JP6223048B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope.

  Conventionally, a laser scanning microscope that observes the function of cells and the like by irradiating a specimen such as a living body with ultrashort pulse laser light from its surface and detecting multiphoton fluorescence emitted from the specimen surface or inside. Is known (for example, see Patent Document 1).

  A light beam having a large NA among the ultrashort pulse laser beams has a long optical path length within the sample and is easily affected by scattering, so that it cannot focus on the deep part of the sample and hardly contributes to two-photon excitation. On the other hand, a light beam having a small NA among the ultrashort pulse laser light has a short optical path length within the sample and is not easily affected by scattering, and thus tends to focus on the deep part of the sample. In the laser scanning microscope described in Patent Document 1, the short pulse laser beam is incident on the specimen with a light beam smaller than the pupil of the objective lens, so that the optical path length in the specimen is shortened and affected by scattering. The excitation efficiency is improved.

Japanese Patent No. 4759425

  However, in the laser scanning microscope described in Patent Document 1, it is effective to make the luminous flux of the short pulse laser beam smaller than the pupil of the objective lens according to the scattering conditions in the specimen and the depth of the focal position of the objective lens. In this case, an image is formed with an effective NA smaller than the nominal NA of the objective lens.

  Here, when acquiring a continuous optical slice image of each focal position while moving the focal position of the objective lens in the sample in the depth direction, if the NA of the short pulse laser beam is large, the sample in the depth direction of the sample is obtained. Since the resolution is high and the observation region layer is thin, the focal position of the objective lens must be switched small in the depth direction. On the other hand, if the NA of the short pulse laser beam is small, the resolution in the depth direction is low and the layer in the observation region is thick, so the focal position of the objective lens may be switched greatly in the depth direction.

  Therefore, in the laser scanning microscope described in Patent Document 1, when the optical slice image is acquired while changing the focal position in the depth direction of the sample by the resolution in the depth direction calculated by the nominal NA, the depth direction There is a problem that the distance between the focal positions adjacent to each other becomes too narrow, and the acquisition time of the XYZ / XYZt image or the XZ / XZt image becomes long. In addition, there is a problem that a sample such as a cell is continuously irradiated with laser light for a long time. Furthermore, there are problems that the number of times of acquiring images is unnecessarily increased, the area for storing the image data is wasted, the image drawing circuit is burdened, and the image display takes extra time.

  The present invention has been made in view of the above-described circumstances, and aims to shorten the time required for an experiment, realize a minimally invasive measurement of a specimen, reduce waste of an image data storage area, and improve a drawing speed of image display. An object of the present invention is to provide a laser scanning microscope capable of performing

In order to solve the above problems, the present invention employs the following means.
The present invention relates to a light source that emits a pulsed laser beam, a scanning unit that scans the laser beam emitted from the light source, and an objective lens that collects the laser beam scanned by the scanning unit and irradiates the sample A detection unit that detects fluorescence generated from the vicinity of the focal position of the objective lens in the specimen by being irradiated with laser light, and outputs a light intensity signal corresponding to the detected luminance of the fluorescence; an image generating unit that generates an image based on the output light intensity signal, and the focus position changing unit that moves a focal position of the objective lens in the depth direction of the target present in the specimen, a focal position of the front Symbol objective lens wherein as the focal point position as the depth becomes deeper in the specimen is switched largely in the depth direction of, and a focus position change control unit that controls the focus position changing section, by the focal position changing unit For each of the focus position that is exposed, the detector the fluorescent said light intensity signal is detected is output by the laser scanning microscope in which the image is generated based on the light intensity signal by the image generating unit provide.

  According to the present invention, when pulsed laser light emitted from a light source is scanned by a scanning unit and irradiated on a specimen by an objective lens, fluorescence is generated near the focal position of the objective lens due to the multiphoton excitation effect in the specimen. Occur. Then, the focal position changing unit moves the focal position of the objective lens in the specimen in the depth direction, so that the fluorescence emitted from the vicinity of the focal position different in the depth direction is detected by the detecting unit, and the image generating unit An image at each focal position is generated based on a light intensity signal corresponding to the detected fluorescence intensity. Thereby, the stereo image of a sample is acquirable.

