JP6205140B2 - Scanning laser microscope equipment - Google Patents

Description

  The present invention relates to a scanning laser microscope apparatus.

  Conventionally, a scanning confocal microscope is known (see Patent Document 1). In a scanning confocal microscope, when the scanning speed is increased by a scanner, the scanner moves from when the excitation light is scanned on the specimen until the fluorescence returns due to the fluorescence delay until the fluorescence is generated in the specimen. There is a problem that it will not pass through the pinhole. On the other hand, the confocal microscope described in Patent Document 1 has an elongated shape in which the shape of the pinhole is expanded along the scanning direction of the excitation light, so that fluorescence without delay and fluorescence with delay (afterglow). ) Can pass through the pinhole.

JP-A-8-5927

  However, in the confocal microscope described in Patent Document 1, since the pinhole is expanded, light without delay such as leakage light of excitation light or reflected light from the specimen passes through the pinhole, and the image quality deteriorates. There is a problem. In addition, when trying to perform reciprocal scanning using the confocal microscope described in Patent Document 1, the direction of detection light deviation is reversed between the going and returning of the scanner, so the pinhole is expanded in both scanning directions. There is a need to. Therefore, there is a problem that the pinhole is enlarged to a size twice as large as originally required, resulting in deterioration of the confocal effect and the optical sectioning capability.

  It is an object of the present invention to provide a scanning laser microscope apparatus that can acquire a good image by preventing image deterioration during high-speed scanning.

In order to achieve the above object, the present invention provides the following means.
The first aspect of the present invention has a scanning member that can swing around a predetermined swing axis , and changes the scanning speed by deflecting the excitation light emitted from the light source by the scanning member and scanning on the specimen. Possible scanning unit, objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample, and ON arranged in a position conjugate with the focal position of the objective lens Array type that has a plurality of micro detection elements that can be individually switched on and off, and that can selectively detect the return light condensed by the objective lens and descanned by the scanning unit by the micro detection elements in the ON state a detecting unit, based on the scanning speed of the scanning unit, shifting the region of the micro-detection element for oN the positional deviation direction of descanning by the scanning unit due to the delay of the return light and / or control to extend Is a scanning laser microscope apparatus comprising and.
According to a second aspect of the present invention, there is provided a scanning member having a scanning member capable of swinging around a predetermined swinging axis, deflecting excitation light emitted from a light source by the scanning member, and scanning the sample. While irradiating the sample with the excitation light scanned by the scanning unit, the objective lens that collects the return light from the sample and ON / OFF arranged in a conjugate position with the focal position of the objective lens can be switched individually An array type detection unit capable of selectively detecting return light condensed by the objective lens and descanned by the scanning unit by the micro detection element in an ON state, and the scanning unit And a control unit that shifts and / or expands the region of the micro detection element that is turned on in the direction of de-scanning by the scanning unit due to the delay of the return light in response to a change in the scanning direction. It is over The microscope apparatus.

  According to the present invention, when the excitation light emitted from the light source is scanned by the scanning unit and irradiated onto the sample by the objective lens, return light such as fluorescence generated in the sample or reflected light from the sample is transmitted by the objective lens. The light is condensed, descanned by the scanning unit, and detected by the micro detection element in the ON state of the array type detection unit. Since each micro detection element is arranged at a position conjugate with the focal position of the objective lens, by turning on only the micro detection element arranged at the collection position of the return light, the focus detection position of the objective lens on the specimen is changed. Only light can be detected accurately.

  In this case, if the scanning speed of the excitation light by the scanning unit is increased, the delay of the fluorescence from when the excitation light is scanned on the specimen to the generation of fluorescence in the specimen and the optical path length difference of the reflected light with respect to the optical path of the excitation light The fluorescence and reflected light are delayed due to the influence of the above. For this reason, the scanning member of the scanning unit may oscillate before the return light returns, and the position where the return light is descanned may be shifted, resulting in a shift in the confocal position of the return light.

  On the other hand, the control unit shifts the region of the micro detection element that is turned on based on the scanning speed and / or the scanning direction of the scanning unit in the direction of the positional deviation of the descanning by the scanning unit due to the delay of the return light and / or Alternatively, by performing the expansion control freely, it is possible to accurately detect light from the focal position of the objective lens on the specimen even if the return light is delayed. As a result, it is possible to prevent image degradation during high-speed scanning and obtain a good image.

In the second aspect , the control unit shifts the region of the minute detection element that is turned on in accordance with the change in the scanning direction of the scanning unit, and the direction in which the scanning unit descans due to the delay of the return light. And, when the scanning unit scans the excitation light in a reciprocating manner of the swinging motion of the scanning member, the region of the micro detection element that is turned on is changed depending on whether the scanning member swings or returns. The direction may be reversed.
By configuring in this way, it is possible to detect the return light from the sample scanned with the excitation light in the reciprocating motion of the scanning member while maintaining the predetermined confocal effect and prevent the occurrence of fringe noise. Can do.
In the aspect described above, the control unit further shifts the region of the minute detection element that is turned on in accordance with the change in the scanning direction of the scanning unit to the direction in which the scanning unit descans due to the delay of the return light. In addition, when the scanning unit scans the excitation light in a reciprocating manner of the swinging motion of the scanning member, the region of the micro detection element that is turned on when the scanning member swings and returns is expanded. It is good also as reversing the direction to do.
In the second aspect, the control unit shifts the region of the minute detection element that is turned on in accordance with the change in the scanning direction of the scanning unit, and the direction in which the scanning unit descans due to the delay of the return light. In addition, when the scanning unit scans the excitation light in a reciprocating manner of the swinging motion of the scanning member, the region of the micro detection element that is turned on when the scanning member swings and returns is expanded. It is good also as reversing the direction to do.
In the first aspect, the region of the micro detection element that is turned on may be shifted in the direction of the descan position by the scanning unit due to the delay of the return light based on the scanning speed of the scanning unit.

In the first aspect and the second aspect , the scanning unit is a resonant scanner that is driven by a sine wave and has a high scanning speed near the center of the scanning range , and a low scanning speed near the end of the scanning range , and the control The unit may further shift and / or expand the area of the minute detection element that is turned on based on the scanning speed that changes in accordance with the swing angle of the scanning member.
Resonant scanner is a sinusoidal driving, near the center of the scanning range has a high scan rate, while the vicinity of an end of the scanning range that a slow scan rate. Therefore, with this configuration, when detecting return light from the vicinity of the center of the scanning range, the amount of shifting and / or expanding the area of the micro detection element that is turned on is increased, while the vicinity of the end of the scanning range is increased. In the case of detecting the return light from, a good image can be obtained over the entire scanning range by reducing the amount of shifting and / or the amount of expansion of the region of the micro detection element to be turned on.

In the first aspect and the second aspect , a light source that oscillates a pulsed laser beam is provided, and the control unit further shifts a region of the microdetection element that is turned on based on characteristics of a fluorescent label applied to the specimen and / or Or it is good also as extending.
The fluorescence delay characteristics are different for each fluorescent label. Therefore, with this configuration, when detecting fluorescence from a fluorescent label having a large fluorescence delay as return light, the amount of shifting and / or expanding the area of the micro detection element to be turned on is increased, while the fluorescence delay is increased. When detecting fluorescence from a fluorescent label with a small size as return light, the amount of shift and / or expansion of the area of the micro detection element that is turned on is reduced, so that the fluorescence delay characteristics change according to the fluorescent label used. Even in this case, it is possible to continue to obtain good images.

