WO2017221356A1

WO2017221356A1 - Microscope

Info

Publication number: WO2017221356A1
Application number: PCT/JP2016/068560
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: 2016-06-22
Filing date: 2016-06-22
Publication date: 2017-12-28
Also published as: JPWO2017221356A1

Abstract

For the purpose of automatically setting an optimum point that allows a confocal pinhole and a light collection point of excitation light to be in an optically non-conjugate positional relationship, and alleviating a burden on an operator, a microscope (1) according to the present invention includes: a scanner (7) that irradiates excitation light for scanning from a light source (2); an objective optical system (8) that collects, at a sample (A), excitation light for scanning and collects, in each scanning position, fluorescent light generated at the sample; a detector (12) that detects the collected fluorescent light; a light-shielding member (11) that is provided between the detector and the objective optical system and partly shields the fluorescent light collected by the objective optical system; a switching unit (3) that temporally switches, between an optically conjugate positional relationship and an optically non-conjugate positional relationship, a positional relationship between the light-shielding member and the light collection point of excitation light at the sample; a fluorescent light distribution acquiring unit (18) that acquires a fluorescent light distribution in an optically conjugate position with respect to the light collection point; and a setting unit (16) that sets a non-conjugate positional relationship in the switching unit on the basis of the acquired fluorescent light distribution.

Description

microscope

The present invention relates to a microscope.

In order to prevent the observation of the deep part of the sample due to leakage of out-of-focus fluorescence into the confocal pinhole, the positional relationship between the confocal pinhole and the focal point of the excitation light is optically determined. A microscope is known that detects fluorescence by switching between a conjugate position and a non-conjugated position, and calculates the difference between the obtained fluorescence signals (see, for example, Patent Document 1).

WO2015 / 163261

However, in the microscope of Patent Document 1, regarding the setting of the optimum point where the confocal pinhole and the condensing point of the excitation light are in an optically non-conjugative positional relationship, the operator manually captures the sample while photographing it. It was complicated because it was necessary to adjust the position of the focal pinhole.
The present invention has been made in view of the above-described circumstances, and automatically sets an optimal point where the confocal pinhole and the condensing point of the excitation light are in an optically non-conjugate positional relationship. It aims at providing the microscope which can reduce this burden.

In one embodiment of the present invention, a scanner that scans excitation light from a light source, and the excitation light that is scanned by the scanner is collected on a sample, while signal light generated in the sample is collected at each scanning position. An objective optical system, a detector for detecting the signal light collected by the objective optical system, and the signal light arranged between the detector and the objective optical system and condensed by the objective optical system And a positional relationship between the light shielding member and the condensing point of the excitation light in the sample in terms of an optically conjugate positional relationship and an optically non-conjugated positional relationship. A switching unit that automatically switches, a fluorescence distribution acquisition unit that acquires a fluorescence distribution at a position optically conjugate with the condensing point, and the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquisition unit. Set the non-conjugated positional relationship A microscope and a that setting section.

According to this aspect, the excitation light from the light source is scanned by the scanner and condensed on the sample by the objective optical system, whereby the fluorescent material is excited at the collection point of the excitation light in the sample to generate fluorescence. The generated fluorescence and the reflected light of the excitation light from the sample are collected by the objective optical system and then detected by the detector, and a fluorescence image is generated by associating the detected fluorescence intensity with the scanning position.

In this case, the fluorescence distribution acquisition unit acquires a fluorescence distribution at a position optically conjugate with the condensing point, and the setting unit sets a non-conjugated positional relationship in the switching unit based on the acquired fluorescence distribution. The When the positional relationship between the light blocking member provided in the previous stage of the detector and the condensing point of the excitation light in the sample is switched to a conjugate positional relationship by the operation of the switching unit, the focus fluorescence and the out-of-focus fluorescence are detected. Detected by the instrument.