  Here, if the focal position of the objective lens in the specimen is shallow, the optical path length in the specimen is short and it is difficult to be affected by scattering, so the resolution in the depth direction is high and the observation area layer is thin. As the focal position becomes deeper, the optical path length in the sample becomes longer and it becomes more susceptible to scattering, so that the resolution in the depth direction decreases and the layer in the observation region becomes thicker.

  In this case, under the control of the focal position change control unit, the focal position changing unit determines the focal position of the objective lens so that the focal position is largely switched in the depth direction as the depth of the focal position of the objective lens in the specimen increases. By moving the sample in the depth direction, the distance between the layers in the observation region adjacent in the depth direction increases as the depth of the sample increases, and it is possible to prevent redundant detection of the fluorescence from the observation regions adjacent to each other. Can do. Thereby, a clear image can be acquired for each desired observation region having a different depth in the specimen.

  Therefore, the acquisition time of the image is prevented and the time required for the experiment is shortened, and the specimen such as a biological specimen is prevented from being continuously irradiated with the laser beam for a long time. Can be realized. Further, it is possible to prevent an unnecessary increase in the number of image acquisitions, reduce waste of the image data storage area, reduce the burden on the image drawing circuit, and improve the image display drawing speed.

  In the above-described configuration, the image generation unit causes the pixel in the cross direction to intersect the depth direction in the sample image as the focal position of the objective lens in the sample becomes deeper by the focus position changing unit. The number may be reduced.

  With this configuration, even when the focal position of the objective lens in the sample becomes deeper, the resolution becomes lower, and even if blurring occurs around the focal position, each pixel in the intersecting direction that intersects the depth direction in the image It is possible to increase the size and generate an image that matches the resolution.

In the above configuration, the image generation unit may generate a plurality of other images of the focal positions having different depths according to the resolution of the focal position having the lowest resolution.
By configuring in this way, it is possible to acquire a stereoscopic image having no variation in sharpness over a plurality of observation regions having different specimen depths.

  In the above configuration, the light beam diameter adjusting unit capable of adjusting the light beam diameter of the laser light, and the light beam diameter of the laser light gradually becomes larger than the pupil of the objective lens as the depth of the focal position in the sample increases. It is good also as providing the light beam diameter adjustment control part which controls the said light beam diameter adjustment part so that it may become small.

  Pulsed laser light with a large NA has a long optical path length within the sample and is easily affected by scattering, so it is difficult to reach the deep part of the sample, whereas pulsed laser light with a small NA Since the optical path length is short and hardly affected by scattering, it easily reaches the deep part of the specimen.

  With this configuration, the light beam diameter adjustment control unit controls the light beam diameter adjustment unit so that the light beam diameter of the laser beam becomes smaller than the pupil of the objective lens as the focal position depth in the sample increases. When observing the deep part of the specimen, it is not necessary to irradiate the specimen with extra light that does not reach the focal position of the deep part, and the influence of the laser light on the specimen can be suppressed.

  According to the present invention, it is possible to shorten the time required for an experiment, to realize a minimally invasive measurement of a specimen, to reduce waste of an image data storage area and to improve an image display drawing speed.

1 is a schematic configuration diagram of a laser scanning microscope according to an embodiment of the present invention. It is a figure explaining the relationship between the magnitude | size of NA of a laser beam, and the influence of scattering. It is a figure which shows the relationship between the resolution (FWHM) of two-photon excitation, and effective NA. It is a figure which shows an example of the resolution approximate table of 2 photon excitation supposing the minute point. It is a figure explaining the surface layer part, intermediate | middle part, and deep layer part which are different observation areas in the Z-axis direction of a sample.

Hereinafter, a laser scanning microscope according to an embodiment of the present invention will be described with reference to the drawings.
The laser scanning microscope 100 according to the present embodiment is a two-photon excitation microscope, for example. As shown in FIG. 1, the laser scanning microscope 100 includes a laser light source 1 that emits a pulsed ultrashort pulse laser beam and a collimator lens optical that collimates the ultrashort pulse laser beam emitted from the laser light source 1. A two-dimensional scanning mechanism (scanning unit) 5 that deflects the ultrashort pulse laser light that has passed through the collimating lens optical system 3 and scans the sample S two-dimensionally, and two-dimensional scanning The sample S is irradiated with the ultrashort pulse laser beam deflected by the mechanism 5, while the objective lens 7 that collects the fluorescence generated in the sample S and the fluorescence collected by the objective lens 7 are converted into the ultrashort pulse laser beam. A dichroic mirror 9 branched from the optical path and a detection device (detection unit) 11 for detecting fluorescence branched by the dichroic mirror 9 are provided.

