JPWO2017221356A1 - microscope - Google Patents

Abstract

  A microscope according to the present invention for the purpose of reducing the burden on the operator by automatically setting the optimum point at which the confocal pinhole and the focusing point of the excitation light are in an optically non-conjugated positional relationship. 1) a scanner (7) for scanning excitation light from a light source (2) and focusing excitation light to be scanned onto a sample (A), while focusing fluorescence generated in the sample at each scanning position An objective optical system (8), a detector (12) for detecting collected fluorescent light, and a detector and an objective optical system arranged to partially block the fluorescent light collected by the objective optical system Switching unit that temporally switches the positional relationship between the light blocking member (11) and the light blocking 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 ( 3) and fluorescence distribution to obtain fluorescence distribution at a position optically conjugate with the focusing point It comprises obtain unit (18), based on the obtained fluorescence distribution, setting unit for setting a non-conjugate relationship in the switching section (17).

Description

  The present invention relates to a microscope.

  The positional relationship between the confocal pinhole and the focusing point of the excitation light is optically set in order to prevent the observation of the deep part of the sample from being difficult due to the leakage of the out-of-focus fluorescence into the confocal pinhole. There is known a microscope which switches between a conjugated position and a non-conjugated position, detects fluorescence respectively, and calculates a difference between acquired fluorescence signals (see, for example, Patent Document 1).

WO 2015/163261

However, in the microscope of Patent Document 1, with regard to setting of the optimum point where the confocal pinhole and the focusing point of the excitation light are in an optically non-conjugated positional relationship, the operator manually photographs the sample while making a joint. The position of the focus pinhole has to be adjusted, which is complicated.
The present invention has been made in view of the above-described circumstances, and an operator automatically sets an optimum point at which the confocal pinhole and the focusing point of the excitation light have an optically non-conjugated positional relationship. An object of the present invention is to provide a microscope capable of reducing such burden.

  One aspect of the present invention is a scanner for scanning excitation light from a light source, and condensing the excitation light scanned by the scanner on a sample while collecting signal light generated on the sample at each scanning position An objective optical system, a detector for detecting the signal light collected by the objective optical system, the signal light disposed between the detector and the objective optical system and collected by the objective optical system The position relationship between the light blocking member that partially blocks the light, the light blocking member and the focusing point of the excitation light in the sample is an optically conjugate position relationship and an optically non-conjugated position relationship. In the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquiring unit, and a fluorescence distribution acquiring unit acquiring the fluorescence distribution at a position optically conjugate with the focusing point, and Set the non-conjugated positional relationship A microscope and a setting unit.

  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 condensing point of the excitation light on 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 detected by the detector, and a fluorescence image is generated by correlating the detected fluorescence intensity with the scanning position.

  In this case, the fluorescence distribution acquiring unit acquires the fluorescence distribution at a position optically conjugate with the condensing point, and the setting unit sets the non-conjugated positional relationship in the switching unit based on the acquired fluorescence distribution. Ru. Then, when the positional relationship between the light shielding member provided in the front stage of the detector and the focusing point of the excitation light in the sample is switched to a conjugate positional relationship by the operation of the switching unit, the focal fluorescence and the out-of-focus fluorescence are detected Detected by the

Further, when the switching unit is switched to the non-conjugated positional relationship set by the setting unit, the focal fluorescence can not pass through the light shielding member, and only the out-of-focus fluorescence is detected by the detector. Then, by calculating the difference between the 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.
That is, according to the present aspect, since the optimal non-conjugated positional relationship can be obtained based on the fluorescence distribution at a position optically conjugate with the focusing point, the operator does not have to perform complicated setting operations. It is possible to obtain focal fluorescence in which the external fluorescence is 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 acquiring unit into focal fluorescence and off-focus fluorescence, and the focal fluorescence becomes equal to or less than a predetermined threshold value and the focusing point. The positional relationship closest to the optically conjugate position may be set as the non-conjugated positional relationship in the switching unit.