In the first aspect , a light source that oscillates pulse laser light may be provided, and the scanning unit may be a resonant scanner, and the scanning speed may be changed according to the resonance frequency to be applied.
The resonant scanner has a high scanning speed when the applied resonance frequency is high, while the scanning speed tends to be low when the applied resonance frequency is low. Therefore, with this configuration, when the resonance frequency applied to the scanning unit is high, the amount of shifting and / or expanding the region of the micro detection element that is turned on is increased, and the resonance frequency applied to the scanning unit is increased. When the frequency is low, the amount of shifting and / or expanding the area of the micro detection element that is turned on is reduced, so that a good image can be obtained even if the resonance frequency applied to the scanning unit is changed.

In the first and second aspects, comprising a measured value detector for detecting a shape of a confocal position, and / or confocal position prior Symbol return light, wherein the control unit is further the measured values detected On the basis of the detection result of the part, the area of the micro detection element that is turned on may be shifted and / or expanded in the direction of the positional deviation of the descan by the scanning part due to the delay of the return light .

According to the present invention, the region of the micro detection element that is turned on by the control unit is shifted and / or based on the confocal position of the return light detected by the actual measurement value detection unit and / or the actual measurement value of the shape at the confocal position. By extending, the light from the focal position of the objective lens on the sample can be detected with higher accuracy even if the return light is delayed.
Therefore, for example, even when a resonant scanner is used as the scanning unit, a good image is always acquired even if the resonance frequency applied changes in temperature or the fluorescence delay characteristics change for each fluorescent label applied to the specimen. can do.

The third aspect of the present invention has a scanning member that can swing around a predetermined swinging axis , and changes the scanning speed by deflecting the excitation light emitted from the light source by the scanning member and scanning the sample. A possible scanning unit, an objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample, and is disposed at a position conjugate to the focal position of the objective lens, A light detection unit that detects return light collected by the objective lens and descanned by the scanning unit, and a plurality of minute deflections that can be individually switched ON / OFF arranged at a position conjugate to the focal position of the objective lens has a device, with a small deflection element array capable deflected return beam selectively directed to the photo detecting portion with fine small deflection element in the oN state, on the basis of the scanning speed of the scanning unit, for oN the micro The deflection element region Ri is a scanning laser microscope apparatus and a positional displacement direction shifting and / or expansion control unit of descanning by the scanning unit due to the optical delay.
According to a fourth aspect of the present invention, there is provided a scanning member having a scanning member capable of swinging around a predetermined swinging axis, and scanning the sample by deflecting the excitation light emitted from the light source by the scanning member; While irradiating the sample with the excitation light scanned by the scanning unit, the objective lens that collects the return light from the sample and the focal position of the objective lens are arranged in a conjugate position with the objective lens. A light detection unit that detects return light descanned by the scanning unit, and a plurality of minute deflection elements that can be individually switched ON / OFF arranged at a position conjugate with the focal position of the objective lens, and in an ON state A micro-deflection element array capable of selectively deflecting return light toward the photodetection unit by the micro-deflection element, and a region of the micro-deflection element that is turned on in response to a change in the scanning direction of the scanning unit. Due to the delay of the return light That is a scanning laser microscope apparatus and a shifting to the displacement direction of the descanned and / or expansion control unit according to the scanning unit.

  According to the present invention, when the excitation light emitted from the light source is scanned by the scanning unit and irradiated onto the sample by the objective lens, return light such as fluorescence generated in the sample or reflected light from the sample is transmitted by the objective lens. After being condensed and descanned by the scanning unit, it is deflected by the micro deflecting element in the ON state of the micro deflecting element array and detected by the light detecting unit. Since the micro deflection element and the light detection unit are arranged at a position conjugate with the focal position of the objective lens, the focus of the objective lens on the specimen is turned on by turning on only the micro deflection element arranged at the condensing position of the return light. Only the light from the position can be detected by accurately entering the light detection unit.

  In this case, based on the scanning speed and / or scanning direction of the scanning unit, the control unit shifts the area of the micro deflection element to be turned on in the direction of the descanning position of the scanning unit due to the delay of the return light and / or By expanding, it is possible to accurately detect and return only the return light from the focal position of the objective lens on the specimen to the light detection section even if the return light is delayed. As a result, it is possible to prevent image degradation during high-speed scanning and obtain a good image.

In the fourth aspect , the control unit shifts the region of the micro deflection element that is turned on in accordance with the change in the scanning direction of the scanning unit to the direction in which the scanning unit descans due to the delay of the return light. And, when the scanning unit scans the excitation light in a reciprocating manner of the swinging motion of the scanning member, the region of the micro deflection element that is turned ON / OFF in the swinging motion of the scanning member The direction may be reversed.
By configuring in this way, it is possible to detect the return light from the specimen scanned with the excitation light by the reciprocation of the swinging motion of the scanning member, by making it incident on the light detection unit while maintaining a predetermined confocal effect. Therefore, generation of fringe noise can be prevented.
In the aspect described above, the control unit further shifts the region of the micro deflection element that is turned on in accordance with the change in the scanning direction of the scanning unit to the direction in which the scanning unit descans due to the delay of the return light. In addition, when the scanning unit scans the excitation light by the reciprocation of the swinging motion of the scanning member, the region of the micro deflection element that is turned on when the scanning member swings and returns is expanded. It is good also as reversing the direction to do.
In the fourth aspect, the control unit shifts the region of the micro deflection element that is turned on in accordance with the change in the scanning direction of the scanning unit to the direction in which the scanning unit descans due to the delay of the return light. In addition, when the scanning unit scans the excitation light by the reciprocation of the swinging motion of the scanning member, the region of the micro deflection element that is turned on when the scanning member swings and returns is expanded. It is good also as reversing the direction to do.
In the third aspect, the control unit shifts the area of the micro deflection element that is turned on in the de-scanning position shift direction due to the delay of the return light based on the scanning speed of the scanning unit. It is good as well.

In the third aspect and the fourth aspect , the scanning unit is a resonant scanner that is driven by a sine wave and has a high scanning speed near the center of the scanning range , and a low scanning speed near the end of the scanning range , and the control The unit may further shift and / or expand the area of the micro deflection element that is turned on based on the scanning speed that changes according to the swing angle of the scanning member.
With this configuration, when detecting the return light from the vicinity of the center of the scanning range, the amount of shifting and / or expanding the area of the micro deflection element that is turned on is increased, while the amount from the vicinity of the end of the scanning range is increased. When returning light is detected, a good image can be obtained over the entire scanning range by reducing the amount of shifting and / or expanding the area of the micro deflection element to be turned on.

In the third and fourth aspects , a light source that oscillates pulsed laser light is provided, and the control unit further shifts the area of the micro deflection element that is turned on based on the characteristics of the fluorescent label applied to the specimen and / or Or it is good also as extending.
With this configuration, when detecting fluorescence from a fluorescent label with a large fluorescence delay, the amount of shifting and / or expanding the area of the micro deflection element to be turned on is increased, while the fluorescence label with a small fluorescence delay is detected. When detecting the fluorescence, the amount of displacement and / or expansion of the area of the micro deflection element that is turned on is reduced, so that a good image can be obtained even if the fluorescence delay characteristics change according to the fluorescent label used. Can continue.

In the third aspect , a light source that oscillates pulsed laser light may be provided, and the scanning unit may be a resonant scanner, and the scanning speed may be changed according to the applied resonance frequency.
With this configuration, when the resonance frequency applied to the scanning unit is high, the amount of shifting and / or expanding the region of the micro deflection element that is turned on is increased, while the resonance frequency applied to the scanning unit is low. By reducing the amount of shifting and / or expanding the area of the micro deflection element that is turned on, a good image can be obtained even if the resonance frequency applied to the scanning unit is changed.

