 and L2 are condensed on the sample X at two points at the same time to detect fluorescence is described, but three or more points may be condensed at the same time. Further, although the point scan method using the scanner 13 has been exemplified, the present invention is applied to a light sheet microscope for photographing fluorescence generated by sheet-like illumination light incident on different positions along the optical axis of the objective lens 14 with a camera. May be.

Further, as a result of being separately modulated by the two

modulators

10a and 10b, when the polarization states of the two illumination lights L1 and L2 are different, the sampling ratio by the beam sampler 18 may be different for each of the illumination lights L1 and L2. Can do. In such a case, even if the two illumination lights L1 and L2 have the same intensity, they are detected as having different intensities in the single point detector 19, so that the single point detector is detected by the correction coefficient obtained in advance. A signal correction unit (not shown) that corrects the intensity signals of the illumination lights L1 and L2 detected by the projector 19 may be provided.
Further, although the modulation control unit 11 corrects the modulation intensity by the two

modulators

10a and 10b, the modulation intensity may be corrected by only one modulator 10a.

In each of the above embodiments, the timing of correcting the modulation intensity of the illumination lights L1 and L2 is not particularly limited. For example, it is automatically performed before the fluorescence observation of the sample X is performed. do it. The intensity correction of the illumination lights L1 and L2 can be performed without using the sample X, and can be performed without damaging the sample X.

1,20 Image acquisition device 2 Laser light source (light source)
3 Modulator 4 Microscope optical system (illumination optical system)
5 Light detector (Signal light detector)
6 Detector (illumination light detector)
7, 21a, 21b Demodulator 8 Image generator 9 First beam splitter (branch unit)
L1, L2 Illumination light X Sample

Claims

A modulator that generates illumination light in which two different states are periodically switched by modulating light from the light source;
An illumination light detector that detects the illumination light generated by the modulator;
An illumination optical system for irradiating a sample with the illumination light generated by the modulation unit;
A signal light detection unit that collects and detects signal light generated at an irradiation position of the illumination light in the sample by the illumination optical system;
A demodulator that demodulates the modulation of the intensity signal of the signal light detected by the signal light detector and the intensity signal of the illumination light detected by the illumination light detector;
An image generation unit that generates a signal light image based on an intensity signal of the signal light demodulated by the demodulation unit;
The image acquisition device in which the modulation unit corrects the modulation intensity of the illumination light based on the intensity signal of the illumination light demodulated by the demodulation unit.
The two states are states in which time integrated intensity is equal,
The demodulator outputs a difference in time integrated intensity of the illumination light demodulated for each state,
The image acquisition apparatus according to claim 1, wherein the modulation unit corrects the modulation intensity so that the difference becomes zero.
The image acquisition device according to claim 1 or 2, wherein the two states are states in which the illumination light is irradiated to different positions of the sample.
3. The image acquisition apparatus according to claim 1, wherein the two states are a state where the signal light generated in the sample is saturated and a state where the signal light is not saturated.
A branching portion for branching light from the light source into two optical paths;
The modulation unit modulates the light of each of the optical paths branched by the branching unit with different frequencies, and the intensity ratio of the two illumination lights demodulated by the demodulation unit becomes a preset value. The image acquisition apparatus according to claim 1, wherein the modulation intensity of the illumination light is corrected.
6. The image acquisition apparatus according to claim 1, further comprising a signal correction unit that corrects an intensity signal of the illumination light detected by the illumination light detection unit using a preset correction coefficient.