  As the position deviates from the conjugate positional relationship, the degree of non-conjugateness becomes stronger, and the acquired focal fluorescence approaches zero, but the acquired out-of-focus fluorescence is an optical property of the light-shielding member and the condensing point of the sample. The out-of-focus fluorescence detected by the detector in the case of a substantially conjugate positional relationship is different. By doing this, when the light blocking member and the light condensing point in the sample are in an optically conjugate positional relationship, the fluorescence which is equal to the out-of-focus fluorescence detected by the detector and does not contain the focal fluorescence is detected It is possible to obtain focused fluorescence from which off-focus fluorescence has been precisely removed by subtraction.

In the above aspect, the setting unit may separate the focal fluorescence and the off-focus fluorescence by applying the fluorescence distribution acquired by the fluorescence distribution acquisition unit to a predetermined distribution model.
By so doing, it is possible to easily separate the in-focus fluorescence and the out-of-focus fluorescence only by applying the fluorescence distribution to a predetermined distribution model, and to set an appropriate non-conjugated positional relationship.

Further, in the above aspect, the signal light detected by the detector when the fluorescence distribution acquiring unit changes the positional relationship between the condensing point of the excitation light in the sample and the light shielding member. The fluorescence distribution may be obtained based on the intensity of the fluorescence.
By doing this, the fluorescence distribution can be obtained by the basic configuration of the microscope.

Further, in the above aspect, when the positional relationship between the light blocking member and the focusing point of the excitation light in the sample is in a conjugate positional relationship, the fluorescence distribution acquiring unit has two different non-conjugated positions. The fluorescence distribution may be acquired based on the intensity of the fluorescence acquired when in a positional relationship.
By doing this, it is necessary to obtain fluorescence intensities at least at three points of the conjugate positional relationship, the high non-conjugated positional relationship, and the non-conjugated positional relationship close to the conjugated positional relationship. Accordingly, it is possible to easily set a non-conjugated positional relationship in which the focusing fluorescence and the out-of-focus fluorescence can be separated with high accuracy by the setting unit.

Further, in the above aspect, the fluorescence distribution acquisition unit is based on the theoretical size of the spot diameter of the excitation light at the focusing point determined by the objective optical system and the wavelength of the excitation light. The positional relationship between the light shielding member for acquiring the intensity of the fluorescence and the focusing point of the excitation light in the sample may be set.
In this way, the degree of non-conjugation can be determined by the theoretical size of the spot diameter, whereby non-conjugation that allows the setting portion to separate focal fluorescence and out-of-focus fluorescence accurately The positional relationship can be set easily.

In the above aspect, the fluorescence distribution acquisition unit may be a two-dimensional sensor disposed at a position optically conjugate with the focusing point of the excitation light in the sample.
In this way, the fluorescence distribution can be acquired at one time, and the setting unit can quickly set a non-conjugated positional relationship that can accurately separate the focal fluorescence and the out-of-focus fluorescence.

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.
By doing this, it is possible to easily set a non-conjugated positional relationship capable of accurately separating the focal fluorescence and the out-of-focus fluorescence by the setting unit without acquiring a fluorescence image, and excitation of the sample is achieved. The light irradiation time can be reduced to reduce the damage to the sample.

  According to the present invention, it is possible to automatically set the optimum point at which the confocal pinhole and the focusing 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 showing a microscope concerning one embodiment of the present invention. It is a partial longitudinal cross-sectional view explaining the shift amount of the fluorescent light beam in a pinhole of the microscope of FIG. 1, and a pinhole. It is a graph which shows the relationship between the shift amount of FIG. It is a graph which shows an example of the relationship of the fluorescence intensity and shift amount acquired with the microscope of FIG. 1, and the shift amount detected in setting mode. It is a figure which shows an example of the irradiation pattern of one excitation light of two types of excitation light set by the setting part in the imaging | photography mode of the microscope of FIG. It is a figure which shows an example of the irradiation pattern of the other excitation light of two types of excitation light set by the setting part in the imaging | photography 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 disc which has a 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.
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 emitted alternately, as shown in FIG. A microscope main body 4 that detects fluorescence generated in the sample A by irradiating the sample A with two types of excitation light; and an operation unit 5 that generates an image by calculation using the fluorescence detected in the microscope main body 4; And a monitor 6 for displaying the image generated by the calculation unit 5.