In the above-described third embodiment and fourth embodiment, with the measured value detector for detecting a shape of a confocal position, and / or confocal position prior Symbol return light, wherein the control unit is further the measured values detected On the basis of the detection result of the part, the area of the micro deflection element that is turned on may be shifted and / or expanded in the direction of the positional deviation of the descanning by the scanning part due to the delay of the return light .

  According to the present invention, based on the confocal position of the return light detected by the actual measurement value detection unit and / or the actual measurement value of the shape at the confocal position, the region of the micro deflection element that is turned on by the control unit is shifted and / or By extending, it is possible to accurately detect light from the focal position of the objective lens on the sample and make it incident on the light detection section even if there is a delay in the return light. Thereby, it is possible to always obtain a good image.

  According to the present invention, it is possible to obtain an excellent image by preventing image deterioration during high-speed scanning.

1 is a schematic configuration diagram illustrating a scanning laser microscope apparatus according to a first embodiment of the present invention. FIG. 2 is an enlarged view of the array type detection unit in FIG. 1. (A) shows a state where the detection region is shifted, (b) shows a state where the detection region is expanded, and (c) shows a state where the detection region is shifted and expanded. (A) shows a state in which the detection area is shifted in the scanning for the swing operation of the XY galvano mirror, and (b) shows a state in which the detection area is shifted in the opposite direction in the scanning after the swing operation of the XY galvano mirror. FIG. (A) shows a state in which the detection region is expanded in the scanning for the swing operation of the XY galvano mirror, and (b) shows a state in which the detection region is expanded in the opposite direction in the scan after the swing operation of the XY galvano mirror. FIG. (A) shows a state in which the detection area is shifted and expanded in the scan for the swing operation of the XY galvano mirror, and (b) is a shift in the opposite direction in the scan after the swing operation of the XY galvano mirror. It is a figure which shows the state expanded with. (A) In the scanning laser microscope apparatus according to the first modification of the first embodiment of the present invention, when detecting fluorescence from the vicinity of the center of the scanning range, the amount of shift and the amount of expansion are increased. The state is shown, and (b) is a diagram showing a state in which the amount of shifting the detection region and the amount of expansion are reduced when detecting fluorescence from the vicinity of the end of the scanning range. (A) shows a detection region in the case of detecting fluorescence from the vicinity of the left end of the scanning range in the scanning of the XY galvanometer mirror, and (b) shows the scanning range of the scanning in the same direction of the oscillating operation. The detection area in the case of detecting the fluorescence from the vicinity of the left end is shown. (C) shows the detection area in the case of detecting the fluorescence from the vicinity of the left end of the scanning range in the same scan of the swing operation. The detection area in the case of detecting fluorescence from the vicinity of the left end of the scanning range in the return scan of the swing operation of the XY galvanometer mirror is shown, and (e) shows the fluorescence from the vicinity of the left end of the scan range in the return scan of the swing operation. (F) is a diagram showing a detection region in the case of detecting fluorescence from the vicinity of the left end of the scanning range in the return scan of the swinging operation. It is a graph which shows the relationship between the intensity | strength of the fluorescence which generate | occur | produced in the sample, and generation | occurrence | production time in the scanning laser microscope apparatus which concerns on the 2nd modification of 1st Embodiment of this invention. 6 is a graph showing the relationship between the intensity of fluorescence generated in a specimen and the amount of positional deviation of descanning by a resonant scanner in a scanning laser microscope apparatus according to a third modification of the first embodiment of the present invention. It is a figure which shows the structure which has arrange | positioned the actual value detection part and the array type | mold detection part so that replacement | exchange is possible in the scanning laser microscope apparatus which concerns on the 4th modification of 1st Embodiment of this invention. In the scanning laser microscope apparatus of FIG. 11, it is a figure which shows the structure arrange | positioned so that light can enter into both by the optical path switching mirror, without replacing an actual value detection part and an array type | mold detection part. It is a schematic block diagram which shows the scanning laser microscope apparatus which concerns on 2nd Embodiment of this invention.

[First Embodiment]
A scanning laser microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a scanning laser microscope 100 according to the present embodiment includes a laser unit 10 that generates laser light (excitation light), and a single mode fiber 11 that guides laser light emitted from the laser unit 10. Any of the collimating lens 13 that shapes the guided laser beam into parallel light, the three excitation dichroic mirrors 15A, 15B, and 15C that can reflect the shaped laser beam, and the excitation dichroic mirrors 15A, 15B, and 15C. A galvano scanner (scanning unit) 17 that two-dimensionally scans the laser beam reflected by the sample S, a pupil projection lens 19 that relays the laser beam from the galvano scanner 17, and the relayed laser beam. An imaging lens 23 that forms an image by light, and the sample S is irradiated with the condensed laser light. Fluorescence generated and an objective lens 25 for condensing. Reference numeral 21 denotes a reflection mirror that reflects the laser light relayed by the pupil projection lens 19 toward the imaging lens 23.

  The laser unit 10 can output each laser beam of the excitation wavelength to the specimen S stained with a fluorescent dye. The laser unit 10 includes, for example, an Ar laser device (light source) 1A that oscillates laser light with an excitation wavelength of 488 nm, a HeNe-G laser device (light source) 1B that oscillates a laser with an excitation wavelength of 543 nm, and a laser with an excitation wavelength of 633 nm. A HeNe-R laser device (light source) 1C that oscillates light.

  Further, the laser unit 10 includes a reflection mirror 3 that reflects the laser light emitted from the Ar laser apparatus 1A, a dichroic mirror 5 that synthesizes laser light having two wavelengths of 488 nm and 543 nm, and a wavelength of 488 nm. A dichroic mirror 7 that synthesizes laser light having three wavelengths of 543 nm and 633 nm, and an acoustooptic device (AOTF) 9 that selects laser light having an arbitrary wavelength among the wavelengths 488 nm, 543 nm, and 633 nm are provided. .

  The three excitation dichroic mirrors 15 </ b> A, 15 </ b> B, and 15 </ b> C are selectively detachably disposed on the optical path of the laser light that has passed through the collimating lens 13. Each of these excitation dichroic mirrors 15A, 15B, and 15C has a characteristic of reflecting the laser light from the collimating lens 13 and transmitting the fluorescence from the specimen S.

  Specifically, the excitation dichroic mirror 15A reflects laser beams having respective excitation wavelengths of 488 nm, 543 nm, and 633 nm, and transmits fluorescence from the specimen S excited by these laser beams. The excitation dichroic mirror 15B has a characteristic of reflecting laser light having an excitation wavelength of 488 nm and transmitting light having a wavelength longer than the excitation wavelength of 488 nm. The excitation dichroic mirror 15C has a characteristic of reflecting laser light having an excitation wavelength of 543 nm and transmitting light having a wavelength longer than the excitation wavelength 543 nm.

  These excitation dichroic mirrors 15A, 15B, and 15C are selectively used depending on the type of specimen S to be observed. For example, the excitation dichroic mirror 15A is used when performing fluorescence observation using only laser light with an excitation wavelength of 633 nm or when performing multiple fluorescence observation using a plurality of excitation wavelengths. When performing fluorescence observation using only laser light having an excitation wavelength of 488 nm, the excitation dichroic mirror 15B is used. When performing fluorescence observation using only laser light having an excitation wavelength of 543 nm, the excitation dichroic mirror 15C is used. Thereby, the fluorescence uptake efficiency can be increased.

  The galvano scanner 17 includes a pair of X galvanometer mirrors (scanning members) 17A and Y galvanometer mirrors (scanning members) 17B that can swing around swing axes orthogonal to each other. The X galvanometer mirror 17A scans the laser beam in the horizontal direction, and the Y galvanometer mirror 17B scans the laser beam in the vertical direction.