When the non-conjugated positional relationship set by the setting unit is switched by the operation of the switching unit, the focused fluorescence cannot pass through the light shielding member, and only the out-of-focus fluorescence is detected by the detector. Then, by calculating the difference between these detected fluorescence signals, it is possible to obtain the focused fluorescence from which the out-of-focus fluorescence has been removed, and to obtain a clear fluorescence image.
In other words, according to this aspect, since an optimal non-conjugated positional relationship can be obtained based on the fluorescence distribution at a position optically conjugate with the condensing point, the operator can perform focusing without performing complicated setting work. It is possible to obtain focused fluorescence from which external fluorescence has been accurately removed. Therefore, the burden on the operator can be reduced.

In the above aspect, the setting unit separates the fluorescence distribution acquired by the fluorescence distribution acquisition unit into a focus fluorescence and an out-of-focus fluorescence, and the focus fluorescence falls below a predetermined threshold and The positional relationship closest to the optically conjugate position may be set as the non-conjugated positional relationship in the switching unit.

The greater the deviation from the conjugate positional relationship, the stronger the degree of non-conjugation, and the acquired focused fluorescence approaches zero, but the acquired out-of-focus fluorescence is such that the light-shielding member and the focal point of the sample are optical. This is different from the out-of-focus fluorescence detected by the detector when the positional relationship is conjugate. In this way, the fluorescence that is equal to the out-of-focus fluorescence detected by the detector and does not include the focused fluorescence when the light shielding member and the focal point of the sample are in an optically conjugate positional relationship is detected. Therefore, it is possible to obtain the focused fluorescence from which the out-of-focus fluorescence is accurately removed by the difference.

Moreover, in the said aspect, the said setting part may apply the said fluorescence distribution acquired by the said fluorescence distribution acquisition part to a predetermined distribution model, and may isolate | separate a focus fluorescence and an out-of-focus fluorescence.
In this way, it is possible to easily separate the focused fluorescence and the out-of-focus fluorescence simply by applying the fluorescence distribution to a predetermined distribution model, and an appropriate non-conjugated positional relationship can be set.

Moreover, in the said aspect, when the said fluorescence distribution acquisition part changes the positional relationship of the said condensing point of the said excitation light in the said sample, and the said light-shielding member, the said signal light detected by the said detector The fluorescence distribution may be acquired based on the fluorescence intensity.
By doing in this way, fluorescence distribution is acquirable with the basic composition of a microscope.

Further, in the above aspect, the fluorescence distribution acquisition unit is configured such that the positional relationship between the light shielding member and the condensing point of the excitation light in the sample is a conjugate positional relationship, and two different non-conjugated conjugates. You may acquire the said fluorescence distribution based on the intensity | strength of the fluorescence acquired when it exists in positional relationship.
In this way, fluorescence intensity is acquired at least at three points: a conjugate positional relationship, a positional relationship with a high degree of non-conjugation, and a positional relationship with a low degree of non-conjugation that is close to the conjugate positional relationship. Thus, it is possible to easily set a non-conjugated positional relationship in which the focused fluorescence and the out-of-focus fluorescence can be accurately separated by the setting unit.

Further, in the above aspect, the fluorescence distribution acquisition unit is based on a theoretical size of the spot diameter of the excitation light at the condensing point determined by the objective optical system and the wavelength of the excitation light. You may set the positional relationship of the said light shielding member which acquires the intensity | strength of the said fluorescence, and the said condensing point of the said excitation light in the said sample.
In this way, the degree of non-conjugation can be determined by the theoretical size of the spot diameter, and thereby, the non-conjugated can be accurately separated from the focused fluorescence and the off-focus fluorescence by the setting unit. The positional relationship can be set easily.

Moreover, in the said aspect, the two-dimensional sensor arrange | positioned in the position optically conjugate with the said condensing point of the said excitation light in the said sample may be sufficient as the said fluorescence distribution acquisition part.
By doing in this way, fluorescence distribution can be acquired at once, and the non-conjugated positional relationship which can isolate | separate focus fluorescence and out-of-focus fluorescence with a setting part accurately can be set at high speed.

In the above aspect, the fluorescence distribution acquisition unit acquires the fluorescence distribution at a plurality of points, and the setting unit determines the non-conjugated positional relationship in the switching unit based on an average of the plurality of fluorescence distributions. It may be set.
In this way, it is possible to easily set a non-conjugated positional relationship in which the focused fluorescence and the out-of-focus fluorescence can be accurately separated by the setting unit without acquiring a fluorescence image, and excitation to the sample. It is possible to reduce the damage of the sample by reducing the light irradiation time.