  The microscope main body 4 two-dimensionally scans the excitation light from the switching unit 3 and the excitation light scanned by the scanner 7 on the sample A to condense the fluorescence (signal light) from the sample A An objective lens (objective optical system) 8 for condensing light, a dichroic mirror 9 for condensing light returned by the objective lens 8 and returning light passing through the scanner 7 from an optical path of excitation light, and branched by the dichroic mirror 9 Light for detecting the fluorescence passing through the pinhole 11 (light shielding member) disposed at a position optically conjugate with the focal position of the objective lens 8 A detector (detector) 12 is provided.

  In addition, although the pinhole 11 is illustrated as a light shielding member, in place of this, when disposed at a position optically conjugate with the focal position of the objective lens 8, the focal fluorescence is allowed to pass and disposed at a nonconjugated position Any light shielding member that shuts off the focal fluorescence when it is used may be employed. In addition, as a light shielding member, a micro mirror device or a spatial light modulator may be mentioned.

The scanner 7 is, for example, a proximity galvano mirror formed by arranging two galvano mirrors swingable around non-parallel axes in close proximity.
The photodetector 12 is, for example, a photomultiplier tube (PMT).

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

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

When the setting mode is selected from the input unit 17, the setting unit 16 operates the drive control unit 14 to drive the movable mirror 13 and shift the laser light flux by a preset shift amount. When the photographing mode is selected, the drive control unit 14 functions as a frequency oscillator that oscillates a predetermined frequency.
Here, as shown in FIG. 2A and FIG. 3, the shift amount x set in advance is the first shift amount of the shift amount zero where the focusing point and the pinhole 11 are in an optically conjugate positional relationship. x1, a second shift amount x2 in which the focusing point and the pinhole 11 are in a partially optically non-conjugated positional relationship, and the focusing point and the pinhole 11 are in a sufficiently optically non-conjugated positional relationship And 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 to an angle that can achieve three preset shift amounts, and the movable mirror 13. The distribution acquisition unit 18 stores the intensity of the fluorescence detected by the light detector 12 at each angle of.
The distribution acquiring unit 18 is configured to generate a fluorescence distribution in which shift amounts x that can achieve each of the three angles of the movable mirror 13 correspond to fluorescence intensities acquired at each angle.

  That is, when the positional relationship between the condensing point of the excitation light by the objective lens 8 and the pinhole 11 is disposed at an optically conjugate position, the fluorescence detected by the light detector 12 is shown in FIG. 2B. As shown, while the focal fluorescence generated from the focal point is largely dominated, the extrafocal fluorescence generated at locations other than the focal point is also included. Then, when the positional relationship between the focal point and the pinhole 11 shifts 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 focusing point and the pinhole 11 that can detect fluorescence that contains little focal fluorescence and is dominated by out-of-focus fluorescence from the fluorescence distribution acquired by the distribution acquisition unit 18 The angle of the movable mirror 13, that is, the achievable angle of the shift amount of the code x _P in FIG. 2B is calculated.
As a method of calculating the angle capable of achieving the shift amount of the code x _P, a method of searching for the optimum position by fitting the fluorescence distribution consisting of three points generated by the distribution acquisition unit 18 to a distribution model such as a Gaussian function, for example It can be mentioned.

That is, when fitting to a Gaussian function is performed, 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 flux, and w ² is the dispersion.

Thereby, it is possible to separate the focal fluorescence of the first term and the off-focus fluorescence of the second term, and for example, the minimum shift amount x = x at which the magnitude of the first term is equal to or less than threshold value 0.01P. _P can be set as an optimal non-conjugated positional relationship by the switching unit 3. In addition, although 0.01 means that focal fluorescence reduced to 1%, this value can be set arbitrarily.

