JP2012208442A - 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 120, an objective lens 115, a scanner unit 114, a pin hole array 113, an optical separator 112, a light quantity detector 116 and a position detecting system 117. The light source unit 120 emits beam pulses from plural radiation points in turn repeatedly. The objective lens 15 images the beam pulses on a sample. The scanner unit 114 scans the sample with the beam pulses imaged by the objective lens. The pin hole array 113 is pin holes shaped on at least one image formation position of a reflected light reflected by the sample and fluorescence. The optical separator 112 isolates at least one of the reflected light and the fluorescence from a light path of a transmitted light by deflecting. The light quantity detector 116 detects light quantity of the reflected light or the fluorescence. The position detecting system 117 detects an irradiation position of an irradiation beam.

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 single mode oscillation laser 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 proportional to the reflectance of the sample 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)

  Incidentally, even in such a multi-beam scanning microscope suitable for high-speed observation, there is a problem in observation of a sample accompanied by scattering. A scanning microscope can select and acquire signal light (for example, fluorescence) only in the vicinity of the laser spot by placing a pinhole at a position conjugate with the laser spot condensed in the material. 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 repeatedly emits light pulses sequentially from a plurality of emission points;
An objective lens that separately collects the light pulses emitted from a plurality of emission points on a sample;
A scanner unit for moving the scanning points of a plurality of light pulses collected by the objective lens relative to the sample; and
A pinhole array in which a pinhole is formed at a position conjugate with the focal point of the light pulse collected by the objective lens;
An optical separator provided on the optical path of the light pulse between the light source unit and the objective lens, and separating the signal light emitted from the sample by irradiation of the light pulse from the light pulse;
A light amount detector for detecting the light amount of the signal light separated by the optical separator;
A position detection system that detects a scanning point at the time of detection of the amount of light by the light amount detector;
An image reconstruction device that reconstructs an image of a sample based on a light amount and a scanning point includes an output device that outputs the light amount and an irradiation position.

The operation type microscope system according to the first aspect of the present invention comprises:
A light source unit that repeatedly emits light pulses sequentially from a plurality of emission points;
An objective lens that separately collects light pulses emitted from a plurality of emission points on a sample;
A scanner unit for moving the scanning points of a plurality of light pulses collected by the objective lens relative to the sample; and
A pinhole array in which a pinhole is formed at a position conjugate with the focal point of the light pulse collected by the objective lens;
An optical separator provided on the optical path of the light pulse between the light source unit and the objective lens, and separating the signal light emitted from the sample by irradiation of the light pulse from the light pulse;
A light amount detector for detecting the light amount of the signal light separated by the optical separator;
A position detection system that detects a scanning point at the time of detection of the amount of light by the light amount detector;
An image reconstruction device for reconstructing an image of the sample based on the amount of light and the scanning point;
The image reconstruction apparatus includes a monitor that displays an image of the reconstructed sample.

  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 block diagram schematically illustrating a configuration of a light source unit in FIG. 1. It is a timing chart which shows the outgoing time of the excitation light pulse which injected into the 1st-4th optical waveguide. It is a figure which shows the scanning range of the sample observed with a scanning microscope. It is a timing chart which shows the time change of the irradiation position of an excitation light pulse. It is a figure which shows the scanning range of the sample when the 1st-4th output end is not arranged on a straight line. 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 the 2nd Embodiment of this invention.

  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. 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 irradiation position of the spot-like excitation light is detected by the scanning microscope 110. The amount of fluorescence and the irradiation position are transmitted from the scanning microscope 110 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 unit 120, first to fifth lenses 111a to 111e, a dichroic mirror 112, a pinhole array 113, a scanner unit 114, an objective lens 115, a PMT 116 (light quantity detector, output device), and position detection. The unit 117 (output device) and the control unit 118 are configured.

  As shown in FIG. 2, the light source unit 120 includes an LD 121, a modulator 122, a branching device 123, a delay device 124, and a position restricting body 125.