According to the present invention, it is possible to automatically set the optimum point where the confocal pinhole and the condensing point of the excitation light are in an optically non-conjugated positional relationship, thereby reducing the burden on the operator. Play.

It is a block diagram which shows the microscope which concerns on one Embodiment of this invention. It is a partial longitudinal cross-sectional view explaining the shift amount of the fluorescent light beam and the pinhole in the pinhole of the microscope of FIG. It is a graph which shows the relationship between the shift amount of FIG. 2, focus fluorescence, and out-of-focus fluorescence. It is a graph which shows an example of the relationship between the fluorescence intensity acquired by the microscope of FIG. 1, and a shift amount, and the shift amount detected in setting mode. FIG. 2 is a diagram illustrating an example of an irradiation pattern of one of the two types of excitation light set by the setting unit in the imaging mode of the microscope of FIG. 1. FIG. 3 is a diagram illustrating an example of an irradiation pattern of the other of the two types of excitation light set by the setting unit in the imaging mode of the microscope of FIG. It is a block diagram which shows the 1st modification of the microscope of FIG. It is a block diagram which shows the 2nd modification of the microscope of FIG. It is a top view which shows the disk which has the pinhole of the microscope of FIG. 6A. It is a block diagram which shows the 3rd modification of the microscope of FIG. It is a block diagram which shows the 4th modification of the microscope of FIG. It is a block diagram which shows the 5th 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 is switched by the switching unit 3 that switches the excitation light from the laser light source 2 to two types of excitation light that are alternately emitted, and the switching unit 3. A microscope body 4 that irradiates the sample A with two types of excitation light and detects the fluorescence generated in the sample A, and a calculation unit 5 that generates an image by calculation using the fluorescence detected in the microscope body 4; And a monitor 6 for displaying an image generated by the calculation unit 5.

The microscope body 4 condenses the excitation light scanned by the scanner 7 in a two-dimensional manner and the excitation light scanned by the scanner 7 on the sample A, and the fluorescence (signal light) from the sample A. A converging objective lens (objective optical system) 8, a dichroic mirror 9 for diverging fluorescence collected by the objective lens 8 and returning via the scanner 7 from the optical path of the excitation light, and branched by the dichroic mirror 9 An imaging lens 10 that condenses the fluorescent light, a pinhole (light-shielding member) 11 that is optically conjugate with the focal position of the objective lens 8, and light that detects the fluorescent light that has passed through the pinhole 11 And a detector (detector) 12.

In addition, although pinhole 11 was illustrated as a light shielding member, it replaces with this, and when it arrange | positions in the position optically conjugate with the focus position of the objective lens 8, it passes a focus fluorescence and arrange | positions in a non-conjugated position. Any light shielding member that blocks the focal fluorescence when the light is emitted may be used. In addition, examples of the light shielding member include a micromirror device and a spatial light modulator.

The scanner 7 is, for example, a proximity galvanometer mirror in which two galvanometer mirrors that can be swung around a non-parallel axis line are arranged close to each other.
The photodetector 12 is, for example, a photomultiplier tube (PMT).

The laser light source 2 is a light source that continuously emits excitation light.
As shown in FIG. 1, the switching unit 3 includes a movable mirror (deflection element) 13 that changes the swing angle, and a drive control unit 14 that drives the movable mirror 13.

A setting unit 16 is connected to the drive control unit 14, and an input unit 17 and a distribution acquisition unit (fluorescence distribution acquisition unit) 18 are connected to the setting unit 16. The input unit 17 is configured by a keyboard, a mouse, or a GUI that performs input for selecting two operation modes of a setting mode and a shooting mode. Based on the input from the input unit 17, the setting unit 16 sets the drive control unit 14 to operate in one of two operation modes.