Then, the setting unit 16 corresponds to the shift amount x = 0 capable of achieving the angle of the movable mirror 13 and the conjugate positional relationship corresponding to the shift amount x = x _P capable of achieving the calculated optimal non-conjugate positional relationship. 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 imaging mode, the drive control unit 14 oscillates a predetermined frequency, and switches the angle of the movable mirror 13 alternately to the two angles set by the setting unit 16 in synchronization with the oscillated frequency. It has become. Reference numeral 15 in the figure is a mirror. Although the movable mirror 13 is exemplified as the deflection element, a device such as an acousto-optic deflector or an electro-optic deflector can also be used.

  As a result, in the imaging mode, two types of excitation light with different incident angles are alternately made to be incident on the microscope main body 4. That is, as shown in FIGS. 4A and 4B, the two types of excitation light incident on the microscope main body 4 are formed in a rectangular wave shape having inverted timing. In addition, although the rectangular wave light which has the timing inverted as two types of excitation light was illustrated here, it replaces with this and it has arbitrary repeating shapes, such as a sine wave, and employ | adopts excitation light from which a phase differs. It is also good.

  One excitation light shown in FIG. 4A focuses on a position optically conjugate with the pinhole 11 through the scanner 7 and the objective lens 8, and the other excitation light shown in FIG. 4B is the scanner 7 and the objective. The lens 8 focuses on a position optically non-conjugated to the pinhole 11. The switching frequency of the incident angle is set to a frequency at which two types of excitation light can be flickered at least once at each pixel position.

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

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

The operation of the microscope 1 according to the present 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 disposed on a stage (not shown) of the microscope main body 4 so that the focal position of the objective lens 8 coincides with the sample A In the state adjusted to, continuous excitation light is generated from the laser light source 2.

  Then, when the setting mode is selected in the input unit 17, the setting unit 16 changes the shift amount x1 at which the light condensing point and the pinhole 11 become conjugate with respect to the drive control unit 14 and the degree of non-conjugation 2 The angles of the movable mirror 13 are set at three angles corresponding to two shift amounts x 2 and x 3, and the fluorescence intensity and shift amount x detected by the light detector 12 at each angle are correlated by the distribution acquisition unit 18 And a fluorescence distribution is generated.

The generated fluorescence distribution is sent to the setting unit 16. In the setting unit 16, the transmitted fluorescence distribution is applied to a Gaussian distribution, and corresponds to a shift amount x = 0 which is a conjugate positional relationship and a shift amount x = x _P which is an optimal non-conjugate positional relationship. The angle of the movable mirror 13 is calculated.
When the photographing mode is selected in the input unit 17, the setting unit 16 sets the calculated angle of the movable mirror 13 as the angle of the movable mirror 13 switched by the drive control unit 14.

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

  As a result, since the focal fluorescence contained in one excitation light is condensed at a position in the sample A conjugate to the pinhole 11, 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, they are separated by the dichroic mirror 9, collected by the imaging lens 10, passed through the pinhole 11, and detected by the light detector 12.

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

  Further, in particular, when the NA of the excitation light incident on the sample A is increased to perform high-definition observation, the region through which the excitation light passes before the focal position is increased. Fluorescence increases. In addition, similarly, the region through which the excitation light passes also increases during deep observation, so that the out-of-focus fluorescence increases. Furthermore, for deep observation, it is necessary to intensify the excitation light in order to compensate for the effects of scattering, and the effects of out-of-focus fluorescence are particularly remarkable.

  Among the fluorescence generated in the sample A, the fluorescence generated from the focal position of the objective lens 8 is easily detected by the light detector 12 as signal light because it easily passes through the pinhole 11 arranged at the optically conjugate position. However, fluorescence generated from a site other than the focal position is scattered by the sample, and a part of the fluorescence passes through the pinhole 11 and is detected as noise by the photodetector 12. Therefore, for the fluorescence detected by the irradiation of one excitation light, the focal fluorescence to be acquired as a signal generated at the focal position of the objective lens 8 and the out-of-focus generated at another portion not to be acquired as a signal Fluorescent and are included.