  When the living body is irradiated, excitation light that emits fluorescence is emitted from the LD 121. The excitation light is converted by the modulator 122 into excitation light pulses having a repetition frequency R and a pulse width T satisfying the expression (1).

N × (T + τ) <1 / R (1)
In the equation (1), τ is the fluorescence lifetime until light emission from the living body irradiated with excitation light is stopped. N is the number of columns of light branched by the branching device 123, and is 4 in this embodiment.

  The pumping light pulses are branched into four rows by the branching device 123 so that the intensity and the phase are substantially equal, and are incident on the delay device 124. The branching device 123 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 delay device 124 is provided with first to fourth delay paths 124a to 124d. The four rows of pumping light pulses branched by the branching device 123 are incident on separate delay paths 124 a to 124. The lengths of the delay paths 124a to 124d are adjusted so that the timings at which the excitation light pulses having the repetition frequency R are emitted from the delay paths 124a to 124d are shifted. For example, the second, third, and fourth delay paths 124b, 124c, and 124d are more than Δ (= c / (4nR), c is the speed of light, and n is the first to fourth speeds than the first delay path 124a. The optical waveguides 124a to 124d have a refractive index of 2Δ and 3Δ).

  With such a configuration, as shown in FIG. 3, the excitation light pulses that are simultaneously incident on the incident ends of the first to fourth delay paths 124a to 124d at the repetition frequency R have a repetition frequency different from that of the adjacent delay paths. 4R is repeatedly emitted in order from the first to fourth emission ends t1 to t4 of the first to fourth delay paths 124a to 124d. The first to fourth emission ends t1 to t4 are fixed by the position restricting body 125 so that the chief rays of the excitation light pulses that are arranged in a straight line at a constant interval and are emitted in parallel to each other.

  In FIG. 1, the excitation light pulses emitted from the first to fourth emission ends t1 to t4 are four pins formed in the pinhole array 113 in a telecentric state by the first and second lenses 111a and 111b. It is condensed separately in the hall. The excitation light pulse collected in the pinhole passes through the pinhole and passes through the third lens 111c, the scanner unit 114, the fourth and fifth lenses 111d and 111e, and the objective lens 115, and the sample surface to the sample. It is condensed inside.

  The third and fourth lenses 111c and 111d form a relay optical system. The fourth lens 111d places the scanner unit 114 and the pupil position of the objective lens 115 at the conjugate position. By using the fourth lens 111d, the excitation light pulse that has passed through the pinhole can be incident on the objective lens 115 at the correct position and angle. As will be described later, the scanner unit 114 includes first and second galvanometer mirrors 114a and 114b that rotate about axes orthogonal to each other, and the first and second galvanometer mirrors 114a and 114b are positioned at the pupil position. By arranging in the vicinity of the conjugate position, the scanner unit 114 and the pupil position can be arranged at the conjugate position. Further, by providing a relay lens between the first and second galvanometer mirrors 114a and 114b so that the first and second galvanometer mirrors 114a and 114b and the pupil position are conjugate, the scanner unit 114 and the pupil position are arranged. May be arranged in a conjugate position.

  The scanner unit 114 scans the sample surface or the inside of the sample with an excitation light pulse in a two-dimensional plane orthogonal to the optical axis. As described above, the excitation light pulse is emitted from the first to fourth emission ends t1 to t4 and irradiated onto the sample in a spot shape. Therefore, the scanner unit 114 sequentially scans the surface of the sample or the inside of the sample with four rows of excitation light pulses. As shown in FIG. 4, the entire field of view ea of the scanning microscope 100 (hereinafter, referred to as an entire irradiation region) is irradiated with four rows of excitation light pulses by scanning.