When the setting mode is selected from the input unit 17, the setting unit 16 operates the drive control unit 14 so as to drive the movable mirror 13 and shift the laser beam by a preset shift amount. When the photographing mode is selected, the drive control unit 14 is caused to function as a frequency oscillator that oscillates a predetermined frequency.
Here, as shown in FIGS. 2A and 3, the preset shift amount x is the first shift amount with zero shift amount in which the condensing point and the pinhole 11 are in an optically conjugate positional relationship. x1, the second shift amount x2 where the condensing point and the pinhole 11 are partially optically non-conjugated, and the positional relationship where the condensing point and the pinhole 11 are sufficiently optically non-conjugated. The third shift amount x3.

More specifically, in the setting mode, the setting unit 16 instructs the drive control unit 14 to set the movable mirror 13 at an angle at which three preset shift amounts can be achieved, and the movable mirror 13. The intensity of the fluorescence detected by the photodetector 12 at each angle is stored in the distribution acquisition unit 18.
The distribution acquisition unit 18 generates a fluorescence distribution in which the shift amount x that can achieve each of the three angles of the movable mirror 13 is associated with the fluorescence intensity acquired at each angle.

In other words, when the positional relationship between the focal point of the excitation light by the objective lens 8 and the pinhole 11 is arranged at an optically conjugate position, the fluorescence detected by the photodetector 12 is shown in FIG. As shown, the out-of-focus fluorescence generated at a position other than the focus is also included, while the focused fluorescence generated from the focus is largely dominant. When the positional relationship between the focal point and the pinhole 11 deviates from the optically conjugate position, the focal fluorescence decreases, but the out-of-focus fluorescence does not change much.

The setting unit 16 can achieve the positional relationship between the condensing point and the pinhole 11 that can detect the fluorescence in which the out-of-focus fluorescence is dominant and hardly includes the focused fluorescence from the fluorescence distribution acquired by the distribution acquisition unit 18. angle of a movable mirror 13, that is, in order to calculate the achievable angle of the shift amount of the code x _P in Figure 2B.
The method of calculating the shift amount of achievable angle of the code x _P is the fluorescence distribution of three points generated by the distribution obtaining unit 18, for example, how to find an optimum position by fitting the distribution model such as a Gaussian function Can be mentioned.

That is, when fitting to a Gaussian function, it can be expressed by the following equation.
L = Pexp (−x ² / 2w ² ) + Q
Here, P is the in-focus fluorescence intensity, and Q is the out-of-focus fluorescence intensity. x is the shift amount of the excitation light beam, w ² is the variance.

As a result, the focal fluorescence of the first term and the out-of-focus fluorescence of the second term can be separated. _P can be set as an optimal non-conjugated positional relationship by the switching unit 3. Although 0.01 means that the focal fluorescence has decreased to 1%, this value can be set arbitrarily.

The setting unit 16 corresponds to the shift amount x = 0 achievable angle and conjugate positional relationship of the movable mirror 13 corresponding to the calculated optimum nonconjugated positional relationship on the achievable shift amount x = x _P The angle of the movable mirror 13 to be set is set in the drive control unit 14 as the angle of the movable mirror 13 in the photographing mode.

In the photographing mode, the drive control unit 14 oscillates a predetermined frequency, and in synchronization with the oscillated frequency, the angle of the movable mirror 13 is alternately switched between the two angles set by the setting unit 16. It has become. Reference numeral 15 in the figure denotes a mirror. In addition, although the movable mirror 13 was illustrated as a deflection | deviation element, devices, such as an acousto-optic deflector and an electro-optic deflector, can also be used.

Thereby, in the imaging mode, two types of excitation light having different incident angles are incident on the microscope body 4 alternately. That is, the two types of excitation light incident on the microscope body 4 are formed in a rectangular wave shape having inverted timings as shown in FIGS. 4A and 4B. In addition, although the rectangular wave-like light which has the timing reversed as two types of excitation light was illustrated here, instead of this, it has arbitrary repetitive shapes, such as a sine wave shape, and employ | adopts the excitation light from which a phase differs Also good.

One excitation light shown in FIG. 4A is focused on a position optically conjugate with the pinhole 11 via the scanner 7 and the objective lens 8, and the other excitation light shown in FIG. The focal point is focused on a position optically unconjugated with the pinhole 11 via the lens 8. The incident angle switching frequency is set to a frequency at which two types of excitation light can be blinked at least once at each pixel position.