  The collimating lens optical system 3 is provided so as to be movable along the optical axis direction on the optical axis of the ultrashort pulse laser beam. The collimating lens optical system 3 changes the collimating relationship of the ultrashort pulse laser light by changing the position in the optical axis direction of the ultrashort pulse laser light, thereby changing the beam diameter at the pupil position of the objective lens 7. Be able to.

  The two-dimensional scanning mechanism 5 is, for example, a two-axis galvanometer mirror, and is configured by opposing two galvanometer mirrors (not shown) that can swing around mutually orthogonal axes. This two-dimensional scanning mechanism 5 swings two galvanometer mirrors to deflect the ultrashort pulse laser beam, and scans the sample in two dimensions (X-axis direction and Y-axis direction). Can be done.

  The dichroic mirror 9 reflects the ultra-short pulse laser beam from the two-dimensional scanning mechanism 5 toward the objective lens 7, and detects fluorescence that is condensed by the objective lens 7 and returns on the optical path of the ultra-short pulse laser beam. It can be transmitted toward

  The detection device 11 detects the fluorescence generated from the vicinity of the focal position of the objective lens 7 in the specimen S by being irradiated with the ultrashort pulse laser beam, and outputs a light intensity signal corresponding to the luminance of the detected fluorescence. It has become.

  The laser scanning microscope 100 includes an electric drive unit (light beam diameter adjusting unit) 13 that moves the collimating lens optical system 3 in the optical axis direction, and a laser control device (light beam diameter adjustment control unit) that controls the electric drive unit 13. ) 21, a Z-axis drive mechanism (focus position changing unit) 25 that moves the objective lens 7 in the optical axis direction, a two-dimensional scanning mechanism 5, a laser control device 21, and a scanning control device that controls the Z-axis driving mechanism 25 ( And a computer (image generation unit) 29 that controls the laser control device 21 and the scanning control device 27, performs image processing, and the like.

  The electric drive unit 13 includes a linear motion rail 15 that guides the movement of the collimating lens optical system 3 in the optical axis direction, a stepping motor 17 having a rack and pinion gear, and a motor driver 19 that drives the stepping motor 17.

  The motor driver 19 controls the stepping motor 17 based on a control command from the laser control device 21. The collimating lens optical system 3 moves on the linear motion rail 15 by a moving amount corresponding to the control amount of the stepping motor 17.

  The laser control device 21 includes an SRAM (memory device) 23 that stores a lookup table in which the depth of the focal position of the objective lens 7 in the sample S is associated with the position of the collimating lens optical system 3 in the optical axis direction. Yes. In the look-up table, the focal point of the objective lens 7 is set so that the beam diameter of the ultrashort pulse laser beam gradually becomes smaller than the pupil of the objective lens 7 as the depth of the focal position of the objective lens 7 in the sample S increases. The position and the position of the collimating lens optical system 3 are associated with each other.

  Further, the laser control device 21 refers to the look-up table of the SRAM 23 based on the synchronization signal from the scanning control device 27 to determine the position of the collimating lens optical system 3 corresponding to the depth of the focal position of the objective lens 7. A control command for acquiring and moving the collimating lens optical system 3 to the acquired position is sent to the electric drive unit 13.

  Here, as shown in FIG. 2, the ultrashort pulse laser beam having a large NA has a long optical path length in the sample S and is easily affected by scattering, so that it is difficult to reach the deep part of the sample S. On the other hand, an ultrashort pulse laser beam having a small NA has a short optical path length within the sample S and is not easily affected by scattering, and therefore easily reaches a deep portion of the sample S.

  The ultrashort pulse laser beam passes through the collimating lens optical system 3 positioned by the electric drive unit 13 based on the control command from the laser control device 21, thereby deepening the focal position of the objective lens 7 in the sample S. As the beam diameter of the ultrashort pulse laser beam gradually becomes smaller than the pupil of the objective lens 7. Thereby, when observing the deep part of the sample S, it is not necessary to irradiate the sample S with an extra ultrashort pulse laser beam that does not reach the focal position of the deep part.

