JP2012208361A - Scanning type microscope and scanning type microscope system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire a low noise image in high speed in a scanning type microscope.SOLUTION: A scanning type microscope comprises a light source unit, first scanning means 114a, a first beam restriction body 115a and a light quantity detector 118. The light source unit emits irradiation beams to irradiate to a sample from first and second radiation points. The first scanning means 114a changes deflection direction of the irradiation beam. The first beam restriction body 115a has plural pores 115as to pass the irradiation beam. The light quantity detector 118 detects the reception quantity of a signal beam caused by the irradiation to the sample. The first scanning means 114a can move a scanning point along the first scanning direction by changing the deflection direction of the irradiation beam. The plural pores 115as are placed so that the irradiation beams emitted from the first and second radiation points pass through the pore 115as at different time depending on the continuous variation of the deflection direction by the first scanning means 114a.

Description

  The present invention relates to a scanning microscope and a scanning microscope system that perform confocal scanning.

  Conventionally, many types of confocal scanning microscopes have been proposed (see Non-Patent Document 1) and commercialized. These confocal scanning microscopes are roughly classified into two types: a microscope using a single beam scanning device and a microscope using a multi-beam scanning device (see Patent Documents 1 to 3). The basic configuration of the two types of microscopes will be briefly described below.

  First, a basic configuration of a scanning microscope (hereinafter referred to as a single beam scanning microscope) using a single beam scanning device will be described. In a single beam scanning microscope, a light beam emitted from a light source is condensed at one point by a condenser lens. A transmission image of the first minute aperture arranged at the condensing position is formed on the sample via the first relay lens and the objective lens. The light beam reflected by the sample is deflected in a direction different from the direction of the first minute aperture by a beam splitter disposed between the objective lens and the first relay lens. The deflected light beam is condensed by the second relay lens on the second minute aperture at the conjugate position of the first minute aperture. The intensity of light passing through the second minute aperture is detected by a detector such as a photomultiplier.

  A scanning device is disposed between the objective lens and the beam splitter. This scanning device has, for example, a galvanometer mirror or a polygon mirror, and scans the image of the first minute aperture on the sample surface. The controller connected to the scanning device and the detector detects the imaging position of the first minute aperture on the sample from the deflection amount of the light beam by the scanning device. Based on the detection position and the intensity of light detected by the detector, a specimen image in a wide area is reconstructed and displayed on an image display device such as a television monitor. When a femtosecond pulse light source is used as the light source, the first minute aperture described above may be omitted.

  Next, a basic configuration of a scanning microscope using a Nipo disk as an example of a scanning microscope using a multi-beam scanning device (hereinafter referred to as a multi-beam scanning microscope) will be described. In a multi-beam scanning microscope, a light beam emitted from a light source is irradiated onto a Nipo disk by a condenser lens. A plurality of light beams divided by transmitting through a plurality of small apertures provided in the Nipo disk are condensed on one point on the sample via a relay lens and an objective lens, respectively. The light beam reflected from the sample is condensed again on the small opening of the Nipo disk through the objective lens and the relay lens.

  The beam splitter disposed between the Nipo disk and the condenser lens deflects the transmitted light from the Nipo disk in a direction different from that of the light source. The deflected transmitted light is collected on the image sensor by the photographing lens. With such a configuration, an image of the specimen is formed on the image sensor. The formed image is detected by the image sensor and displayed on the image display device. Since the plurality of small openings provided in the Nipo disk are provided at predetermined intervals, the illumination that hits the sample at a time is multi-spot illumination. However, since the Nipo disk is rotated at high speed by the motor, all specimen surfaces can be scanned in a short time, and confocal observation with the naked eye becomes possible.

  Thus, as can be understood from the difference between the basic configurations of the two, the single beam scanning microscope has good illumination efficiency of the light source, but the scanning time of the entire field of view of the microscope is longer than that of the multi-beam scanning microscope. In recent years, it has been required to observe high-speed time changes such as the appearance of a sample. For example, in order to acquire an image at 100 fps that is equal to or higher than the video rate, the scanning device needs high-speed performance equivalent to several tens of KHz. However, it is difficult to satisfy high-speed performance while keeping the size of the reflection portion provided in the scanning device large. On the other hand, in the multi-beam scanning microscope including the one using the Nipo disk as described above, since the scanning time is very short, an image can be acquired at a high speed at 1000 fps, and the time required for the appearance of the sample is high. Suitable for observing changes.