Since the two types of excitation light are alternately applied to the sample A, the generated fluorescence is also detected by the photodetector 12 at different times.
The calculation unit 5 calculates the difference in the intensity of the fluorescence generated by the two types of excitation light detected by the photodetector 12 at the same pixel position. The computing unit 5 includes, for example, a lock-in amplifier (not shown).

The lock-in amplifier calculates the difference between the two types of fluorescence signals output from the photodetector 12 in hardware in synchronization with the frequency generated by the drive control unit 14 for blinking the excitation light. ing.
Then, the calculation unit 5 generates an image by storing the difference calculated for each pixel and the scanning position by the scanner 7 in association with each other.

The operation of the microscope 1 according to this embodiment configured as described above will be described below.
In order to perform fluorescence observation of the sample A using the microscope 1 according to the present embodiment, the sample A is arranged on the stage (not shown) of the microscope body 4 so that the focal position of the objective lens 8 coincides with the sample A. Then, continuous excitation light is generated from the laser light source 2.

When the setting mode is selected in the input unit 17, the setting unit 16 is different from the drive control unit 14 in the shift amount x1 in which the condensing point and the pinhole 11 are conjugate and the degree of nonconjugation is different. The angle of the movable mirror 13 is set to three angles corresponding to the two shift amounts x2 and x3, and the fluorescence intensity detected by the photodetector 12 at each angle is associated with the shift amount x by the distribution acquisition unit 18. And a fluorescence distribution is generated.

The generated fluorescence distribution is sent to the setting unit 16. In setting unit 16, the fluorescence distribution sent is being fitted to a Gaussian distribution, corresponding to the shift amount x = x _P in which the shift amount x = 0 and optimal nonconjugated positional relation of conjugate positional relationship The angle of the movable mirror 13 is calculated.
The setting unit 16 sets the calculated angle of the movable mirror 13 as the angle of the movable mirror 13 that is switched by the drive control unit 14 when the photographing mode is selected by the input unit 17.

In the photographing mode, the drive control unit 14 alternately changes the angle of the movable mirror 13 to two angles set by the setting unit 16 according to a predetermined frequency oscillated by the drive control unit 14. Two types of excitation light having different incident angles are alternately incident on the main body 4.

As a result, the focal fluorescence contained in one excitation light is condensed at a position in the sample A conjugate with the pinhole 11, so that the fluorescence generated in the vicinity of the focal position is condensed by the objective lens 8, On the way back through the scanner 7, it is separated by the dichroic mirror 9, condensed by the imaging lens 10, passes through the pinhole 11, and is detected by the photodetector 12.

In this case, when the sample A is irradiated with the excitation light, the excitation light excites the fluorescent substance by passing through the sample A even in the middle of the path to the focal position of the objective lens 8. This occurs not only in the focal position of the lens but also in the middle of the path to the focal position. In particular, when the sample A is made of a scattering material, the fluorescence tends to be generated at a site other than the focal position due to scattering of the excitation light.

In particular, if the NA of the excitation light incident on the sample A is increased for high-definition observation, the area through which the excitation light passes until reaching the focal position increases. Fluorescence increases. Similarly, since the region through which excitation light passes increases during deep observation, the out-of-focus fluorescence increases. Further, in deep observation, it is necessary to increase the excitation light in order to compensate for the influence of scattering, and the influence of out-of-focus fluorescence is particularly remarkable.

Of the fluorescence generated in the sample A, the fluorescence generated from the focal position of the objective lens 8 easily passes through the pinhole 11 disposed at the optically conjugate position, and thus is detected as signal light by the photodetector 12. However, the fluorescence generated from the part other than the focal position is scattered by the sample, and a part thereof passes through the pinhole 11 and is detected as noise by the photodetector 12. Therefore, the fluorescence detected by the irradiation of one excitation light includes the focal fluorescence generated at the focal position of the objective lens 8 and to be acquired as a signal, and the out-of-focus that is generated at the other part and should not be acquired as a signal. Fluorescence and are included.