For example, the resolution of two-photon excitation (FWHM (Full Width at Half Maximum): full width at half maximum) can be estimated by the following equation.
The resolution in the Z-axis direction (Z resolution: FWHMz) is expressed by equation (1).
FWHMz = 0.62 × λ / (n-SQRT (n ² −NA ² )) (1)
The resolution in the X-axis direction and the Y-axis direction (XY resolution: FWHMxy) is expressed by equation (2).
FWHMxy = 0.36 × λ / NA (2)
Here, λ is an excitation wavelength, n is a refractive index (usually using a refractive index of water of 1.33), and NA is an effective NA.

  Further, the relationship between the resolution (FWHM) of two-photon excitation and the effective NA is as shown in FIG. 3, for example. In FIG. 3, the vertical axis represents the resolution (FWHM), and the horizontal axis represents the effective NA. An example of a resolution estimation table for two-photon excitation assuming a minute point is shown in FIG. According to FIGS. 3 and 4, the higher the effective NA, the higher the resolution, and the smaller the resolution (FWHM) value. It can also be seen that when the effective NA becomes small, the XY resolution does not deteriorate so much, whereas the Z resolution significantly deteriorates.

  The Z-axis drive mechanism 25 can move the focal position of the objective lens 7 in the specimen S in the Z-axis direction of the specimen S by changing the distance in the optical axis direction between the objective lens 7 and the specimen S. It has become.

  The scanning control device 27 controls the two-dimensional scanning mechanism 5, the laser control device 21, and the Z-axis drive mechanism 25 based on a control command from the computer 29. Specifically, the scanning control device 27 oscillates the galvanometer mirror of the two-dimensional scanning mechanism 5 to scan the ultrashort pulse laser beam in the XY direction on the focal plane of the objective lens 7 in the sample S. Yes.

  In addition, the scanning control device 27 drives the Z-axis drive mechanism 25 so that the focal position of the objective lens 7 in the sample S becomes deeper in the Z-axis direction as the depth of the focal position becomes deeper. Further, the scanning control device 27 outputs a synchronization signal to the laser control device 21 at the timing of driving the Z-axis drive mechanism 25.

  The amount by which the focal position of the objective lens 7 is switched in the Z-axis direction can be determined, for example, by measuring in advance the brightness and characteristics in the Z-axis direction of different parts of the cranial nerve of a biological sample, And the brightness of each fluorescent bead of the phantom and the characteristic in the Z-axis direction may be measured in advance, and the obtained Z half width may be set as a reference. The setting may be performed by the user, or may be performed by the manufacturer during the manufacturing process or setup of the laser scanning microscope 100.

  The computer 29 has a user interface such as a GUI, and outputs control commands to the laser control device 21 and the scanning control device 27 based on an observation schedule set by the user. .

  The computer 29 can generate an image based on the light intensity signal output from the detection device 11 and display the image on a monitor (not shown). Thereby, the observation image of the focal position of the objective lens 7 in the sample S can be provided to the user.

The operation of the laser scanning microscope 100 configured as described above will be described.
According to the laser scanning microscope 100 according to the present embodiment, the ultrashort pulse laser light emitted from the laser light source 1 is collimated by the collimating lens optical system 3 and deflected by the two-dimensional scanning mechanism 5. Then, the ultrashort pulse laser light is condensed by the objective lens 7 via the dichroic mirror 9 and irradiated on the focal position of the objective lens 7 in the sample S. As a result, the ultrashort pulse laser beam is scanned two-dimensionally on the focal plane of the sample S according to the swing angle of the pair of galvanometer mirrors of the two-dimensional scanning mechanism 5. Further, the focal position of the objective lens 7 on the sample S is moved in the Z-axis direction by the Z-axis drive mechanism 25.

  When fluorescence is generated in the vicinity of the focal position of the objective lens 7 due to the multiphoton excitation effect in the sample S due to the irradiation with the ultrashort pulse laser beam, the fluorescence is condensed by the objective lens 7 and the ultrashort pulse laser beam is collected. , Passes through the dichroic mirror 9 and is detected by the detection device 11. Then, the computer 29 generates an image of the focal plane of the sample S based on the light intensity signal corresponding to the luminance of the fluorescence output from the detection device 11.