  These XY galvanometer mirrors 17A and 17B are arranged on the reflection light path of the laser beam by the excitation dichroic mirrors 15A, 15B and 15C, and each laser beam having an excitation wavelength of 488 nm, 543 nm and 633 nm is two-dimensionally oriented on the sample S ( (XY direction) can be scanned.

  The objective lens 25 irradiates the sample S with the laser light transmitted through the imaging lens 23, and condenses the fluorescence generated in the sample S when irradiated with the laser light. The fluorescence condensed by the objective lens 25 returns to the optical path of the laser light through the imaging lens 23, the reflection mirror 21, and the pupil projection lens 19, and is descanned by the galvano scanner 17 to be excited dichroic mirrors 15A and 15B on the optical path. , 15C.

  Further, the scanning laser microscope apparatus 100 includes a confocal lens 27 that condenses the fluorescence transmitted through any of the excitation dichroic mirrors 15A, 15B, and 15C, and a CMOS that detects the fluorescence collected by the confocal lens 27. An array type detection unit 30 such as an image sensor, and a control unit 31 that controls the acoustooptic device 9, the galvano scanner 17, and the array type detection unit 30 are provided.

  As shown in FIG. 2, the array-type detection unit 30 includes a plurality of minute detection elements 29 that are two-dimensionally arranged at a position conjugate with the focal position of the objective lens 25. For example, the array-type detection unit 30 is arranged so that the vicinity of the substantial center of the plurality of minute detection elements 29 is positioned at a normal fluorescence confocal position (confocal spot).

  In addition, the array type detection unit 30 switches ON / OFF of the micro detection elements 29 individually, and the fluorescence from the specimen S collected by the confocal lens 27 is selectively selected by the micro detection elements 29 in the ON state. It is detected and not detected by the micro detection element 29 in the OFF state.

  The control unit 31 operates the Ar laser device 1A, the HeNe-G laser device 1B, or the HeNe-R laser device 1C from the laser unit 10 and selectively emits laser light of that wavelength by the acoustooptic device 9. ing. The control unit 31 scans and drives the XY galvanometer mirrors 17A and 17B. For example, the control unit 31 scans the laser beam during the swing operation of the XY galvano mirrors 17A and 17B of the galvano scanner 17.

  The control unit 31 normally detects the fluorescence by turning on the minute detection element 29 arranged near the center of the array type detection unit 30. Further, the control unit 31 shifts and / or expands the area of the minute detection element 29 to be turned on in the descanning position shift direction by the galvano scanner 17 due to the fluorescence delay based on the scanning speed and the scanning direction of the galvano scanner 17. It is supposed to be. An example of the delay in fluorescence is a delay in the time from when the laser beam is scanned on the specimen S until the fluorescence is generated when the scanning speed of the laser beam by the galvano scanner 17 is increased.

The operation of the scanning laser microscope apparatus 100 configured as described above will be described.
In order to observe the specimen S with the scanning laser microscope apparatus 100 according to the present embodiment, a fluorescent label is attached to the specimen S, and the AOTF 9, the galvano scanner 17, and the array type detection section 30 are operated by the control unit 31.

  For example, the control unit 31 issues a selection command for the Ar laser apparatus 1A to the acoustooptic element 9 of the laser unit 10, and the acoustooptic element 9 selects laser light having an excitation wavelength of 488 nm output from the Ar laser apparatus 1A. And emitted from the laser unit 10.

  Laser light having an excitation wavelength of 488 nm emitted from the laser unit 10 is transmitted through the single mode fiber 11 and guided to the collimating lens 13, is shaped into parallel light by the collimating lens 13, and is reflected by the excitation dichroic mirror 15. The laser light reflected by the excitation dichroic mirror 15 is deflected by the galvano scanner 17, passes through the pupil projection lens 19, is reflected by the reflection mirror 21, and passes through the imaging lens 23 and the objective lens 25 on the sample S. It is imaged as a light spot.

  The light spot imaged on the specimen S is scanned in the horizontal direction by the X galvanometer mirror 17A of the galvano scanner 17, and then scanned by one pixel in the vertical direction by the Y galvanometer mirror 17B, and again horizontal by the X galvanometer mirror 17A. Scanning in the direction is repeated.

  When, for example, fluorescence having a center wavelength of 520 nm is generated by scanning the laser beam on the specimen S, the fluorescence is collected by the objective lens 25, and the imaging lens 23, the reflection mirror 21, the pupil projection lens 19, and the galvano scanner. The light passes through the excitation dichroic mirror 15 </ b> A via 17 </ b> A and 17 </ b> B and is collected from the confocal lens 27.

  For example, if there is no delay, the fluorescence collected from the confocal lens 27 is incident near the approximate center of the plurality of micro detection elements 29 that are the original positions of the confocal spots. Since each micro detection element 29 is two-dimensionally arranged at a position conjugate with the focal position of the objective lens 25, the sample can be obtained by turning on only the micro detection element 29 arranged at the fluorescence condensing position. Only light from the focal position of the objective lens 25 on S can be accurately detected.

  In this case, if the scanning speed of the laser light by the galvano scanner 17 is increased, the fluorescence is delayed due to the delay from when the laser light is scanned on the specimen S until the fluorescence is generated. For this reason, the XY galvano mirrors 17A and 17B of the galvano scanner 17 are swung before the fluorescence returns, so that the position where the fluorescence is descanned may be shifted and the confocal position of the fluorescence may be shifted.

  In contrast, in the present embodiment, the control unit 31 causes the galvano scanner 29 to be turned on from the vicinity of the center of the region of the micro detection element 29 that is turned on based on the scanning speed and / or the scanning direction of the galvano scanner 17. It is shifted and / or expanded in the direction of misalignment of the descan by the scanner 18.

  For example, when the position of fluorescence descanning by the galvano scanner 17 is shifted by ΔX to one side in the horizontal direction, the controller 31 causes the position shift in the descanning position shift direction from the approximate center of the plurality of micro detection elements 29. The minute detection element 29 in the region shifted by the distance is turned on. As a result, as shown in FIG. 3A, a circular detection region K is set at a position shifted by ΔX from the center of the array-type detection unit 30.

  Further, when the position of fluorescence descanning by the galvano scanner 17 is shifted by ΔL in one of the horizontal directions, for example, when the fluorescence position shift is tailed due to the fluorescence lifetime, the control unit 31 causes a plurality of minute scans. The micro-detecting element 29 in the area expanded from the approximate center of the detecting element 29 in the descanning position shift direction is turned on. Thereby, as shown in FIG. 3B, an oval detection region K extending by ΔL from the center of the array-type detection unit 30 is set.

Furthermore, when the position of the fluorescence descanning by the galvano scanner 17 is greatly shifted in one of the horizontal directions, for example, when the fluorescence position shift is tailed due to the fluorescence lifetime, the control unit 31 performs a plurality of minute detections. The micro-detecting element 29 in the area shifted (for example, ΔX) from the approximate center of the element 29 (for example, ΔX) and expanded (for example, ΔL) is turned on. Thereby, as shown in FIG. 3C, an ellipse-shaped detection region K that is shifted by ΔX from the center of the array-type detection unit 30 and further extended by ΔL is set.
Thereby, even if the fluorescence is delayed, the light from the focal position of the objective lens 25 on the sample S can be detected with high accuracy.

  As described above, according to the scanning laser microscope apparatus 100 according to the present embodiment, even if a position shift occurs in the descan of the galvano scanner 17 due to the fluorescence delay during high-speed scanning, the array type is placed at the confocal position of the delayed fluorescence. The detection region K of the detection unit 30 can be shifted and / or expanded to detect light from the focal position of the objective lens 25 on the sample S with high accuracy. Accordingly, it is possible to acquire a good confocal image while preventing image deterioration during high-speed scanning.