The other excitation light is condensed at a position unconjugated with the pinhole 11 in the sample A, and excites the fluorescent substance in the middle of the path to the focal position and the focal position of the objective lens 8. This generates fluorescence.
In this case, the fluorescence generated at the non-conjugated focal position cannot be passed through the pinhole 11 and is blocked, while a part of the fluorescence generated from the part other than the focal position is scattered by the sample. Similarly to the step, the light passes through the same pinhole 11 and is detected by the photodetector 12.

According to the microscope 1 according to the present embodiment, since the non-conjugated position is optimally set, the focal fluorescence in the fluorescence detected by the photodetector 12 is reduced by 99% and the focal point is reduced. Outer fluorescence is substantially equivalent to out-of-focus fluorescence detected by the photodetector 12 in a conjugate positional relationship.
Therefore, the calculation unit 5 calculates the difference between the fluorescence detected by the irradiation of these two types of excitation light, thereby generating a focal point that should not be acquired as a signal, generated in a part other than the focal position of the objective lens 8. The focal fluorescence from which the external fluorescence is removed can be obtained with high accuracy.

The range in which fluorescence is generated by irradiating two types of excitation light is not exactly the same, but it is the same in many parts, and detection is performed using the same pinhole 11 Thus, most of the out-of-focus fluorescence can be removed by subtracting as it is. In particular, when high-definition observation is performed by increasing the NA of excitation light, the overlap ratio of the fluorescence generation range increases, so that the out-of-focus fluorescence can be more effectively removed.

Thus, according to the microscope 1 according to the present embodiment, the fluorescence generated at the focal position of the objective lens 8 can be detected with a high S / N ratio, and a clear image with little noise can be acquired. There are advantages. In particular, the effect is high at the time of high-definition observation with a large NA of excitation light and when the sample A is a strong scattering material and easily generates out-of-focus fluorescence. Since the optimum non-conjugate position is automatically set, the operator does not need to perform the troublesome work of manually adjusting the positional relationship between the pinhole 11 and the fluorescent light beam while photographing the sample A. There is an advantage that the burden can be reduced.

In the present embodiment, since the difference between two types of fluorescence acquired with a very short time difference is calculated for each pixel, a fluorescent image with less blur can be acquired even with the sample A moving at high speed. There is an advantage that you can.

In the present embodiment, the pinhole 11 is exemplified as the light shielding member, but instead of this, when the focus hole is arranged at a position optically conjugate with the focus position of the objective lens 8, the focused fluorescence is passed, An arbitrary light shielding member that blocks the focal fluorescence when arranged at a conjugate position may be employed. Examples of the other light shielding member include a micromirror device and a spatial light modulator.

In the present embodiment, the signal light from the sample A detected by the photodetector 12 is exemplified using only fluorescence, but in addition to this, reflected light of excitation light from the sample A is used. May be. By imaging the warped light of the excitation light from the sample A, the refractive index distribution can be imaged.

Although the movable mirror 13 is exemplified as the deflecting element constituting the switching unit 3, as shown in FIG. 5, the acousto-optic deflector (acousto-optic element, beam moving unit) 19 or the electro-optic deflector (electro-optic element, beam). A device such as (moving unit) 20 can also be used. These

devices

19 and 20 also switch the voltage input according to a predetermined frequency oscillated by the drive control unit 14, so that the fluorescent light beam enters the imaging lens 10 in synchronization with the input voltage in the same manner as the movable mirror 13. The angle can be changed.

The setting unit 16 switches the voltage generated by the drive control unit 14 during the setting mode, causes the distribution acquisition unit 18 to acquire the fluorescence distribution, and inputs the fluorescence distribution to the

devices

19 and 20 during the imaging mode based on the acquired fluorescence distribution. Set the voltage. Since these

devices

19 and 20 do not include a movable part such as the movable mirror 13, the device can be configured to be compact and have a long life.

In the present embodiment, the incident position of the fluorescent light beam incident on the fixed light shielding member 11 is switched over time. Instead, the fluorescent light beam is fixed and the light shielding member 21 is fixed. May be moved in a direction crossing the optical axis S of the fluorescent light beam.
That is, as shown in FIG. 6B, a disk-shaped disk having a plurality of pinholes 22 arranged at intervals in the circumferential direction is adopted as the light shielding member 21, and a motor is used as shown in FIG. 6A. The (switching unit) 23 may rotate the disk 21 around the central axis.