  The first galvanometer mirror 114a scans the sample surface or the inside of the sample along the first scanning direction. Further, the sample surface or the inside of the sample is scanned along the second scanning direction perpendicular to the first scanning direction by the second galvanometer mirror 114b. 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 pulses ep1 corresponding to the first emission end t1. Similarly, the second to fourth partial regions ua2 different from the first partial region ua1 by the excitation light pulses ep2 to ep4 in the second to fourth columns corresponding to the second to fourth emission ends t2 to t4. ... Ua4 is raster scanned. For example, when the entire irradiation area ea is scanned by 512 scanning lines, the first to 128th scanning lines are scanned by the excitation light pulse ep1 of the first column, and the 129th to 256th scanning lines are scanned. Scanning is performed with two rows of excitation light pulses, the 257th to 384th scanning lines are scanned with the third row of excitation light pulses, and the 385th to 512th scanning lines are scanned with the fourth row of excitation light pulses.

  As described above, since the excitation light pulses in adjacent columns have a shift of 1 / (4 × R), as shown in FIG. 5, the partial regions ua1 to ua4 are excited while shifting the time instead of simultaneously. Light pulses (see symbol ep) are repeatedly irradiated in order.

  As described above, the first to fourth emission ends t1 to t4 are scanned so that the first to fourth partial regions ua1 to ua4 are scanned by the excitation light pulses of the first to fourth columns, respectively. An interval is defined. In other words, the virtual planes perpendicular to the optical axis of the excitation light pulse at the first to fourth emission ends t1 to t4 are equally spaced on a straight line perpendicular to the first corresponding direction corresponding to the first scanning direction. In this way, the positions of the first to fourth emission ends t1 to t4 are determined.

  When the excitation light pulse is irradiated, the sample is excited in the vicinity of the condensing point of the irradiated excitation light pulse, and fluorescence (signal light) is generated. The objective lens 115 and the pinhole array 113 are arranged so that the condensing point by the objective lens 115 and the pinhole are in a conjugate position, and only the fluorescent component generated at the condensing point is each pin of the pinhole array 113. Form an image in the hole. Since the fluorescent component generated in the vicinity of the condensing point protrudes around the pinhole and forms an image, passage is restricted. Therefore, only the fluorescent component at the condensing point passes through the pinhole.

  The fluorescence that has passed through the pinhole array 113 passes through the second lens 111 b and reaches the dichroic mirror 112. The fluorescence is reflected by the dichroic mirror 112 and separated from the reflected light from the sample S of the excitation light that passes through the dichroic mirror 112. The reflected fluorescence is received by the PMT 116. A pixel signal corresponding to the amount of received light is generated by the PMT 116 and transmitted to the image processing apparatus 101.

  Note that the fluorescence corresponding to the excitation light pulse in each column is widely distributed on the light receiving portion of the PMT 116 via the second lens 111b and the dichroic mirror 112. A PMT 116 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 pulse detected by the position detection unit 117 is transmitted to the image processing apparatus 101 as position information as described below. 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. Further, the position detection unit 117 is transmitted with the emission end from which the excitation light pulse detected by the control unit 118 is emitted. Note that the emission end during emission may be detected by any method. For example, the control unit 118 can detect from which exit end the excitation light pulse is emitted based on the emission start time of the excitation light pulse and the elapsed time after the emission start. The position detection unit 117 detects the position of the scanning point irradiated with the excitation light pulse based on the deflection direction and the emission end during emission.

  The light source unit 120, the scanner unit 114, and the position detection unit 117 emit excitation light pulses based on the control of the control unit 118, perform scanning by the first and second galvanometer mirrors 114a and 114b, and perform excitation. The irradiation position of the light pulse is detected.

  The image processing apparatus 101 reconstructs a two-dimensional sample image 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. As described above, also in this embodiment, the fluorescence in the sample S can be incident on a pinhole other than the corresponding pinhole by scattering, that is, spatial crosstalk can occur. However, according to the scanning microscope of this embodiment, it is possible to separate the irradiation time of the multi-beams emitted from a plurality of emission ends onto the sample S and the generation time of fluorescence from the sample S. By separating the generation time of fluorescence, detection of fluorescence at another irradiation position is prevented even if spatial crosstalk occurs, so that a low-noise sample image can be reconstructed. In addition, since the sample is scanned by four multi-beams, the scanning speed can be improved while using an existing galvanometer mirror. Therefore, a sample image can be collected at high speed.