Next, the case where the sample S is observed with the laser scanning microscope 100 according to the present embodiment will be described.
First, pre-scan and create a lookup table. In pre-scanning, the Z-axis drive mechanism 25 is driven, and the focal position of the objective lens 7 is set at the upper limit (hereinafter referred to as “surface layer portion”) in the Z-axis direction of specimen observation input by the user. An image is acquired by irradiating an ultrashort pulse laser beam and displayed on a monitor.

  Next, the collimating lens optical system 3 is gradually moved in the optical axis direction, and the beam diameter of the ultrashort pulse laser beam is gradually reduced. The user confirms the observation image displayed on the monitor and causes the computer 29 to record the position of the collimating lens optical system 3 from which a clear image can be obtained.

Subsequently, the Z-axis drive mechanism 25 is driven, the focal position of the objective lens 7 is set at the lower limit in the Z-axis direction of specimen observation (hereinafter referred to as “deep layer portion”), and laser light is irradiated in this state. The image is acquired and displayed on the monitor. Then, in the same manner as in the case of the surface layer portion, the collimating lens optical system 3 is adjusted to gradually reduce the beam diameter of the ultrashort pulse laser beam, and the user can confirm the observation image to obtain a clear image. The computer 29 records the position of the lens optical system 3.
When the surface layer portion is set on the surface of the specimen S, a light beam system capable of producing the original performance of the objective lens 7 is automatically set, so that the position recording in the pre-scan as described above is not necessary.

  If the position of the collimating lens optical system 3 in the surface layer portion and the deep layer portion of the sample S is specified in this way, the laser light intensity is increased as the depth of the focal position of the objective lens 7 in the sample S is increased using these values. The positions of the collimating lens optical systems 3 corresponding to a plurality of layers having different depths in the intermediate portion between the surface layer portion and the deep layer portion are obtained so that the beam diameter is gradually smaller than the pupil of the objective lens 7.

  For determining the position of the collimating lens optical system 3 corresponding to each layer in the intermediate portion, a commonly used interpolation method, a method of performing pre-scanning on all the depths of the focal position for observation, or the like can be used. .

  With such a method, collimation corresponding to the depth of each focal position is set so that the beam diameter of the laser beam gradually becomes smaller than the pupil of the objective lens 7 as the depth of the focal position of the objective lens 7 becomes deeper. The position of the lens optical system 3 is determined. Then, the computer 29 creates a lookup table by associating these pieces of information with each other and writes the lookup table in the SRAM 23 of the laser control device 21.

  Accordingly, the laser control device 21 controls the electric drive unit 13 based on the look-up table, and adjusts the position of the collimating lens optical system 3 in accordance with the depth of the focal position of the objective lens 7. As the depth of the focal position at S increases, the NA of the ultrashort pulse laser beam can be made gradually smaller than the pupil of the objective lens 7.

Next, this observation is performed.
First, the Z-axis drive mechanism 25 is driven by the scanning control device 27 so that the focal position of the objective lens 7 is arranged on the surface layer portion of the sample S, and a synchronization signal is sent from the scanning control device 27 to the laser control device 21.

  In the laser control device 21, the position of the collimating lens optical system 3 corresponding to the surface layer portion is acquired from the look-up table stored in the SRAM 23 by inputting the synchronization signal from the scanning control device 27, and is electrically driven. A control command is sent to the unit 13.

  In the electric drive unit 13, the motor driver 19 drives the stepping motor 17 based on the control command from the laser control device 21, and the collimating lens optical system 3 is guided by the linear motion rail 15 and corresponds to the surface layer portion. Move to.

  As a result, the ultrashort pulse laser light emitted from the laser light source 1 passes through the collimating lens optical system 3 and is converted into an NA smaller than the pupil of the objective lens 7 corresponding to the surface layer portion, and is two-dimensionally scanned. The specimen S is irradiated by the objective lens 7 through the mechanism 5.

  Then, the ultrashort pulse laser beam is scanned two-dimensionally on the surface layer portion of the sample S by the two-dimensional scanning mechanism 5, and the fluorescence generated on the surface layer portion is detected by the detection device 11, and the computer 29 Based on the light intensity signal corresponding to the luminance of the fluorescence, an image of the surface layer portion that is the focal position of the objective lens 7 is generated.