By doing so, the fluorescence condensing position by the imaging lens 10 is made coincident with the disk 21, and the disk 21 is rotated by the motor 23 so that the pinhole 22 coincides with the optical axis S of the fluorescence. And a state that does not match can be alternately repeated. That is, in a state where any one of the pinholes 22 coincides with the fluorescence optical axis S, the focal point of the excitation light in the sample A and the pinhole 22 are in an optically conjugate positional relationship. On the other hand, in a state where none of the pinholes 22 is coincident, the condensing point of the excitation light in the sample A and the pinhole 22 are in an optically non-conjugated positional relationship.

In the present embodiment, the fluorescence distribution is calculated based on the fluorescence intensities acquired at the three positions of the position where the pinhole 22 is aligned with the optical axis S of the fluorescence, the position where the pinhole 22 is partially matched, and the position which is not completely matched. By setting the optimal position of the disk 21 that is generated and set as a non-conjugated position based on the generated fluorescence distribution, the out-of-focus fluorescence can be accurately removed.

Thereby, an optically conjugate positional relationship and an optically non-conjugated positional relationship are alternately formed in time, and two types of fluorescence for accurately detecting the focused fluorescence are detected by the same photodetector 12. Can be detected sequentially. Further, there is an advantage that the two positional relationships can be switched at a higher speed by rotating the disk 21 at a higher speed.

In the present embodiment, the case where the fluorescent light beam is moved in the direction intersecting the optical axis S with respect to the pinhole 22 is exemplified, but instead, the pinhole 22 is moved with respect to the fluorescent light beam. It may be.
Further, as shown in FIG. 7, the non-conjugated positional relationship may be achieved by making the incident angle of the excitation light incident on the sample A from the laser light source 2 different. In that case, when the incident angle of the excitation light is changed by the movable mirror 30 arranged between the laser light source 2 and the dichroic mirror 9, the scanner 7 is moved so that the position of the condensing point in the sample A does not shift. It is necessary to operate in conjunction. In this case, when the drive control unit 14 drives the movable mirror 30, the movable mirror 30 is swung to shift the laser beam. Reference numeral 31 denotes a mirror for forming an optical path of excitation light from the laser light source 2 to the movable mirror 30.

Also, the distribution model for fitting is not limited to a Gaussian function, and a Lorentz function or a Forked function may be used. In addition, the fluorescence intensity is detected at three points, ie, the shift amount x1 = 0 which is a conjugate positional relationship and the shift amounts x2 and x3 which are a non-conjugated positional relationship. A fluorescence distribution may be generated by detecting the fluorescence intensity in the quantity. Thereby, the precision of fitting to a distribution model can be improved.

Further, the fluorescence distribution may be acquired at one location in the sample A, or the fluorescence distribution may be generated by averaging the fluorescence intensities acquired at two or more locations.
In addition, x = x2 and x3 are set in advance, but as a guide, Airy (AIRY) which is a unit representing the theoretical size of the spot diameter of the excitation light may be used.

That is, when the focal spot size (spot diameter of excitation light) and the pinhole size determined by the NA of the objective lens 8 and the wavelength λ are 1 air, the shift amount x2 = 0.5 air, the shift amount x3 = 5 air You can set it to. In this way, it is possible to calculate the shift amount that optimizes the non-conjugated positional relationship with high accuracy by the three fluorescence distributions.

In the present embodiment, the fluorescence intensity detected using the photodetector 12 provided in the microscope body 4 is also used when setting the shift amount in the setting mode. Thereby, the fluorescence distribution can be generated without adding a new device to the basic configuration of the microscope body 4.

Instead, as shown in FIG. 8, a half mirror 24 that branches a part of the fluorescence from the sample A, and a camera that images the branched fluorescence at a position optically conjugate with the pinhole 11 ( A two-dimensional sensor such as a fluorescence distribution acquisition unit 25 may be provided. In the figure, reference numeral 26 denotes a condenser lens.
In this way, a two-dimensional fluorescence distribution can be acquired at once by the camera 25, and when the fluorescence distribution is acquired in the setting mode, it is not necessary to shift the fluorescent light flux or the pinhole 11, so that processing can be performed in a short time. There is an advantage that you can.