  Further, according to the scanning microscope of the first embodiment, the first to fourth emission ends t1 to t4 are arranged as described above to control the scanner unit 114 in a configuration in which the irradiation timing is separated. It is possible to maximize the frame rate while simplifying. For example, as a configuration different from the present embodiment, the excitation light pulses emitted from the first, second, third, and fourth emission ends t1, t2, t3, and t4 are (4n-3) th (4n- 2) A configuration in which scanning is performed along the (4n−1) th and 4nth scanning lines (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 114 can be kept constant regardless of the deflection direction of the first galvanometer mirror 114a, and thus the control of the scanner unit 114 is simple. It becomes.

  Further, according to the scanning microscope of the first embodiment, it is possible to effectively use the fluorescence received by the PMT 116 by arranging the first to fourth emission ends t1 to t4 as described above. is there. When the first to fourth emission ends t1 to t4 are not arranged on a straight line perpendicular to the first corresponding direction, as shown in FIG. 6, the excitation light pulses of each column along the first scanning direction The scanning range changes. Therefore, when reproducing as a rectangular image, a region that is not used for image reproduction (see the shaded area) is also scanned, and fluorescent light that is not used may be received. On the other hand, according to the configuration of the present embodiment, only the region used for image reproduction is scanned, so that it is possible to effectively utilize the received fluorescence.

  Further, according to the scanning microscope of the first embodiment, the excitation light pulse irradiated on the sample S is provided by providing the pinhole array 113 between the second lens 111b and the scanner unit 114 as described above. It is possible to share a pinhole for limiting and a pinhole for obtaining a confocal effect. The configuration can be simplified by sharing the pinhole.

  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 configuration between the light source unit 120 and the pinhole array 113 and between the dichroic mirror 112 and the PMT 116. 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. 7, the light source unit 120, the third to fifth lenses 111c to 111e, the dichroic mirror 112, the pinhole array 113, the scanner unit 114, and the objective lens in the scanning microscope 200 to which the second embodiment is applied. 115, the position detection unit 117, and the control unit 118 have the same functions and configurations as 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.

  In the second embodiment, first and second lens arrays 211a and 211b are provided instead of the first and second lenses 111a and 111b. Four lenses are provided in the first and second lens arrays 211a and 211b. The excitation light pulses in each column are converted into parallel light by passing through the four lenses in the first lens array 211a. The parallel light passes through the dichroic mirror 112 and passes through the four lenses in the second lens array 211b, thereby being condensed in the pinhole formed in the pinhole array 113.

  As in the first embodiment, the fluorescence generated in the sample S reaches the pinhole array 113 by the excitation light pulse of each column that has passed through the pinhole. Unlike the first embodiment, the fluorescence that has passed through the pinhole is converted into parallel light by the second lens array 211b. The converted parallel light is separated by the dichroic mirror 112 and reflected toward the PMT 116. A condensing lens 119 is provided between the dichroic mirror 112 and the PMT 116. The condensing lens 119 condenses the fluorescence corresponding to each column at one point of the PMT 116.

  Similar to the first embodiment, a pixel signal corresponding to the amount of received light is generated in the PMT 116 and transmitted to the image processing apparatus 101. Further, as in the first embodiment, the image processing apparatus 101 reconstructs a two-dimensional sample image based on the pixel signal and the position signal transmitted from the position detection unit 117.

  Even with the scanning microscope of the second embodiment configured as described above, a low-noise image can be acquired at high speed. Similarly to the first embodiment, the scanning microscope of the second embodiment can also achieve the highest frame rate, effective use of fluorescence, and simplification of the configuration.