Also, the other excitation light is condensed at a position that is not conjugate 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 It generates fluorescence by doing.
In this case, the fluorescence generated at the non-conjugated focal position is blocked without being able to pass through the pinhole 11, while a part of the fluorescence generated from a site other than the focal position is scattered by the sample and Similarly to the step, the light passes through the same pinhole 11 and is detected by the light detector 12.

And according to the microscope 1 which concerns on this embodiment, since the nonconjugated position is set optimally, the focal fluorescence in the fluorescence detected by the photodetector 12 is reduced 99%, and the focal point The external fluorescence is approximately equivalent to the out-of-focus fluorescence detected by the light detector 12 in a conjugate positional relationship.
Therefore, by calculating the difference between the fluorescence detected by the irradiation of these two types of excitation light in the operation unit 5, a focus that should not be acquired as a signal is generated at a site other than the focal position of the objective lens 8. It is possible to accurately obtain the focal fluorescence from which the external fluorescence has been removed.

  Although the range in which the fluorescence is generated by the irradiation of the two types of excitation light is not exactly the same, in many parts, the detection is performed using the same pinhole 11 as well. By this, it is possible to remove most of the out-of-focus fluorescence even by subtraction as it is. In particular, when performing high-resolution observation by increasing the NA of the excitation light, the overlapping ratio of the fluorescence generation range is increased, so that the out-of-focus fluorescence can be more effectively removed.

  As described above, according to the microscope 1 according to the present embodiment, it is possible to detect fluorescence generated at the focal position of the objective lens 8 at a high S / N ratio, and to obtain a clear image with little noise. There is an advantage. In particular, the effect is high at the time of high definition observation where the NA of the excitation light is large, and when the sample A is a strong scattering material and off-focus fluorescence is likely to occur. Then, since the optimum non-conjugated 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, which requires the operator There is an advantage that the burden can be reduced.

  Further, in the present embodiment, since the difference between the two types of fluorescence acquired with an extremely short time difference is calculated for each pixel, it is possible to acquire a fluorescence image with less blurring even with the sample A moving at high speed. It has the advantage of being able to

  In the present embodiment, the pinhole 11 is illustrated as the light shielding member, but instead, when it is disposed at a position optically conjugate with the focal position of the objective lens 8, the focal fluorescence is allowed to pass and Any light blocking member that blocks focal fluorescence when placed at a conjugate position may be employed. Other light blocking members include micro mirror devices and spatial light modulators.

  Moreover, in this embodiment, although what used only fluorescence was illustrated as signal light from sample A which photodetector 12 detects, in addition to this, reflected light of excitation light from 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.

  The movable mirror 13 is illustrated as a deflection element constituting the switching unit 3, but as shown in FIG. 5, an acousto-optic deflector (acousto-optic element, luminous flux moving part) 19 or an electro-optic deflector (electro-optical element or luminous flux) A device such as the mobile unit 20 can also be used. In these devices 19 and 20 as well as the movable mirror 13, the fluorescent light flux is incident on the imaging lens 10 in synchronization with the input voltage by switching the voltage to be input according to the predetermined frequency oscillated by the drive control unit 14 The angle can be changed.

  The setting unit 16 switches the voltage generated by the drive control unit 14 in 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 in the imaging mode based on the acquired fluorescence distribution. Set the voltage. According to these devices 19 and 20, since the movable portion such as the movable mirror 13 is not included, the device can be configured to be compact and have a long life.

Further, in the present embodiment, the incident position of the fluorescent light flux incident on the fixed light shielding member 11 is temporally switched, but instead of this, the fluorescent light flux 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 flux.
That is, as shown in FIG. 6B, a disk-shaped disc having a plurality of pinholes 22 arranged at intervals in the circumferential direction is adopted as the light shielding member 21, and as shown in FIG. The (switching unit) 23 may rotate the disc 21 about the central axis.