In the present embodiment, the case where the fluorescent light beam and the pinhole 11 are relatively moved in the direction intersecting the optical axis S of the fluorescent light beam has been described. Instead, as shown in FIG. The fluorescent light beam and the pinhole 11 may be relatively moved in the direction along the optical axis S of the fluorescent light beam.

That is, in the example shown in FIG. 9, an acousto-optic lens 27 is employed instead of the acousto-optic deflector 19 or the electro-optic deflector 20. The acousto-optic lens 27 is a lens that changes the refractive power in accordance with the input voltage. By switching the refractive power in synchronization with the frequency of the frequency oscillator 28, the imaging position of the fluorescence by the imaging lens 10 is changed. The position can be switched between a position matching the pinhole 11 and a position shifted from the pinhole 11 in the optical axis direction.
Even in this case, the optimum non-conjugated positional relationship exists, and the out-of-focus fluorescence can be accurately removed by setting the optimal positional relationship.

1 microscope 2 laser light source (light source)
3 Switching unit 7 Scanner 8 Objective lens (objective optical system)
11 Pinhole (shading material)
12 Light detector (detector)
16 Setting unit 18 Distribution acquisition unit (fluorescence distribution acquisition unit)
23 Motor (switching part)
25 Camera (two-dimensional sensor, fluorescence distribution acquisition unit)
A Sample

Claims

A scanner that scans excitation light from a light source;
An objective optical system that condenses the excitation light scanned by the scanner on the sample, and condenses the signal light generated in the sample at each scanning position;
A detector for detecting the signal light collected by the objective optical system;
A light blocking member that is disposed between the detector and the objective optical system and partially blocks the signal light collected by the objective optical system;
A switching unit that temporally switches the positional relationship between the light shielding member and the condensing point of the excitation light in the sample to an optically conjugate positional relationship and an optically non-conjugated positional relationship;
A fluorescence distribution acquisition unit for acquiring a fluorescence distribution at a position optically conjugate with the condensing point;
A microscope comprising: a setting unit that sets the non-conjugated positional relationship in the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquisition unit.
The setting unit separates the fluorescence distribution acquired by the fluorescence distribution acquisition unit into focused fluorescence and out-of-focus fluorescence, and the focused fluorescence is equal to or less than a predetermined threshold and is optically conjugate with the condensing point. The microscope according to claim 1, wherein the positional relationship closest to the position is set as a non-conjugated positional relationship in the switching unit.
The microscope according to claim 1, wherein the setting unit applies the fluorescence distribution acquired by the fluorescence distribution acquisition unit to a predetermined distribution model and separates the focused fluorescence and the out-of-focus fluorescence.
Based on the intensity of the fluorescence that is the signal light detected by the detector when the fluorescence distribution acquisition unit changes the positional relationship between the condensing point of the excitation light and the light shielding member in the sample. The microscope according to any one of claims 1 to 3, wherein the fluorescence distribution is acquired.
Acquired when the fluorescence distribution acquisition unit has a conjugate positional relationship between the light-shielding member and the condensing point of the excitation light in the sample, and when it is in a non-conjugate positional relationship between two different points. The microscope according to claim 4, wherein the fluorescence distribution is acquired based on the intensity of the emitted fluorescence.
The fluorescence distribution acquisition unit acquires the intensity of the fluorescence based on a theoretical size of the spot diameter of the excitation light at the condensing point determined by the objective optical system and the wavelength of the excitation light. The microscope according to claim 4 or 5, wherein a positional relationship between the light shielding member and the condensing point of the excitation light in the sample is set.
The microscope according to any one of claims 1 to 3, wherein the fluorescence distribution acquisition unit is a two-dimensional sensor disposed at a position optically conjugate with the condensing point of the excitation light in the sample.
The fluorescence distribution acquisition unit acquires the fluorescence distribution at a plurality of points,
The microscope according to any one of claims 1 to 7, wherein the setting unit sets the non-conjugated positional relationship in the switching unit based on an average of a plurality of the fluorescence distributions.