JP-T-1-503493 Japanese Patent Laid-Open No. 5-60980 Japanese Patent Laid-Open No. 8-21296

T.R.Corle and G.S.Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press (1966)

  By the way, even in such a multi-beam scanning microscope suitable for high-speed observation, there is a problem in observation of tissue accompanied by scattering. The scanning microscope can select and acquire signal light (for example, fluorescence) only in the vicinity of the laser spot by arranging a pinhole at a position conjugate with the laser spot focused in the sample. It is characterized by tightening. Therefore, when it can be considered that light scattering by the sample is not large, the effect is exhibited. However, particularly when a biological tissue is used as a sample, the signal light in the vicinity of the laser spot is quickly scattered due to the strong scattering property of the biological tissue. Therefore, even if a large number of multi-beams generated by the Nipo disk are collected on the specimen, the signal light generated by the multi-beams is strongly scattered, and the signal light is dyed into a small aperture different from the corresponding small aperture. Crosstalk occurs. Since the small aperture also transmits signal light in a minute region other than the optically conjugate minute region, the noise component increases. In order to reduce the crosstalk, it is conceivable to increase the spatial interval of the multi-beams. However, when the influence of scattering is very large, crosstalk can occur even if the number of multi-beams is two.

  Therefore, in the present invention made in view of the above problems, a scanning microscope and an operation microscope that can acquire an image with little noise even when spatial crosstalk occurs in signal light generated by a multi-beam. The purpose is to provide a system.

In order to solve the above-described problems, the scanning microscope according to the first invention is
A light source unit that has at least first and second emission points on the same plane and emits irradiation light to irradiate the sample from the first and second emission points so that chief rays are parallel to each other;
First scanning means for changing a deflection direction of irradiation light emitted from the light source unit;
A first light restrictor having a first irradiation surface to which the irradiation light deflected by the first scanning means is irradiated, and having a plurality of holes through which the irradiation light passes. When,
An objective lens for condensing the irradiation light that has passed through the light restrictor on the sample;
A light amount detector that detects the amount of signal light received by irradiating the sample with irradiated light; and
An optical separator provided between the sample and the light amount detector, for separating the signal light from the irradiation light;
A position detector for detecting the position of the scanning point on the sample of the irradiated light at the time of detection of the light amount by the light amount detector;
An image processing apparatus that reconstructs an image of a sample based on the amount of light and the position of the scanning point, and an output device that outputs the amount of received light and the position of the scanning point,
The first scanning means is movable along the first scanning direction by changing the scanning direction of the irradiation light irradiated on the sample by changing the deflection direction of the irradiation light,
The plurality of hole portions pass through different hole portions where the irradiation light emitted from the first and second emission points passes through the different hole portions according to the continuous change in the deflection direction by the first scanning unit, and the first The irradiation light emitted from the second emission point is arranged to pass through the hole at different times.

The operation type microscope system according to the first aspect of the present invention comprises:
A light source unit that has at least first and second emission points on the same plane and emits irradiation light to irradiate the sample from the first and second emission points so that chief rays are parallel to each other;
First scanning means for changing a deflection direction of irradiation light emitted from the light source unit;
A first light restrictor having a first irradiation surface to which the irradiation light deflected by the first scanning means is irradiated, and having a plurality of holes through which the irradiation light passes are formed on the first irradiation surface. When,
An objective lens for condensing the irradiation light that has passed through the light restrictor on the sample;
A light amount detector for detecting the amount of signal light received by irradiation of the sample with irradiation light;
An optical separator provided between the sample and the light amount detector, for separating the signal light from the irradiation light;
A position detector for detecting the position of the scanning point on the sample of the irradiated light at the time of detection of the light amount by the light amount detector;
An output device that outputs the amount of received light and the position of the scanning point to an image processing device that reconstructs an image of the sample based on the amount of light and the position of the scanning point;
An image processing device for reconstructing an image of the sample based on the amount of light and the irradiation position;
A monitor for displaying an image of the sample,
The first scanning means is movable along the first scanning direction by changing the scanning direction of the irradiation light irradiated on the sample by changing the deflection direction of the irradiation light,
The plurality of hole portions pass through different hole portions where the irradiation light emitted from the first and second emission points passes through the different hole portions according to the continuous change in the deflection direction by the first scanning unit, and the first The irradiation light emitted from the second emission point is arranged to pass through the hole at different times.

  According to the scanning microscope according to the present invention configured as described above, an image with less noise can be acquired using a multi-beam.