  Subsequently, when the scanning of the ultrashort pulse laser beam on the surface layer is completed, the scanning control device 27 drives the Z-axis drive mechanism 25 to switch the focal position of the objective lens 7 on the specimen S to the next depth. . Further, a synchronization signal is output from the scanning control device 27 to the laser control device 21, and the collimating lens optical system 3 moves to a position corresponding to the depth of the next focal position by the electric drive unit 13.

  In this case, under the control of the scanning control device 27, the Z-axis drive mechanism 25 is switched greatly in the Z-axis direction as the focal position of the objective lens 7 in the sample S becomes deeper as shown in FIG. The focal position of the objective lens 7 is moved.

  Next, an ultrashort pulse laser beam whose NA has been converted by the collimating lens optical system 3 is irradiated by the objective lens 7 through the two-dimensional scanning mechanism 5 to the focal position of the next depth. Then, the fluorescence generated in the vicinity of the focal position is detected by the detection device 11, and an image of an intermediate portion that is the focal position of the objective lens 7 is generated by the computer 29.

  In this way, the depth of the focal position of the objective lens 7 in the specimen S is switched stepwise by the Z drive mechanism 25, and an image is generated based on the fluorescence generated in the vicinity of each focal position. Thereby, the three-dimensional image of the sample S can be acquired.

  Here, if the focal position of the objective lens 7 in the sample S is shallow, the optical path length in the sample S is short and hardly affected by scattering, so that the resolution in the Z-axis direction is high and the layer of the observation region is thin. On the other hand, as the focal position of the objective lens 7 in the specimen S becomes deeper, the optical path length in the specimen S becomes longer and it is more susceptible to scattering, so that the resolution in the Z-axis direction is lowered and the layer in the observation region is reduced. Become thicker.

  According to the laser scanning microscope 100 according to the present embodiment, the focal position of the objective lens 7 increases in the Z-axis direction as the depth of the focal position of the objective lens 7 in the sample S increases under the control of the scanning control device 27. Since the sample moves, the deeper the sample S, the distance between the layers in the observation region adjacent to each other in the Z-axis direction can be separated, and the fluorescence emitted from the observation regions adjacent to each other can be prevented from being detected redundantly. Thereby, a clear image can be acquired for each desired observation region having a different depth in the specimen S.

  Accordingly, it is possible to prevent the image acquisition time from being lengthened and reduce the time required for the experiment, and to prevent the specimen S such as a biological specimen from being irradiated with the ultrashort pulse laser light for a long time. A minimally invasive measurement of S can be realized. Further, it is possible to prevent an unnecessary increase in the number of image acquisitions, reduce waste of the image data storage area, reduce the burden on the image drawing circuit, and improve the image display drawing speed.

  In the present embodiment, the Z-axis drive mechanism 25 that moves the objective lens 7 in the optical axis direction is described as an example of the focal position changing unit. However, the focal position of the objective lens 7 in the specimen S is the focal position of the specimen S. Any stage can be used as long as it can be moved in the Z-axis direction. For example, a stage that moves the focal position of the objective lens 7 in the specimen S in the Z-axis direction by moving the specimen S in the optical axis direction. It may be adopted.

  Moreover, the electric drive part 13 is not restricted to the above-mentioned structure, For example, it is good also as employ | adopting an actuator which can be positioned with high precision, such as a piezoelectric actuator.

This embodiment can be modified as follows.
As a first modified example, the computer 29 causes the Z-axis drive mechanism 25 to increase the depth of the focal position of the objective lens 7 in the sample S, and the X-axis direction and the Y-axis intersecting the Z-axis direction in the sample S image. The number of pixels in the axial direction may be reduced.

  In this case, the pixel sizes in the X-axis direction and the Y-axis direction may be measured in advance, for example, by measuring brightness and characteristics in the Z-axis direction at different depths of the cranial nerve of a biological sample, It is also possible to set the pixel size of the sample surface based on the obtained XY half-value width by inserting fluorescent beads and measuring in advance the brightness of the fluorescent beads and the characteristics in the Z-axis direction. The pixel size may be set by the user, or may be set by measurement by the manufacturer during the manufacturing process or setup of the laser scanning microscope 100.