  By doing this, the fluorescent light focusing position by the imaging lens 10 is made to coincide with the disc 21, and by rotating the disc 21 by the motor 23, the pinhole 22 coincides with the optical axis S of the fluorescence. And the non-coincident state can be alternately repeated. That is, in a state where one of the pinholes 22 coincides with the optical axis S of the fluorescence, the focal point of the excitation light in the sample A and the pinhole 22 have an optically conjugate positional relationship, and the optical axis S of the fluorescence On the other hand, when none of the pinholes 22 coincide with each other, the focal point of the excitation light in the sample A and the pinhole 22 have an optically non-conjugated positional relationship.

  In the present embodiment, the fluorescence distribution is determined based on the fluorescence intensities acquired at three positions where the pinhole 22 coincides with the optical axis S of the fluorescence, a position where the pinhole 22 partially matches, and a position where the pinhole 22 does not completely match. By setting the optimum position of the disc 21 to be set as a non-conjugated position based on the generated and generated fluorescence distribution, off-focus fluorescence can be removed with high accuracy.

  As a result, the optically conjugate positional relationship and the optically non-conjugated positional relationship are alternately formed temporally, and two types of fluorescence for detecting focal fluorescence with high accuracy by the same light detector 12 , Can be detected sequentially. Further, by rotating the disk 21 at high speed, there is an advantage that two positional relationships can be switched at higher speed.

Further, in the present embodiment, the case where the fluorescent light flux 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 relative to the fluorescent light flux You may
Further, as shown in FIG. 7, the non-conjugated positional relationship may be achieved by making the incident angles 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 disposed between the laser light source 2 and the dichroic mirror 9, the scanner 7 is used so that the position of the light condensing point on 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 swings 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.

  Further, the distribution model to be fitted is not limited to the Gaussian function, and Lorentz function or Voigt function may be used. In addition, although it has been decided to detect the fluorescence intensity at three points of shift amount x1 = 0 which is a conjugate positional relationship and shift amounts x2 and x3 which is a non-conjugate positional relationship, it is replaced by four or more shifts The fluorescence intensity may be detected in quantity to generate a fluorescence distribution. This can improve the accuracy of fitting to the distribution model.

In addition, the fluorescence distribution may be obtained at one place in the sample A, or the fluorescence intensities obtained at two or more places may be averaged to generate the fluorescence distribution.
In addition, although x = x2 and x3 are set in advance, Airy (AIRY), which is a unit representing the theoretical size of the spot diameter of the excitation light, may be used as a standard.

  That is, when the focal point size (spot diameter of excitation light) and the pinhole size determined by the NA of the objective lens 8 and the wavelength λ and the pinhole size are 1 airy, shift amount x2 = 0.5 airy, shift amount x3 = 5 airy, etc. You can set it to. By doing this, it is possible to calculate the shift amount in which the non-conjugated positional relationship is optimized with high accuracy by the fluorescence distribution of three points.

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

Instead of this, as shown in FIG. 8, a half mirror 24 for branching a part of the fluorescence from the sample A and a camera for imaging 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 is a condenser lens.
In this way, two-dimensional fluorescence distribution can be acquired at one time by the camera 25. Since fluorescence light flux or pinhole 11 shift is unnecessary when acquiring fluorescence distribution in the setting mode, processing can be performed in a short time. It has the advantage of being able to

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

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 refracting power according to the input voltage, and switches the refracting power in synchronization with the frequency by the frequency oscillator 28 so that the fluorescence imaging position by the imaging lens 10 is It is possible to switch between the position coincident with the pinhole 11 and the position displaced from the pinhole 11 in the optical axis direction.
Also in this case, the optimal non-conjugated positional relationship exists, and by setting the optimal positional relationship, the out-of-focus fluorescence can be accurately removed.