1 is a configuration diagram illustrating an optical schematic configuration and an electrical schematic configuration of a scanning microscope system including a scanning microscope according to a first embodiment of the present invention. FIG. 2 is a layout diagram of the branching device, the first galvanometer mirror, and the first slit in FIG. 1. It is a figure explaining the area | region on the sample scanned with the excitation light of each row | line | column. It is a front view of the 1st slit array in FIG. It is a timing chart explaining the time change of the irradiation position of the excitation light which injects into a 1st slit array. It is a block diagram which shows the optical schematic structure and electrical schematic structure of the scanning microscope system containing the scanning microscope which concerns on 2nd Embodiment. It is a block diagram which shows arrangement | positioning and a shape of the 2nd slit array with respect to the 1st slit array in FIG. It is a block diagram which shows the optical schematic structure and electrical schematic structure of the scanning microscope system containing the scanning microscope which concerns on 3rd Embodiment. FIG. 9 is a layout diagram of the branching device, the first galvanometer mirror, and the first slit in FIG. 8. It is a front view which shows the 1st modification of a 1st slit array. It is a front view which shows the 2nd modification of a 1st slit array.

  Hereinafter, embodiments of a scanning microscope to which the present invention is applied will be described with reference to the drawings.

  FIG. 1 is a configuration diagram showing an optical configuration and an electrical schematic configuration of a scanning microscope system including a scanning microscope according to the first embodiment of the present invention.

  The scanning microscope system 100 includes a scanning microscope 110, an image processing apparatus 101, and a monitor 102. As will be described in detail later, the scanning microscope 110 scans the sample S with spot-like excitation light (irradiation light). In addition, the amount of fluorescent light generated by the excitation light applied to the sample S is detected by the scanning microscope 110. Further, the position of the scanning point is detected from the scanning microscope 110. The amount of fluorescent light and the position of the scanning point are transmitted to the image processing apparatus 101. The image processing apparatus 101 reconstructs an image of the sample S based on the amount of fluorescent light and the irradiation position. The reconstructed image is transmitted to the monitor 102 and displayed.

  Next, the configuration of the scanning microscope 110 will be described. The scanning microscope 110 includes a light source 111, a branching device 112, first to fifth lenses 113a to 113e, first and second galvanometer mirrors 114a and 114b (first and second scanning means), and a first slit. By an array 115 (first light restrictor), a dichroic mirror 116 (optical separator), an objective lens 117, a PMT 118 (light quantity detector, output device), and a position detection unit 119 (position detection unit, output device), etc. Composed.

  Excitation light that generates fluorescence when emitted from the light source 111 is emitted as femtosecond pulses. The excitation light is branched into four rows by the branching device 112 so that the intensity is substantially equal. The first to fourth emission ends of the spectroscope 112 are formed so that chief rays of excitation light that are arranged in a straight line at a predetermined interval and are emitted are parallel to each other. The branching device 112 can be formed by, for example, a combination of a half mirror (not shown) and a mirror (not shown), a combination of a fiber coupler (not shown), a diffraction grating, or the like.

  The first and second lenses 113a and 113b are positive lenses and constitute a relay optical system. The first to fourth columns of excitation light emitted from the first to fourth emission ends are relayed telecentrically on both sides by the relay optical system, and are condensed separately on the first slit array 115a. A first galvanometer mirror 114a is disposed at a conjugate position with the pupil position of the objective lens 117 between the first and second lenses 113a and 113b. As shown in FIG. 2, the first galvanometer mirror 114a emits excitation light in a deflection direction rotated about a straight line parallel to the first straight line L1 connecting the first to fourth emission ends t1 to t4. Reflect. By continuously changing the deflection direction, the sample S is scanned along the first scanning direction (see FIG. 3) on the sample S by the excitation light.

  A second lens 113b and a first slit array 115a are arranged in the deflection direction of the excitation light by the first galvanometer mirror 114a. The first slit array 115a is a flat plate in which a large number of first slits 115as (holes) are formed, and is arranged so that the plate surface is parallel to the first straight line L1. As shown in FIG. 4, the first slit array 115a is formed with first slits 115as corresponding to the number of pixels in the first scanning direction of the acquired image. The first slits 115as are arranged in a second direction d2 perpendicular to the first straight line L1 on the plate surface. Note that the second direction d2 corresponds to the first scanning direction.

  All the first slits 115as have a shape inclined with respect to the first direction d1 perpendicular to the second direction d2 on the plate surface. Further, while the single column of excitation light (see EL3 in FIG. 4) in the first to fourth columns passes through the first slit 115as, the other three columns of excitation light (FIG. 4). The angle of inclination of the first slit 115as with respect to the first direction d1 is determined so that the plate surface of the first slit array 115a is irradiated on the plate surface of the first slit array 115a. The first slit 115as is formed so that the first to fourth rows of excitation light can pass through the single first slit 115as while being inclined with respect to the second direction d2.