  By doing so, the resolution becomes lower as the focal position of the objective lens 7 in the sample S becomes deeper, and an image is formed by the computer 29 even if the focal position is blurred in the X-axis direction and the Y-axis direction. By increasing the size of each pixel in the X-axis direction and the Y-axis direction, an image that matches the resolution can be generated. For example, when the resolution is halved, the number of pixels in the X-axis direction and the Y-axis direction may be halved and the size of each pixel may be increased accordingly.

  In general, the XYZ half-value width increases as the depth in the sample S increases, so that the XYZ resolution deteriorates as the depth increases. According to this modification, as the XYZ resolution deteriorates as the depth increases, the amount of data to be acquired is kept to the minimum necessary, the time required for the experiment is further reduced, and the specimen S is measured more effectively and less invasively. can do.

  As a second modification, the computer 29 may generate images of other focal positions having different depths according to the resolution of the focal position having the lowest resolution in the sample S. In this case, since the XYZ pixel size (converted to the sample plane) in the deep layer portion of the sample S is usually the largest, an image at other focal positions of the surface layer portion and the intermediate portion is generated depending on the resolution of the deep layer portion. That's fine.

  In this way, it is possible to acquire a stereoscopic image having no variation in sharpness over a plurality of observation regions having different depths of the specimen S. That is, it is possible to display an easy-to-see 3D image with uniform display resolution of the entire stereoscopic image. In addition, it is possible to minimize image data storage and display function such as memory and image graphics, and to improve the image display drawing speed.

  In the present embodiment, the collimating lens optical system 3 is exemplified as the light beam diameter adjusting unit, and the light beam diameter of the ultrashort pulse laser light is adjusted by shifting the position of the collimating lens optical system 3 in the optical axis direction. However, as a third modification, an electric zoom optical system (for example, a focal zoom lens) may be employed as the light beam diameter adjusting unit.

In this case, the light beam diameter of the ultrashort pulse laser beam may be changed without affecting the collimating condition by the collimating lens optical system 3 by the electric zoom optical system. Even in this case, the same effect as the case where the light beam diameter is adjusted by the collimating lens optical system 3 can be obtained without reducing the amount of light.
Moreover, it is good also as employ | adopting as a light beam adjustment part the aperture_diaphragm | restriction which restrict | limits the light beam of the ultrashort pulse laser beam to pass.

1 Laser light source
5 Two-dimensional scanning mechanism (scanning unit)
7 Objective Lens 11 Detector (Detector)
13 Electric drive unit (light beam diameter adjustment unit)
21 Laser controller (light beam diameter adjustment controller)
25 Z-axis drive mechanism (focal position changing unit)
27 Scanning control device (focus position control unit)
29 Computer (image generator)
100 Laser scanning microscope S specimen

Claims (4)

A light source that emits a pulsed laser beam;
A scanning unit that scans the laser beam emitted from the light source;
An objective lens that collects the laser beam scanned by the scanning unit and irradiates the sample;
A detection unit that detects fluorescence generated from the vicinity of the focal position of the objective lens in the specimen by being irradiated with laser light, and outputs a light intensity signal corresponding to the detected luminance of the fluorescence;
An image generation unit that generates an image based on the light intensity signal output from the detection unit;
A focal position changing unit that moves the focal position of the objective lens in the specimen in the depth direction of the specimen;
A focal position change control unit that controls the focal position change unit so that the focal position is largely switched in the depth direction as the depth of the focal position of the objective lens in the sample increases .
For each focal position changed by the focal position changing unit, the detection unit detects the fluorescence and outputs the light intensity signal, and the image generation unit generates the image based on the light intensity signal. laser scanning microscope that.   The image generation unit reduces the number of pixels in the intersecting direction intersecting the depth direction in the sample image as the focal position of the objective lens in the sample becomes deeper by the focal position changing unit. 2. A laser scanning microscope according to 1.   3. The laser scanning microscope according to claim 1, wherein the image generation unit generates images of the plurality of other focal positions having different depths according to the resolution of the focal position having the lowest resolution. A beam diameter adjusting unit capable of adjusting a beam diameter of the laser beam;
And a light beam diameter adjustment control unit configured to control the light beam diameter adjustment unit so that a light beam diameter of the laser light gradually becomes smaller than a pupil of the objective lens as a depth of the focal position in the sample increases. The laser scanning microscope according to any one of claims 1 to 3.

Images