  In the present embodiment, the micro detection elements 29 are arranged two-dimensionally. Instead, the micro detection elements 29 are arranged one-dimensionally at a position conjugate with the focal position of the objective lens 25. It is good to do.

  In this embodiment, the laser beam is scanned during the oscillating operation of the XY galvano mirrors 17A and 17B. Instead, for example, the XY galvano mirrors 17A and 17B are oscillated. It is good also as scanning a laser beam by reciprocation. In this case, the control unit 31 reverses the direction of shifting and / or expanding the region of the micro detection element 29 that is turned on between the return and return of the XY galvano mirrors 17A and 17B.

  For example, when the position of the fluorescence descan by the galvano scanner 17 is shifted by ΔX in the horizontal direction, the control unit 31 detects the fluorescence generated by the scanning of the XY galvanometer mirrors 17A and 17B. As shown in FIG. 4A, a circular detection region K in which the region of the minute detection element 29 to be turned ON is shifted in one direction from the substantially center is set, and the return of the swing operation of the XY galvanometer mirrors 17A and 17B is set. For fluorescence generated by scanning, as shown in FIG. 4B, a circular detection region K is set in which the region of the minute detection element 29 that is turned on is shifted in one direction from the approximate center.

  Further, for example, when the position of the fluorescence descan by the galvano scanner 17 is shifted by ΔL in the horizontal direction, and the position of the fluorescence is shifted due to the fluorescence lifetime, the controller 31 causes the XY galvano mirror 17A, For the fluorescence generated by the scanning of the swing movement of 17B, as shown in FIG. 5 (a), an oval detection region K in which the region of the minute detection element 29 that is turned on is expanded in one side in the horizontal direction. Is set, and the region of the minute detection element 29 that is turned on is located on the other side in the horizontal direction, as shown in FIG. 5B, for the fluorescence generated by the scanning of the return movement of the XY galvanometer mirrors 17A and 17B. An expanded oval detection region K is set.

  Further, for example, when the position of the descan by the galvano scanner 17 is greatly deviated to one side in the horizontal direction, and the position of the fluorescence shifts due to the fluorescence lifetime, the control unit 31 causes the XY galvano mirrors 17A and 17B. As shown in FIG. 6A, the region of the micro detection element 29 that is turned ON is further expanded by shifting the position to one side in the horizontal direction. The detection region K is set, and the region of the micro detection element 29 that is turned on is horizontal as shown in FIG. 6B with respect to the fluorescence generated by the return scanning of the XY galvanometer mirrors 17A and 17B. An ellipse-shaped detection region K that is further expanded by shifting the position in the other direction is set.

  By doing so, it is possible to detect the fluorescence from the sample S scanned with the laser light in the reciprocating motion of the XY galvanometer mirrors 17A and 17B while maintaining a predetermined confocal effect. Thereby, generation | occurrence | production of fringe noise can be prevented.

This embodiment can be modified as follows.
In the present embodiment, the galvano scanner 17 is exemplified as the scanning unit, but as a first modification, a resonant scanner (not shown) may be employed as the scanning unit.

  In this case, the control unit 31 shifts and / or expands the region of the micro detection element 29 that is turned on in the descanning position shift direction by the galvano scanner 17 based on the scanning speed, scanning direction, and swing angle of the resonant scanner. Is desirable. In this way, a detection area corresponding to the scanning speed distribution of the resonant scanner can be set for each scanning range on the sample S.

  That is, the resonant scanner is normally sine wave driven, and the scanning speed tends to be high near the center of the scanning range, while the scanning speed tends to be low near the end of the scanning range. Therefore, according to this modification, for example, as shown in FIG. 7A, when detecting fluorescence from the vicinity of the center of the scanning range, the amount of the micro detection element 29 to be turned on is shifted and / or expanded. A detection region K having an increased amount is set. Further, for example, as shown in FIG. 7B, when detecting fluorescence from the vicinity of the end of the scanning range, a detection region K in which the amount of shifting and / or extending the region of the micro detection element 29 to be turned on is reduced. Set. Thereby, a good confocal image can be obtained over the entire scanning range. In FIG. 7A and FIG. 7B, Xn> X0 and Ln> L0. The same applies to FIGS. 8A to 8F.

  Further, in this modification, when the laser beam is scanned by the reciprocation of the swinging operation of the resonant scanner, as shown in FIGS. A detection region K in which the direction of shifting the region of the minute detection element 29 to be shifted and / or the direction of expansion is reversed is set. 8 (a) to 8 (c) show changes in the detection region K set for the fluorescence generated by the forward scanning of the swing operation, and FIGS. 8 (d) to 8 (f). The change of the detection region K set with respect to the fluorescence which generate | occur | produced by the scanning of the return of rocking | fluctuation operation is shown.

  As a second modified example, the Ar laser device 1A emits a short-time pulse laser beam (excitation laser pulse), or a pulsed laser beam (excitation laser) is arranged by arranging a pulse laser (not shown) instead of the Ar laser device 1A. The control unit 31 shifts and / or expands the area of the micro detection element 29 that is turned on based on the scanning speed and scanning direction of the galvano scanner 17 and the characteristics of the fluorescent label applied to the specimen S. It is good as well.

  The fluorescence delay characteristics are different for each fluorescent label. According to this modification, for example, when detecting fluorescence from a fluorescent label with a large fluorescence delay, the amount of shifting and / or expanding the area of the micro detection element 29 to be turned on is increased while the fluorescence with a small fluorescence delay is detected. When detecting fluorescence from the label, the amount of shifting and / or expanding the region of the micro detection element 29 to be turned on is reduced. Thereby, even if the fluorescence delay characteristic changes according to the fluorescent label to be used, a good confocal image can be continuously acquired.

  Further, as shown in FIG. 9, the fluorescence decay time varies with the fluorescence delay characteristic for each fluorescent label. Therefore, the fluorescence without delay is incident near the center of the array-type detection unit 30 that is the original position of the confocal spot, but the confocal spot tends to move and become elongated according to the fluorescence delay characteristics and decay time. There is. In FIG. 9, the vertical axis indicates the fluorescence intensity (I), and the horizontal axis indicates time (t).

  According to this modification, the optimum detection region according to the fluorescence delay characteristic and the decay time is changed by changing the amount of shifting and / or the amount of expansion of the region of the micro detection element 29 that is turned on based on the property of the fluorescent label. Can be set. Therefore, since the amount of fluorescent light can be taken without waste, the brightness is improved and the deterioration of image quality can be prevented.

  As a third modified example, the Ar laser device 1A irradiates a short-time pulse laser beam (excitation laser pulse) or arranges a pulse laser (not shown) in place of the Ar laser device 1A to provide a pulse laser beam (excitation laser). It is also possible to employ a resonant scanner (not shown) instead of the galvano scanner 17 as the scanning unit, and to change the scanning speed of the resonant scanner in accordance with the resonance frequency to be applied.

  As shown in FIG. 10, the resonant scanner has a high scanning speed when the applied resonance frequency is high, while the scanning speed tends to be slow when the applied resonance frequency is low. In FIG. 10, the vertical axis indicates the fluorescence intensity (I), and the horizontal axis indicates the amount of displacement (X) in the fluorescence descanning by the resonant scanner.

  According to this modification, for example, fluorescence without delay enters near the center of the array-type detection unit 30 that is the original position of the confocal spot, so the minute detection element 29 near the center is turned on. On the other hand, when the resonance frequency applied to the resonant scanner is high, the amount of shifting and / or expanding the region of the micro detection element 29 that is turned on is increased, while when the resonance frequency applied to the resonant scanner is low, the micro detection that is turned on. The amount by which the region of the element 29 is shifted and / or the amount to be expanded is reduced. Therefore, a good confocal image can be obtained even if the resonance frequency (for example, 4 kHz, 8 kHz, 16 kHz) applied to the resonant scanner is changed.