  As described above, the excitation light branched into four rows is condensed on the first slit array 115a so that the principal rays are parallel to each other, so that the first direction d1 is formed on the first slit array 115a. A four-row irradiation spot image is formed along with the excitation light. By scanning the first slit array 115a having such a configuration with the first to fourth columns of excitation light, the first to fourth columns of excitation light pass through the first slit 115as at different times, The sample S is irradiated. As shown in FIG. 5, at the timing t1, only the first row of excitation light EL1 passes through the first slit 115as in the first row, and the second to fourth rows of excitation light EL2 to EL4 pass through the first slit. The plate surface of the array 115a is irradiated. Thereafter, the deflection direction is changed by the first galvanometer mirror 114a, and the irradiation spot image by the excitation light is displaced in the second direction d2. At timing t2, only the second row of excitation light EL2 passes through the first slit 115as of the first row, and the first, third, and fourth rows of excitation light EL1, EL3, and EL4 pass through the first slit array 15a. Irradiated to the plate surface. Similarly, at timings t3 and t4, only the third and fourth rows of excitation light EL3 and EL4 pass through the first slit 115as in the first row, respectively. Only the excitation light EL4 in the fourth row passes through the first slit 115as in the first row, and the irradiation spot image by the excitation light is further displaced in the second direction d2, and only the excitation light in the first row is 2 at the timing t5. It passes through the first slit 115as in the row. Thereafter, similarly, the passage of the excitation light in the first to fourth columns through the first slit 115as in the second, third,.

  As a result, when the first galvanometer mirror 114a reciprocally scans along the first scanning direction at the frequency RHz, the sample is in the optical conjugate position by the 2R / s irradiation spot images by one row of excitation light. S is irradiated. Therefore, the sample S is irradiated with four times the irradiation spot image of 8R / s by four rows of excitation light.

  The excitation light that has passed through the first slit array 115a is telecentrically relayed on both sides by the third and fourth lenses 113c and 113d. Between the third and fourth lenses 113c and 113d, the second galvanometer mirror 114b is arranged at a conjugate position with the pupil position of the objective lens 117. The second galvanometer mirror 114b reflects the excitation light transmitted through the third lens 113c in a deflection direction rotated about a straight line parallel to the second direction d2. By continuously changing the deflection direction, the sample S is scanned along the second scanning direction (see FIG. 3) perpendicular to the first scanning direction on the sample S by the excitation light.

  As shown in FIG. 3, the entire field-of-view range ea (hereinafter referred to as the entire irradiation region) of the scanning microscope 100 is obtained by scanning along the second and first scanning directions by the first and second galvanometer mirrors 114a and 114b, respectively. Is irradiated by the irradiation spot image. The first partial region ua1 obtained by dividing the entire irradiation region ea into four along the second scanning direction is raster-scanned by the first row of excitation light EL1. Similarly, the first to second partial areas ua1 and the second to fourth partial areas ua2 to ua4 are raster-scanned by the second to fourth rows of excitation light EL2 to EL4. For example, when the entire irradiation area ea is scanned with 512 scanning lines, the first to 128th scanning lines are scanned with the first column of excitation light EL1, and the 129th to 256th scanning lines are scanned with the second scanning line. The 257th to 384th scanning lines are scanned with the third column excitation light EL3, and the 385th to 512th scanning lines are scanned with the fourth column excitation light EL4. Note that the intervals between the first to fourth emission ends t1 to t4 are determined so that the first to fourth partial regions ua1 to ua4 are scanned by the first to fourth rows of excitation light EL1 to EL4, respectively. It is done.

  As shown in FIG. 1, the excitation light reflected by the second galvanometer mirror 114b is projected onto the sample S via the fourth and fifth lenses 113d and 113e, the dichroic mirror 116, and the objective lens 117. . The dichroic mirror 116 is formed so as to transmit the excitation light and reflect the fluorescence emitted from the living tissue by the excitation light. Therefore, the excitation light that has passed through the fifth lens 113 e passes through the dichroic mirror 116. The excitation light to be projected is collected on the sample S by the fifth lens 113d and the objective lens 117 which are imaging lenses.