1 microscope 2 laser light source (light source)
3 switching unit 7 scanner 8 objective lens (objective optical system)
11 Pinhole (light blocking member)
12 light detector (detector)
16 setting unit 18 distribution acquisition unit (fluorescence distribution acquisition unit)
23 Motor (switching unit)
25 cameras (two-dimensional sensor, fluorescence distribution acquisition unit)
A sample

One aspect of the present invention is a scanner for scanning excitation light from a light source, and condensing the excitation light scanned by the scanner on a sample while collecting signal light generated on the sample at each scanning position An objective optical system, a detector for detecting the signal light collected by the objective optical system, the signal light disposed between the detector and the objective optical system and collected by the objective optical system The position relationship between the light blocking member that partially blocks the light, the light blocking member and the focusing point of the excitation light in the sample is an optically conjugate position relationship and an optically non-conjugated position relationship. In the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquiring unit, and a fluorescence distribution acquiring unit acquiring the fluorescence distribution at a position optically conjugate with the focusing point, and Set the non-conjugated positional relationship And a setting unit, the setting unit, said fitting fluorescence distribution the fluorescence distribution obtained by the obtaining unit to a predetermined distribution model is a microscope you separate the focal fluorescence and out of focus fluorescence.

Further, it is possible to easily separate the in-focus fluorescence and the out-of-focus fluorescence only by applying the fluorescence distribution to a predetermined distribution model, and to set an appropriate non-conjugated positional relationship .

In the above aspect, the setting unit sets, as the non-conjugated positional relationship in the switching unit, a positional relationship in which the focal fluorescence becomes equal to or less than a predetermined threshold and is closest to a position optically conjugate with the focusing point. You may

As the position deviates from the conjugate positional relationship, the degree of non-conjugateness becomes stronger, and the acquired focal fluorescence approaches zero, but the acquired out-of-focus fluorescence is an optical property of the light-shielding member and the condensing point of the sample. The out-of-focus fluorescence detected by the detector in the case of a substantially conjugate positional relationship is different. By doing this, when the light blocking member and the light condensing point in the sample are in an optically conjugate positional relationship, the fluorescence which is equal to the out-of-focus fluorescence detected by the detector and does not contain the focal fluorescence is detected it can be can Rukoto give focus fluorescence accurately remove off-focal fluorescence by the difference.

Claims (8)

A scanner for scanning excitation light from a light source;
An objective optical system which condenses the excitation light scanned by the scanner onto a sample while condensing signal light generated on the sample at each scanning position;
A detector for detecting the signal light collected by the objective optical system;
A light shielding member disposed between the detector and the objective optical system and partially blocking the signal light collected by the objective optical system;
A switching unit for temporally switching the positional relationship between the light blocking member and the focusing 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 focusing point;
A setting unit configured to set the non-conjugated positional relationship in the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquiring unit.   The setting unit separates the fluorescence distribution acquired by the fluorescence distribution acquisition unit into focused fluorescence and off-focus fluorescence, the focused fluorescence becomes equal to or less than a predetermined threshold, and is optically conjugate with the focusing 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 acquiring unit to a predetermined distribution model to separate focal fluorescence and off-focus fluorescence.   When the fluorescence distribution acquiring unit changes the positional relationship between the condensing point of the excitation light in the sample and the light blocking member, the fluorescence distribution acquiring unit is based on the intensity of fluorescence that is the signal light detected by the detector. The microscope according to any one of claims 1 to 3, wherein the fluorescence distribution is acquired.   The fluorescence distribution acquisition unit acquires when the positional relationship between the light blocking member and the focusing point of the excitation light in the sample is in a conjugate positional relationship and in two different non-conjugated positional relationships. The microscope according to claim 4, wherein the fluorescence distribution is acquired based on the intensity of the fluorescence.   The fluorescence distribution acquisition unit acquires the intensity of the fluorescence based on the theoretical size of the spot diameter of the excitation light at the focusing 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 blocking member and the focusing 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 focusing 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.

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