  In the present embodiment, the control unit 31 shifts and / or expands the area of the micro detection element 29 that is turned on based on the scanning speed and the scanning direction of the galvano scanner 17, but as a fourth modification example, For example, as shown in FIG. 11, the scanning laser microscope apparatus 100 includes an actual value detection unit 33 that detects the confocal position of the fluorescence and the shape at the confocal position, and the control unit 31 detects the actual value. Based on the detection result of the unit 33, the area of the micro detection element 29 to be turned on may be shifted and / or expanded in the direction of the position shift of the descan by the galvano scanner 17 due to the fluorescence delay.

  In this case, as the actual measurement value detection unit 33, a PSD (Position Sensitive Detector) sensor or an image sensor arranged one-dimensionally or two-dimensionally may be employed. Further, the actual measurement value detection unit 33 is replaced with the array type detection unit 30 and disposed at a position conjugate with the focal position of the objective lens 25, and the position and / or shape of the confocal spot when the galvano scanner 17 is scanned at high speed is actually measured. What is necessary is just to detect with the detection part 33. FIG.

  When the detection by the actual value detection unit 33 is completed, the array type detection unit 30 is arranged at a position conjugate with the focal position of the objective lens 25 instead of the actual value detection unit 33, and the actual value detection unit is controlled by the control unit 31. What is necessary is just to turn ON the micro detection element 29 of the area | region corresponding to the position and / or shape of the confocal spot detected by 33. FIG.

  By doing so, delayed fluorescence can be detected with higher accuracy. Therefore, for example, when a resonant scanner is used as the scanning unit, even if the resonance frequency applied changes in temperature, or the fluorescence delay characteristic changes for each fluorescent label applied to the sample S, it is always good confocal. Images can be acquired.

  In this modification, the array type detection unit 30 and the actual measurement value detection unit 33 are replaced. For example, as shown in FIG. 12, on the optical path between the confocal mirror 27 and the array type detection unit 30. An optical path switching mirror 35 that can switch the optical path is arranged, a confocal lens 37 is arranged in a new optical path that is switched by the optical path switching mirror 35, and the actual value detection unit 33 is located at a position conjugate with the focal position of the objective lens 25. It is good also as arranging.

  Even in this case, the position and / or shape of the confocal spot of the light in the new optical path switched by the optical path switching mirror 35 during the high-speed scanning of the galvano scanner 17 is detected by the actual measurement value detection unit 33, and the control unit 31 Then, the micro detection element 29 in the region corresponding to the position and / or shape of the confocal spot detected by the actual measurement value detection unit 33 is turned on, and the fluorescence of the original optical path switched by the optical path switching mirror 35 is detected by the array type detection unit. By detecting with 30, a good confocal image can always be acquired.

[Second Embodiment]
Next, a scanning laser microscope apparatus 200 according to the second embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 13, the scanning laser microscope apparatus 200 according to the present embodiment replaces the array type detection unit 30 with a minute deflection mirror array (minute deflection element array) 121, a reflection mirror 123, and a spectral dichroic mirror. 125, barrier filters 127A and 127B, a first CH side photodetector (light detection unit) 129A, and a second CH side photodetector (light detection unit) 129B, which are different from the first embodiment.
In the following, portions having the same configuration as those of the scanning laser microscope apparatus 100 according to the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  The micro deflection mirror array 121 includes a plurality of micro deflection mirrors (micro deflection mirrors, illustrated in the drawing) that are two-dimensionally arranged at the imaging position of the confocal lens 27, that is, at a position conjugate with the sample S via the confocal lens 27. The angle of these minute deflection mirrors can be freely changed. The minute deflection mirror is formed in a 10 μm square, for example.

  In this micro deflection mirror array 121, the micro deflection mirror is individually switched between the ON state angle and the OFF state angle, and the fluorescence is selectively deflected toward the photodetectors 129A and 129B by the micro deflection mirror in the ON state. It can be changed in the other direction by the micro deflection mirror in the OFF state.

The reflection mirror 123 reflects the fluorescence deflected toward the photodetectors 129 </ b> A and 129 </ b> B by the micro deflection mirror array 121 toward the spectroscopic dichroic mirror 125.
The spectroscopic dichroic mirror 125 transmits or reflects incident fluorescence according to the wavelength, for example, fluorescence having a wavelength shorter than the wavelength of 570 nm (fluorescence acquired by excitation with an excitation wavelength of 488 nm) and wavelength longer than the wavelength of 570 nm. And fluorescence (fluorescence obtained by excitation at an excitation wavelength of 543 nm or 633 nm).

Each of the barrier filters 127A and 127B cuts the reflected light of the laser light out of the light from the spectroscopic dichroic mirror 125 and allows only the fluorescence to pass therethrough.
The first CH side photodetector 129 </ b> A and the second CH side photodetector 129 </ b> B are respectively disposed at positions conjugate to the focal position of the objective lens 25. These photodetectors 129 </ b> A and 129 </ b> B are configured to send a light intensity signal corresponding to the detected fluorescence intensity to the control unit 31.

  The controller 131 controls the reverberation optical element 9, the galvano scanner 17 and the minute deflection mirror array 121. The control unit 131 normally turns on a minute deflection mirror arranged near the center of the minute deflection mirror array 121 to selectively deflect fluorescence toward the photodetectors 129A and 129B. . In addition, the control unit 131 shifts and / or expands the region of the micro deflection mirror that is turned on in the de-scanning position shift direction by the galvano scanner 17 due to fluorescence delay based on the scanning speed and scanning direction of the galvano scanner 17. It is like that.

  In addition, the control unit 131 integrates the light intensity signals transmitted from the photodetectors 129A and 129B for each pixel corresponding to the swing angle of the XY galvano mirrors 17A and 17B of the galvano scanner 17, thereby obtaining an image of the sample S. It is designed to generate.

The operation of the scanning laser microscope apparatus 200 configured as described above will be described.
When the specimen S is observed by the scanning laser microscope apparatus 200 according to the present embodiment, the fluorescence generated in the specimen S, descanned by the galvano scanner 17 and incident on the confocal lens 27 is condensed by the confocal lens 27. Then, an image is formed as a light spot on the minute deflection mirror array 121.

  The fluorescence imaged on the micro deflection mirror array 121 is incident near the approximate center of a plurality of micro deflection mirrors, which is the original position of the confocal spot, for example, if there is no delay. Since each micro deflection mirror is two-dimensionally arranged at a position conjugate with the focal position of the objective lens 25, by turning on only the micro deflection mirror arranged at the fluorescence condensing position, the sample S Only the light from the focal position of the objective lens 25 can be deflected toward the photodetectors 129A and 129B.

  When the fluorescent light is selectively deflected toward the photodetectors 129A and 129B by the minute deflection mirror in the ON state, the fluorescent light is branched according to the wavelength by the spectroscopic dichroic mirror 125 via the reflection mirror 123. For example, fluorescence having a wavelength shorter than 570 nm passes through the barrier filter 127A and is detected by the 1CH side photodetector 129A, and fluorescence having a wavelength longer than 570 nm passes through the barrier filter 127B and is detected by the 2CH side. Detected by instrument 129B.

  Since the photodetectors 129A and 129B are arranged at positions conjugate to the focal position of the objective lens 25, the focal points of the objective lens 25 on the specimen S selectively deflected by the micro deflection mirror array 121 in the ON state. Only light from the position can be detected.

  When fluorescence is detected by the photodetectors 129A and 129B, the light intensity signal is sent to the control unit 31. In the control unit 31, the light intensity signals transmitted from the photodetectors 129A and 129B are integrated for each pixel corresponding to the swing angle of the XY galvano mirrors 17A and 17B of the galvano scanner 17 to generate an image of the sample S. Is done.