  When the sample S is irradiated with the excitation light, the sample S is excited at the condensing point of the irradiated excitation light, and fluorescence (signal light) is generated toward the objective lens 117. Therefore, as described above, when the sample is irradiated at an irradiation speed of 8R / s, fluorescence is sequentially generated at the same generation speed. The generated fluorescence passes through the objective lens 117 and reaches the dichroic mirror 116. The fluorescence is reflected by the dichroic mirror 116, and the reflected light of the excitation light sample S passes through the dichroic mirror 116. Therefore, the fluorescence is separated from the reflected light of the excitation light. The separated fluorescence is received by the PMT 118. The PMT 118 generates a pixel signal converted into an electrical signal corresponding to the fluorescence intensity. The pixel signal is transmitted to the image processing apparatus 101.

  Note that the fluorescence corresponding to the excitation light in each column is widely distributed on the light receiving unit of the PMT 118 via the objective lens 117 and the dichroic mirror 116. A PMT 118 having a light receiving portion wider than the fluorescence incident range is selected and used.

  Note that not only the pixel signal but also the position of the scanning point of the excitation light detected by the position detection unit 119 is transmitted to the image processing apparatus 101 as a position signal. The position detection unit 117 is based on the rotation speeds of the first and second galvanometer mirrors 114a and 114b detected by a rotation speed meter (not shown) provided in the first and second galvanometer mirrors 114a and 114b. 1. The deflection direction of the excitation light pulse of each column by the first and second galvanometer mirrors 114a and 114b is calculated. The position detector 119 detects the position of the scanning point based on the deflection direction.

  The image processing apparatus 101 reconstructs a two-dimensional image of the sample S based on the pixel signal and the position signal. The reconstructed two-dimensional image is transmitted to the monitor 102 and displayed.

  According to the scanning microscope of the first embodiment configured as described above, a low-noise image can be acquired at high speed. Since the sample is scanned with four multi-beams, the scanning speed limited by the scanning frequency of the first galvanometer mirror 114a can be increased by the amount of light beam to be branched. Therefore, a sample image can be collected at high speed. In addition, since the irradiation time of the multi-beam sample S is separated, it is possible to eliminate the influence of spatial crosstalk.

  Further, according to the scanning microscope of the first embodiment, the second galvanometer mirror 114b is configured in the configuration in which the irradiation timings are separated by arranging the first to fourth emission ends t1 to t4 as described above. The frame rate can be maximized while simplifying the control. For example, as a configuration different from the present embodiment, the excitation light emitted from the first, second, third, and fourth emission ends may be (4n-3) th, (4n-2) th, (4n-1). ), A configuration in which scanning is performed along the 4nth scanning line (1 ≦ n ≦ 128) is conceivable. In order to maximize the frame rate in such a configuration, when the scanning point reaches the end along the first scanning direction, it corresponds to four scanning lines in the second scanning direction at high speed. It is necessary to move the scanning point by the distance. Therefore, it is necessary to change the scanning speed by the second galvanometer mirror 114b in accordance with the deflection direction by the first galvanometer mirror 114a. On the other hand, according to the configuration of the present embodiment, the scanning speed of the second galvanometer mirror 114b can be kept constant regardless of the deflection direction of the first galvanometer mirror 114a, and the control is simplified.

  Further, according to the scanning microscope of the first embodiment, a femtosecond pulse laser is used as the excitation light applied to the sample S, and an image is acquired by fluorescence generated by two-photon absorption. Two-photon absorption occurs only at a condensing point with substantially high photon density. Therefore, it is not necessary to apply the confocal effect, that is, no pinhole is required, and the number of parts can be reduced.

  Next, a scanning microscope according to the second embodiment of the present invention will be described. The second embodiment differs from the first embodiment in the type of light source and the arrangement of optical members. The second embodiment will be described below with a focus on differences from the first embodiment. In addition, the same code | symbol is attached | subjected to the site | part which has the same function and structure as 1st Embodiment.

  As shown in FIG. 6, in the scanning microscope 210 to which the second embodiment is applied, the splitter 112, the first to fifth lenses 113a to 113e, the first and second galvanometer mirrors 114a and 114b, the first The functions and configurations of the slit array 115a, the objective lens 117, the PMT 118, and the position detector 119 are the same as those in the first embodiment. The functions and configurations of the image processing apparatus 101 and the monitor 102 are the same as those in the first embodiment.

  Unlike the first embodiment, the light source 211 emits continuous wave (CW) excitation light. The dichroic mirror 216 is provided between the first galvanometer mirror 114a and the second lens 113b. In addition, the second slit array 115b is arranged so as to be stacked on the first slit array 115a. Further, the first and second slit arrays 115a and 115b are disposed at positions conjugate with the focal point of the objective lens 117.