  Furthermore, unlike the first embodiment, the fluorescence emitted from the pinhole array 113 and transmitted through the second lens array 211b is emitted in a parallel direction regardless of the emission end from which the excitation light pulse is emitted. Since the light is emitted in a parallel direction, it is possible to collect light at one point of the PMT 116 by using the condensing lens 119. Therefore, unlike the first embodiment, it is possible to use the PMT 116 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 and second embodiments described above, the dichroic mirror 112 is used to separate fluorescence, but the reflected light of the sample is obtained using a configuration in which a polarizing beam splitter and a λ / 4 plate are combined. May be separated and received by the PMT 116. Any optical separator that transmits light emitted from the light source and separates at least one of reflected light and fluorescence from the light emitted from the light source may be used.

  In the first and second embodiments, the scanner unit 114 is configured to scan the sample by deflecting the excitation light pulse. However, a plurality of excitation light pulses are sampled using a stage scanner that displaces the sample. It may be configured to be scanned by moving it relative to.

  In the first and second embodiments, the delay unit 124 is formed by changing the length of the optical waveguide in the light source unit 120, but any conventionally known delay unit may be used. For example, a delay device having a function similar to that of the present embodiment can be formed by combining a mirror and a half mirror.

  In the first and second embodiments, the excitation light pulse is generated by modulating the excitation light emitted from the LD 121. However, the excitation light source is driven to emit the excitation light pulse. May be. In the first and second embodiments, the pumping light pulse is branched into four rows using the branching device 123, but four light sources are provided directly without using the branching device 123, and the pumping light pulse is provided. The structure which emits may be sufficient. If the light sources are provided separately, the same effects as those of the first and second embodiments can be obtained even when the delay device 123 is omitted and the emission timing of the excitation light pulse is shifted.

  In the first and second embodiments, the scanning point of the excitation light pulse is detected based on the rotational speeds of the first and second galvanometer mirrors 114a and 114b. The structure to detect may be sufficient. 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 and second embodiments, the LD 121 is configured to emit excitation light, but is configured to reconstruct a two-dimensional image of a sample based on reflected light using a light source that emits normal visible light. It may be.

  In the first embodiment, the dichroic mirror 112 is provided between the first and second lenses 111a and 111b. However, the dichroic mirror 112 is provided at any position between the first lens 111a and the objective lens 115. May be. However, the fluorescence detected by the PMT 116 needs to be limited by the pinhole, and the fluorescence received by the PMT 116 by the dichroic mirror 112 needs to pass through the pinhole. Therefore, when the dichroic mirror 112 is provided closer to the objective lens 115 than the pinhole array 113, it is necessary to provide the pinhole array 113 at a position conjugate with the focal point of the objective lens 115 in the reflection direction of the dichroic mirror 112. .

DESCRIPTION OF SYMBOLS 100, 200 Scanning microscope system 101 Image processing apparatus 102 Monitor 110 Scanning microscope 111a-111d 1st-4th lens 112 Dichroic mirror 113 Pinhole array 114 Scanner unit 114a, 114b 1st, 2nd galvanometer mirror 115 Objective Lens 116 PMT
117 Position detector 119 Condenser lens 120 Light source unit 121 LD
122 modulator 123 branching device 124 delay device 124a to 124d first to fourth optical waveguides 125 position restrictors 211a and 211b first and second lens arrays s samples t1 to t4 first to fourth emission ends

Claims (11)