  In this case, based on the scanning speed and scanning direction of the galvano scanner 17, the control unit 31 shifts the area of the micro deflection mirror that is turned on in the descanning position shift direction of the galvano scanner 17 due to fluorescence delay and / or. By extending, even if the fluorescence is delayed, only the fluorescence from the focal position of the objective lens 12 on the specimen S can be accurately incident on the photodetectors 129A and 129B and detected. Therefore, according to the scanning laser microscope apparatus 200 according to the present embodiment, it is possible to prevent image deterioration during high-speed scanning and obtain a good confocal image.

  In the present embodiment, the minute deflection mirror is arranged two-dimensionally. Instead, the minute deflection mirror is arranged one-dimensionally at a position conjugate with the focal position of the objective lens 25. It is good.

  In the present embodiment, the laser beam may be scanned by reciprocation of the swinging motion of the XY galvanometer mirrors 17A and 17B. In this case, similarly to the region of the micro detection element 29 in the ON state that forms the detection region of FIGS. 4 (a), 4 (b), 5 (a), 5 (b), 6 (a), 6 (b). Then, the deflection element control unit 131 reverses the direction of shifting and / or expanding the area of the minute deflection mirror that is turned on between the return and return of the XY galvanometer mirrors 17A and 17B.

  By doing so, the fluorescence from the specimen S scanned with the laser light by the reciprocation of the swinging movement of the XY galvanometer mirrors 17A and 17B is made incident on the photodetectors 129A and 129B while maintaining a predetermined confocal effect. Can be detected. Thereby, generation | occurrence | production of fringe noise can be prevented.

This embodiment can be modified as follows.
In the present embodiment, the galvano scanner 17 is exemplified as the scanning unit, but as a first modification, a resonant scanner (not shown) may be employed as the scanning unit.

  In this case, similarly to the region of the micro detection element 29 in the ON state that forms the detection region in FIGS. 7A, 7B, and 8A to 8C, the control unit 131 scans the resonant scanner. It is desirable to shift and / or expand the area of the minute deflection mirror to be turned on in the direction of the position deviation of the descan by the galvano scanner 17 based on the speed, the scanning direction, and the swing angle. In this way, only the fluorescence from the focal position of the objective lens 12 on the specimen S is accurately incident on the photodetectors 129A and 129B for each scanning range on the specimen S according to the scanning speed distribution of the resonant scanner. Can be made.

  According to this modification, when detecting fluorescence from the vicinity of the center of the scanning range by the resonant scanner, the amount of shifting and / or expanding the area of the micro deflection mirror to be turned on is increased, while from the vicinity of the end of the scanning range. In the case of detecting the fluorescent light, it is possible to obtain a good confocal image over the entire scanning range by reducing the amount of shifting and / or expanding the amount of the micro deflection mirror to be turned on.

  As a second modified example, the Ar laser device 1A emits a short-time pulse laser beam (excitation laser pulse), or a pulsed laser beam (excitation laser) is arranged by arranging a pulse laser (not shown) instead of the Ar laser device 1A. The control unit 131 shifts and / or expands the region of the micro deflection mirror that is turned on based on the scanning speed and scanning direction of the galvano scanner 17 and the characteristics of the fluorescent label applied to the specimen S. It is good.

  According to this modification, when detecting fluorescence from a fluorescent label having a large fluorescence delay, the amount of shifting and / or expanding the area of the micro deflection mirror to be turned on is increased, while the amount from the fluorescent label having a small fluorescence delay is increased. When detecting the fluorescence, the amount of displacement and / or expansion of the area of the micro deflection mirror that is turned on is reduced to obtain a good confocal image even if the fluorescence delay characteristics change according to the fluorescent label used. Can continue. Further, according to this modification, only the fluorescence from the focal position of the objective lens 12 on the sample S can be accurately incident on the photodetectors 129A and 129B according to the fluorescence delay characteristics and decay time.

  As a third modified example, the Ar laser device 1A irradiates a short-time pulse laser beam (excitation laser pulse) or arranges a pulse laser (not shown) in place of the Ar laser device 1A to provide a pulse laser beam (excitation laser). It is also possible to employ a resonant scanner (not shown) instead of the galvano scanner 17 as the scanning unit, and the control unit 131 may change the scanning speed of the resonant scanner in accordance with the applied resonance frequency.

  According to this modification, when the resonance frequency applied to the resonant scanner is high, the amount of shifting and / or expanding the area of the micro deflection array to be turned on is increased, while when the resonance frequency applied to the resonant scanner is low. The amount of shifting and / or the amount of expansion of the area of the micro deflection array to be turned on is reduced. Therefore, even if the resonance frequency (for example, 4 kHz, 8 kHz, 16 kHz) applied to the resonant scanner is changed, a good image can be obtained.

  As a fourth modification, the scanning laser microscope apparatus 200 is actually measured like a PSD sensor that detects the fluorescence confocal position and the shape at the confocal position or an image sensor that is arranged one-dimensionally or two-dimensionally. A value detector (not shown), and based on the detection result of the actual value detector, the controller 131 moves the region of the micro deflection mirror that is turned on in the direction of the descan position by the galvano scanner 17 due to the fluorescence delay. It may be shifted and / or expanded.

In this case, similarly to the array-type detection unit 30 in FIG. 11, the actual measurement value may be detected by replacing the actual measurement value detection unit with the minute deflection mirror array 121. Similarly to the array-type detection unit 30 in FIG. 12, an optical path switching mirror (not shown) that can switch the optical path is used to switch the minute deflection mirror array 121 while leaving the original optical path by the optical path switching mirror. A confocal lens (not shown) may be arranged on a new optical path, and an actual measurement value detection unit may be arranged at a position conjugate with the focal position of the objective lens 25 to detect the actual measurement value.
In this way, even if the fluorescence is delayed, the light from the focal position of the objective lens 25 on the sample S can be accurately incident on the photodetectors 129A and 129B, and can be detected. Images can be acquired.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to each of the above embodiments, and may be applied to embodiments in which these embodiments are appropriately combined, and is not particularly limited.

  In the above embodiment, the detection area of the array type detection unit 30 is changed based on the scanning speed and the scanning direction of the galvano scanner 17 and the resonant scanner. Instead, the galvano scanner 17 and the resonant scanner are used. The detection area of the array type detection unit 30 may be changed based on the scanning speed or the scanning direction.

  In the above embodiment, the case where fluorescence generated in the specimen S is detected as the return light has been described as an example. Instead, the reflected light of the laser beam reflected by the specimen S is detected as the return light. It is good to do. The delay of the reflected light is affected by the difference in the optical path length of the optical path of the reflected light with respect to the optical path of the laser light.