  As shown in FIG. 7, the second slit array 115b is formed with four second slits 115bs extending along the second direction d2. A second slit 115bs is formed at each irradiation position of the excitation light beams EL1 to EL4 in the first to fourth rows passing through the first slit 115as.

  In the above configuration, the first to fourth rows of excitation light EL1 to EL4 reflected by the first galvanometer mirror 114a pass through the dichroic mirror 216 and the second lens 113b. The excitation light to be applied to the sample S is selected by the first slit array 115a from the transmitted first to fourth rows of excitation light. In the second slit array 115b, the excitation light passing through the first slit array 115a is not limited. As will be described later, the second slit array 115b is used to limit the fluorescence emitted from the sample S. The excitation light that has passed through the first and second slit arrays 115a and 115b passes through the third lens 113c, the second galvanometer mirror 114b, the fourth and fifth lenses 113d and 113e, and the objective lens 117. It is focused on and irradiated.

  Fluorescence generated by the irradiation of excitation light reaches the second slit array 115b through the objective lens 117, the fifth and fourth lenses 113e and 113d, the second galvanometer mirror 114b, and the third lens 113c. The first and second slits 115a and 115b block the fluorescence generated in the region other than the focal point, and only the fluorescent light generated at the focal point passes through the first and second slits 115as and 115bs. Therefore, it is possible to obtain a confocal effect by the first and second slits 115as and 115bs. The fluorescence that has passed through the first and second slits 115as and 115bs passes through the second lens 113b, is separated from the excitation light by the dichroic mirror 216, and is received by the PMT 118.

  As in the first embodiment, the pixel signal generated by the PMT 118 and the position signal detected by the position detection unit are transmitted to the image processing apparatus 101, and a two-dimensional image of the sample S is reconstructed.

  Also with the scanning microscope of the second embodiment configured as described above, a low-noise image can be acquired at high speed as in the first embodiment. Further, as in the first embodiment, it is possible to achieve the highest frame rate.

  Unlike the first embodiment, the dichroic mirror 216 is provided between the first galvanometer mirror 114a and the second lens 113b, thereby obtaining a confocal effect by the first and second slits 115as and 115bs. It is possible. Unlike the first embodiment, by using the confocal effect, it is possible to use excitation light that is a continuous wave without limiting the type of excitation light emitted from the light source to the femtosecond pulse laser.

  Next, a scanning microscope according to the third embodiment of the present invention will be described. The third embodiment differs from the second embodiment in the arrangement of optical members and the like. The third embodiment will be described below with a focus on differences from the second embodiment. In addition, the same code | symbol is attached | subjected to the site | part which has the same function and structure as 1st, 2nd embodiment.

  As shown in FIG. 8, in the scanning microscope 310 to which the third embodiment is applied, the light source 211, the branching device 112, the first to fifth lenses 113a to 113e, the first and second galvanometer mirrors 114a and 114b. The functions and configurations of the first and second slit arrays 115a and 115b, the objective lens 117, the PMT 118, and the position detection unit 119 are the same as those in the second embodiment. The functions and configurations of the image processing apparatus 101 and the monitor 102 are also the same as those in the second embodiment.

  Unlike the second embodiment, the dichroic mirror 316 is provided between the branching device 112 and the first lens 113a. A lens array 120 having four collimating lenses 120c and a condenser lens 121 are provided between the dichroic mirror 316 and the PMT 118.

  A collimator lens 120c is disposed at each position where the fluorescence corresponding to the first to fourth columns of excitation light EL1 to EL4 is reflected by the dichroic mirror 316 and incident on the lens array 120. The fluorescent light is converted into parallel light by the collimating lens 120 c and enters the condenser lens 121. The condensing lens 121 condenses the fluorescence corresponding to each column at one point of the PMT 118.

  Similar to the second embodiment, the pixel signal generated by the PMT 118 and the position signal detected by the position detection unit 119 are transmitted to the image processing apparatus 101, and a two-dimensional image of the sample S is reconstructed. .

  Also with the scanning microscope of the third embodiment configured as described above, a low-noise image can be acquired at high speed as in the second embodiment. Further, similarly to the second embodiment, it is possible to maximize the frame rate and apply a light source that emits a continuous wave.

  Furthermore, unlike the second embodiment, the fluorescence transmitted through the lens array 120 is emitted in a parallel direction regardless of the emission end from which the excitation light is emitted. Since the light is emitted in a parallel direction, it is possible to collect light at one point of the PMT 118 by using the condensing lens 121. Therefore, unlike the second embodiment, it is possible to use the PMT 118 having a small detection unit.