A light source unit that repeatedly emits light pulses sequentially from a plurality of emission points;
An objective lens that separately focuses the light pulses emitted from the plurality of emission points on a sample;
A scanner unit for moving the scanning points of the plurality of light pulses collected by the objective lens relative to the sample;
A pinhole array in which a pinhole is formed at a position conjugate with a condensing point of the optical pulse collected by the objective lens;
An optical separator provided on an optical path of the light pulse between the light source unit and the objective lens, and separating signal light emitted from the sample by irradiation of the light pulse from the light pulse;
A light amount detector for detecting the light amount of the signal light separated by the optical separator;
A position detection system for detecting the scanning point at the time of detection of light quantity by the light quantity detector;
An image reconstruction device that reconstructs an image of the sample based on the light amount and the scanning point includes an output device that outputs the light amount and the irradiation position.   2. The scanning microscope according to claim 1, wherein the scanner unit includes a deflector that is provided between the light source unit and the objective lens and changes a deflection direction of the light pulse.   3. The scanning microscope according to claim 1, wherein the light source unit emits the light pulse from the plurality of emission points so that chief rays are parallel to each other. microscope.   4. The scanning microscope according to claim 3, further comprising a first optical system that emits the principal rays of the optical pulses emitted from the plurality of emission points so as to be kept parallel.   5. The scanning microscope according to claim 4, wherein each of the incident positions of the light pulses corresponding to the plurality of emission points emitted from the first optical system matches the position of the pinhole in the pinhole array. A scanning microscope characterized by being arranged. In the scanning microscope according to claim 4 or 5,
A condensing lens for condensing the signal light separated by the optical separator at one point of the light amount detector;
The first optical system is collimated by a first lens group that is provided at an incident position of each of the light pulses emitted from the plurality of emission points and that collimates the light pulse, and the first lens group. A second lens group for condensing each light pulse in the pinhole,
The scanning microscope, wherein the optical separator is provided between the first and second lens groups. In the scanning microscope of any one of Claims 1-6,
The scanner unit is capable of scanning the sample with the light pulse along at least a first scanning direction,
The exit points are arranged such that a line segment connecting any two of the exit points of the plurality of exit points is inclined from the corresponding first corresponding direction on the exit point in the first scanning direction. A scanning microscope characterized by that.   The scanning microscope according to claim 7, wherein the plurality of emission points are arranged perpendicular to the first corresponding direction. The scanning microscope according to claim 8, wherein
The scanner unit is capable of scanning the light pulse along a second scanning direction that is non-parallel to the first scanning direction;
The plurality of emission points have a scanning range along the second scanning direction of the light pulses emitted from all the emission points, and the second of the light pulses emitted from each of the emission points. A scanning microscope characterized by being arranged at equal intervals so as to be equally divided by a scanning range along a scanning direction. In the scanning microscope according to any one of claims 1 to 9,
The light source unit is
A single light source emitting the light pulse;
A branching device for branching a light pulse emitted from the light source into a plurality of light pulses;
A delay device having a plurality of optical waveguides for separately transmitting the optical pulses branched by the branching device to the plurality of emission points, and having an optical path length difference so that a propagation time is different for each of the plurality of optical waveguides When,
The positions of the emission points are defined so that the principal rays of the light pulses emitted from the plurality of emission points are parallel to each other and the plurality of emission points are arranged on a plane perpendicular to the emission direction. A scanning microscope characterized by comprising: a position defining body. A light source unit that repeatedly emits light pulses sequentially from a plurality of emission points;
An objective lens that separately focuses the light pulses emitted from the plurality of emission points on a sample;
A scanner unit for moving the scanning points of the plurality of light pulses collected by the objective lens relative to the sample;
A pinhole array in which a pinhole is formed at a position conjugate with a condensing point of the optical pulse collected by the objective lens;
An optical separator provided on an optical path of the light pulse between the light source unit and the objective lens, and separating signal light emitted from the sample by irradiation of the light pulse from the light pulse;
A light amount detector for detecting the light amount of the signal light separated by the optical separator;
A position detection system for detecting the scanning point at the time of detection of light quantity by the light quantity detector;
An image reconstruction device for reconstructing an image of the sample based on the light quantity and the scanning point;
A scanning microscope system comprising: a monitor that displays an image of the sample reconstructed by the image reconstruction device.

Images