1A Ar laser device (light source)
1B HeNe-G laser device (light source)
1C HeNe-R laser device (light source)
17 Galvano scanner (scanning part)
17A X Galvano mirror (scanning member)
17B Y Galvano mirror (scanning member)
25 Objective lens 30 Array type detection unit 31, 131 Control unit 33 Actual value detection unit 100, 200 Scanning laser microscope apparatus

Claims (20)

A scanning unit having a scanning member capable of swinging around a predetermined swinging axis, deflecting excitation light emitted from a light source by the scanning member and scanning the sample, and a scanning speed changeable ;
An objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample;
It has a plurality of minute detection elements that can be individually switched ON / OFF arranged at a position conjugate with the focal position of the objective lens, and turns on the return light condensed by the objective lens and descanned by the scanning unit An array type detection unit that can be selectively detected by the micro detection element in a state;
Based on the scanning speed of the scanning unit, the scanning and a positional displacement direction shifting and / or expansion control unit of descanning by the scanning unit due to the delay of ON to the microscopic regions the return light detecting element Type laser microscope apparatus. A scanning unit having a scanning member capable of swinging around a predetermined swinging shaft, and deflecting excitation light emitted from a light source by the scanning member and scanning the sample;
An objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample;
It has a plurality of minute detection elements that can be individually switched ON / OFF arranged at a position conjugate with the focal position of the objective lens, and turns on the return light condensed by the objective lens and descanned by the scanning unit An array type detection unit that can be selectively detected by the micro detection element in a state;
In accordance with the change in the査direction run of the scanning unit, and a positional displacement direction shifting and / or expansion control unit of descanning by the scanning unit due to the delay of ON to the microscopic regions the return light detecting element Scanning laser microscope apparatus provided. The control unit shifts the region of the minute detection element that is turned on in accordance with a change in the scanning direction of the scanning unit in a direction of a descan position by the scanning unit due to the delay of the return light, and the scanning unit If There to scan the excitation light with reciprocating oscillating movement of the scanning member, claim reversing towards direction to shift the region of the micro-detection element to oN in the go and return of the rocking motion of the scanning member 2 The scanning laser microscope apparatus described in 1. The control unit further expands the area of the micro detection element that is turned on in response to a change in the scanning direction of the scanning unit in the direction of the displacement of the descanning by the scanning unit due to the delay of the return light, when the scanning unit to scan the excitation light with reciprocating oscillating movement of the scanning member, reversing the direction that extends the area of the micro-detection element to oN in the go and return of the rocking motion of the scanning member The scanning laser microscope apparatus according to claim 3 . The control unit expands the area of the micro detection element that is turned on in response to a change in the scanning direction of the scanning unit in the direction of the positional deviation of the descanning by the scanning unit due to the delay of the return light. If the part is to scan the excitation light with reciprocating oscillating movement of the scanning member, wherein for reversing the direction that extends the area of the micro-detection element to oN in the go and return of the rocking motion of the scanning member Item 3. A scanning laser microscope apparatus according to Item 2 . 2. The scanning according to claim 1, wherein the control unit shifts a region of the minute detection element that is turned on in a direction of a positional deviation of descanning by the scanning unit due to a delay of the return light based on a scanning speed of the scanning unit. Type laser microscope apparatus. The scanning unit is a resonant scanner that is driven by a sine wave and has a high scanning speed near the center of the scanning range , and a low scanning speed near the end of the scanning range ,
3. The scanning according to claim 1, wherein the control unit further shifts and / or expands a region of the minute detection element that is turned on based on the scanning speed that changes in accordance with a swing angle of the scanning member. Type laser microscope apparatus. It has a light source that oscillates pulsed laser light,
3. The scanning laser microscope apparatus according to claim 1, wherein the control unit further shifts and / or expands a region of the micro detection element that is turned on based on characteristics of a fluorescent label applied to the specimen. It has a light source that oscillates pulsed laser light,
The scanning laser microscope apparatus according to claim 1, wherein the scanning unit is a resonant scanner and can change a scanning speed in accordance with an applied resonance frequency. With the measured value detector for detecting a shape of a confocal position, and / or confocal position prior Symbol return light,
The control unit further shifts the region of the minute detection element that is turned on based on the detection result of the actual measurement value detection unit in the direction of the displacement of the descan by the scanning unit due to the delay of the return light and / or査型laser microscope apparatus run according to claim 1 or claim 2 extends. A scanning unit having a scanning member capable of swinging around a predetermined swinging axis, deflecting excitation light emitted from a light source by the scanning member and scanning the sample, and a scanning speed changeable ;
An objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample;
A light detection unit that is disposed at a position conjugate with the focal position of the objective lens, and that detects return light that is collected by the objective lens and descanned by the scanning unit;
A plurality of micro-deflection elements that can be individually switched ON / OFF arranged at a position conjugate with the focal position of the objective lens, and the light detection unit selectively selects return light by the micro-deflection elements in the ON state A micro deflection element array that can be deflected toward the
Based on the scanning speed of the scanning unit, the scanning and a positional displacement direction shifting and / or expansion control unit of descanning by the scanning unit caused the area of the micro-deflection device for ON delay of the return light Type laser microscope apparatus. A scanning unit having a scanning member capable of swinging around a predetermined swinging shaft, and deflecting excitation light emitted from a light source by the scanning member and scanning the sample;
An objective lens that irradiates the sample with excitation light scanned by the scanning unit, and collects return light from the sample;
A light detection unit that is disposed at a position conjugate with the focal position of the objective lens, and that detects return light that is collected by the objective lens and descanned by the scanning unit;
A plurality of micro-deflection elements that can be individually switched ON / OFF arranged at a position conjugate with the focal position of the objective lens, and the light detection unit selectively selects return light by the micro-deflection elements in the ON state A micro deflection element array that can be deflected toward the
In accordance with the change in the査direction run of the scanning unit, and a positional displacement direction shifting and / or expansion control unit of descanning by the scanning unit caused the area of the micro-deflection device for ON delay of the return light Scanning laser microscope apparatus provided. The control unit shifts the region of the micro deflection element that is turned on in accordance with a change in the scanning direction of the scanning unit in a direction of de-scanning by the scanning unit due to the delay of the return light, and the scanning unit If There to scan the excitation light with reciprocating oscillating movement of the scanning member, according to claim 12 for inverting describes the direction to shift the region of the micro-deflection element oN in the go and return of the rocking motion of the scanning member The scanning laser microscope apparatus described in 1. The control unit further expands the region of the micro deflection element that is turned on in response to a change in the scanning direction of the scanning unit in a direction of a positional deviation of descanning by the scanning unit due to the delay of the return light, when the scanning unit to scan the excitation light with reciprocating oscillating movement of the scanning member, reversing the direction that extends the area of the micro-deflection element oN in the go and return of the rocking motion of the scanning member The scanning laser microscope apparatus according to claim 13 . The control unit expands the region of the micro deflection element that is turned on in accordance with a change in the scanning direction of the scanning unit in the direction of the positional deviation of the descanning by the scanning unit due to the delay of the return light. If the part is to scan the excitation light with reciprocating oscillating movement of the scanning member, wherein for reversing the direction that extends the area of the micro-deflection element oN in the go and return of the rocking motion of the scanning member Item 13. A scanning laser microscope apparatus according to Item 12 . 12. The scanning according to claim 11, wherein the control unit shifts the region of the micro deflection element that is turned on in the direction of the position deviation of the descanning by the scanning unit due to the delay of the return light based on the scanning speed of the scanning unit. Type laser microscope apparatus. The scanning unit is a resonant scanner that is driven by a sine wave and has a high scanning speed near the center of the scanning range , and a low scanning speed near the end of the scanning range ,
The scanning laser microscope apparatus according to claim 11 , wherein the control unit further shifts and / or expands a region of the micro deflection element that is turned on based on the scanning speed that changes in accordance with a swing angle of the scanning member. . It has a light source that oscillates pulsed laser light,
The scanning laser microscope apparatus according to claim 11 , wherein the control unit further shifts and / or expands a region of the micro deflection element that is turned on based on a characteristic of a fluorescent label applied to the specimen. It has a light source that oscillates pulsed laser light,
The scanning laser microscope apparatus according to claim 11 , wherein the scanning unit is a resonant scanner and can change a scanning speed according to a resonance frequency to be applied. With the measured value detector for detecting a shape of a confocal position, and / or confocal position prior Symbol return light,
The control unit further shifts the region of the micro deflection element that is turned on based on the detection result of the actual measurement value detection unit in the direction of the position of descanning by the scanning unit due to the delay of the return light and / or The scanning laser microscope apparatus according to claim 11 or 12, which is expanded.

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