  Although the present invention has been described based on the drawings and examples, it should be noted that those skilled in the art can easily make various modifications and corrections based on the present disclosure. Therefore, it should be noted that these variations and modifications are included in the scope of the present invention.

  For example, in the first to third embodiments, the first slit 115as has a shape inclined with respect to the first direction d1, but is not limited to such a shape. For example, as shown in FIG. 9, when the first straight line L1 is inclined with respect to the rotation axis of the first galvanometer mirror 114a, as shown in FIG. 10, it is parallel to the first direction d1. The first slit 115as may be formed in a shape extending in the direction. The first slits 115as are formed so that when one column of excitation light passes through the single first slit 115as, the excitation light of the other column is not incident on all the first slits 115as. In this case, the same effect as in the present embodiment can be obtained.

  In addition, the first slit array 115a has a configuration in which the first slit 115as is formed. However, as shown in FIG. 11, a plurality of hole portions 122 may be formed instead of the slit. The irradiation position of the excitation light in each column is displaced along the second direction by the first galvanometer mirror 114a. According to the continuous displacement of the irradiation position of the excitation light of each row, the excitation light of each row is passed through different hole portions 122, and the excitation light of all rows passes through the hole portions 122 at different times. In addition, if the hole 122 is disposed, it is possible to obtain the same effect as in the present embodiment. Each row of excitation light in each row is passed through different hole portions 122 in accordance with the continuous displacement of the irradiation position of the excitation light in each row, so that each row has an optical image taken as a pixel on the sample S. This is because it becomes possible to irradiate the excitation light. Further, it is possible to separate the irradiation timings by passing the excitation light of all the rows through the holes 112 at different times in accordance with the continuous displacement of the excitation light irradiation positions of the respective rows. Note that. If the hole 122 is formed instead of the first slit 115as, the second slit array 115b is unnecessary in the second and third embodiments.

  In the second and third embodiments, the second slit array 115b is arranged by being stacked on the first slit array 115a. However, the second slit array 115b may not be provided. Even if the second slit array 115b is not provided, it is possible to limit light other than the light beam passing through the first slit by the first slit array 115a.

  In the second and third embodiments, the light source 211 that emits the excitation light and the dichroic mirror 116 are used to separate the fluorescence. However, the light source that emits normal visible light is used, and the deflection beam splitter is used. Instead of the dichroic mirror 116, an optical separator that transmits the light emitted from the light source and separates the reflected light, such as a configuration in which the λ / 4 plate is combined, may be used.

  In the first embodiment, the scanning microscope 110 is configured to be applied to an episcopic microscope, but can be applied to a transmission microscope.

  In the first to third embodiments, the sample S is scanned in the second scanning direction by deflecting the excitation light using the second galvanometer mirror 114b. The sample S may be scanned using the.

  Further, in the first to second embodiments, the pump light is branched into four rows using the branching device 112, but four light sources are provided directly without using the branching device 112, and the pumping light is emitted. It may be a configuration. The branching by the branching unit 112 is not limited to four columns. If branched into two or more rows, the image acquisition is speeded up as compared with a single beam scanning endoscope.

  In the first to third embodiments, the scanning point of the excitation light is detected based on the rotational speeds of the first and second galvanometer mirrors 114a and 114b. However, the scanning point is detected by other methods. It may be configured to. For example, a configuration in which a rotational position is detected using an encoder and a scanning point is detected based on the rotational position may be used.

  In the first embodiment, the dichroic mirror 116 is provided between the objective lens 117 and the fifth lens 113e. However, the dichroic mirror 116 is provided at any position between the branching device 112 and the objective lens 117. Also good. In the second embodiment, the dichroic mirror 116 is provided between the first galvanometer mirror 114a and the second lens 113b. The dichroic mirror 116 is provided between the branching device 112 and the first slit array 115a. It may be provided at any position.

DESCRIPTION OF SYMBOLS 100 Scanning microscope system 101 Image processing apparatus 110, 210, 310 Scanning microscope 111, 211 Light source 112 Branch device 113a-113e 1st-5th lens 114a, 114b 1st, 2nd galvanometer mirror 115a, 115b 1st , Second slit array 115as, 115bs first, second slit 116, 216, 316 dichroic mirror 117 objective lens 118 PMT
119 Position detection unit EL1 to EL4 First to fourth rows of excitation light S Sample t1 to t4 First to fourth emission ends

Claims (7)

A light source unit that has at least first and second emission points on the same plane, and emits irradiation light to irradiate the sample from the first and second emission points so that chief rays are parallel to each other;
First scanning means for changing a deflection direction of the irradiation light emitted from the light source unit;
A first irradiation surface on which the irradiation light deflected by the first scanning unit is irradiated; and a plurality of holes through which the irradiation light passes are formed in the first irradiation surface. Light limiter,
An objective lens for condensing the irradiation light that has passed through the light restrictor on the sample;
A light amount detector that detects the amount of signal light received by irradiating the sample with the irradiation light; and
An optical separator provided between the sample and the light amount detector, for separating the signal light from the irradiation light;
A position detection unit for detecting a position of a scanning point on the sample of the irradiation light at the time of detection of the light amount by the light amount detector;
An image processing device that reconstructs an image of the sample based on the light amount and the position of the scanning point, and an output device that outputs the received light amount and the position of the scanning point.
The first scanning means is movable along the first scanning direction by changing a scanning point of the irradiation light irradiated on the sample by changing a deflection direction of the irradiation light.
The plurality of hole portions pass through the hole portions where the irradiation lights emitted from the first and second emission points are different according to a continuous change in the deflection direction by the first scanning unit. And the scanning microscope characterized by arrange | positioning so that the said irradiation light radiate | emitted from the said 1st, 2nd radiation | emission point may pass the said hole part at a different time.   The scanning microscope according to claim 1, wherein the light source unit emits femtosecond pulsed light as the irradiation light.   The scanning microscope according to claim 1, wherein the light source unit emits a continuous wave as the irradiation light, the optical separator is provided between the light source unit and the first light restrictor, The first light restrictor is arranged so that the hole through which the irradiation light passes and the condensing point of the irradiation light condensed by the objective lens are in a conjugate position. Scanning microscope. The scanning microscope according to claim 3, wherein
The second slit has a second irradiation surface on which the signal light is incident, a second slit is formed at a position conjugate with the condensing point corresponding to the first and second emission points, and the second slit is A second light restricting body formed to extend in the displacement direction of the signal light on the second irradiation surface corresponding to the displacement of the scanning point by the first scanning means;
The hole portion is inclined in both the direction passing through the irradiation position on the first irradiation surface of the irradiation light emitted from the first and second emission points and the first displacement direction. A first slit extending to
The irradiation light corresponding to the irradiation light emitted from the first and second emission points can pass through the same first slit by changing the deflection angle by the first scanning means. A scanning microscope characterized by the above. In the scanning microscope according to any one of claims 1 to 4,
A second scanning unit provided between the light restrictor and the objective lens and relatively moving the scanning point along a second scanning direction different from the first scanning direction;
The first and second emission points are arranged such that the scanning points corresponding to the first and second emission points are displaced in different ranges along the second scanning direction. A scanning microscope. The scanning microscope according to any one of claims 1 to 5,
The light source unit is
A single light source for emitting the irradiation light;
A branching device that branches the irradiation light emitted from the light source at the plurality of emission ends arranged on a plane perpendicular to the emission direction so that chief rays of the irradiation light are parallel to each other; A scanning microscope characterized by the above. A light source unit that has at least first and second emission points on the same plane, and emits irradiation light to irradiate the sample from the first and second emission points so that chief rays are parallel to each other;
First scanning means for changing a deflection direction of the irradiation light emitted from the light source unit;
A first irradiation surface on which the irradiation light deflected by the first scanning unit is irradiated; and a plurality of holes that allow the irradiation light to pass through are formed on the first irradiation surface. Light limiter,
An objective lens for condensing the irradiation light that has passed through the light restrictor on the sample;
A light amount detector that detects the amount of signal light received by irradiating the sample with the irradiation light; and
An optical separator provided between the sample and the light amount detector, for separating the signal light from the irradiation light;
A position detection unit for detecting a position of a scanning point on the sample of the irradiation light at the time of detection of the light amount by the light amount detector;
An output device that outputs the received light amount and the position of the scanning point to an image processing device that reconstructs an image of the sample based on the light amount and the position of the scanning point;
An image processing device for reconstructing an image of the sample based on the light amount and the irradiation position;
A monitor for displaying an image of the sample,
The first scanning means is movable along the first scanning direction by changing a scanning point of the irradiation light irradiated on the sample by changing a deflection direction of the irradiation light.
The plurality of hole portions pass through the hole portions where the irradiation lights emitted from the first and second emission points are different according to a continuous change in the deflection direction by the first scanning unit. And the scanning microscope system characterized by arrange | positioning so that the said irradiation light radiate | emitted from the said 1st, 2nd radiation | emission point may pass the said hole part at a different time.

Images