JPWO2010143375A1 - Microscope device and control program - Google Patents

Abstract

An objective lens, a stage on which the test object is placed, an imaging unit that captures an image of the test object, and a moving unit that changes the relative position of the objective lens and the stage in the optical axis direction of the objective lens; A calculation unit for obtaining a brightness adjustment parameter for each of at least two surfaces in the test object having different positions in the optical axis direction, and a plurality of objects having different positions in the optical axis direction based on the at least two brightness adjustment parameters. When generating a plurality of images having different positions in the optical axis direction of the objective lens by including a determination unit that determines a luminance adjustment parameter when performing imaging by the imaging unit on each of the surfaces in the inspection object, Reduces variations in brightness among a plurality of generated images.

Description

  The present invention relates to a microscope apparatus and a control program.

  Conventionally, a microscope apparatus that generates a plurality of images having different positions in the optical axis direction (so-called Z direction) of an objective lens is known. For example, in the invention of Patent Document 1, a stereoscopic image is constructed from a plurality of images (slice images) having different positions in the Z direction, and a development view of an image projected in the horizontal and vertical directions is constructed.

JP 2005-326601 A

  By the way, as the distance from the objective lens in the Z direction becomes longer, the generated image tends to be darker. In order to cope with such a problem, it is necessary to appropriately specify illumination conditions, imaging conditions, and the like for each slice. Such designation is time consuming and requires experience, and can be a factor that hinders stable observation.

  The present invention has been made in view of the above problems, and an object of the present invention is to suppress variation in brightness in a plurality of generated images when generating a plurality of images having different positions of the objective lens in the optical axis direction. To do.

  The microscope apparatus of the present invention includes an objective lens, a stage on which a test object is placed, an imaging unit that captures an image of the test object, and the objective lens and the stage in the optical axis direction of the objective lens. A moving unit that changes a relative position of the light source, a calculation unit that obtains a luminance adjustment parameter for each of at least two surfaces in the test object that have different positions in the optical axis direction, and at least two of the luminance adjustment parameters And a determination unit that determines a luminance adjustment parameter for performing imaging by the imaging unit on each of the surfaces in the plurality of objects having different positions in the optical axis direction.

  The brightness adjustment parameter may include at least one of an illumination intensity when illuminating the object to be examined, a moving speed of the stage, and a time interval of imaging by the imaging unit.

  In addition, an illumination unit that irradiates the test object with laser light, and the brightness adjustment parameter includes at least one of the intensity of the laser light and the gain when the laser light is generated from a light source as the illumination intensity. May be included.

  Further, the determination unit performs an interpolation calculation based on at least two of the luminance adjustment parameters, and sets the luminance adjustment parameters when performing imaging by the imaging unit on each of a plurality of surfaces having different positions in the optical axis direction. You may decide.

  In addition, the moving unit includes a control unit that moves the stage at a predetermined speed, performs imaging by the imaging unit at predetermined time intervals, and generates a plurality of pre-scan images. Based on the pre-scan image, the surface corresponding to both ends of the test object in the optical axis direction is estimated, and the brightness adjustment parameter is obtained for each of a plurality of surfaces including the surfaces corresponding to the both ends. good.

  Further, the control unit may generate a plurality of observation images by performing imaging by the imaging unit while moving the stage by the moving unit based on the luminance parameter determined by the determining unit.

  Moreover, you may provide the three-dimensional image generation part which produces | generates the three-dimensional image of the said test object based on the said some observation image.

  In addition, a configuration obtained by converting the configuration related to the above invention into a control program for realizing control of the microscope apparatus by a computer is also effective as a specific aspect of the present invention.

It is a figure which shows the structure of the confocal microscope system 1 of this embodiment. It is a flowchart which shows operation | movement of the control part 53 at the time of acquiring an observation image. It is a timing diagram explaining operation | movement of the control part 53 at the time of acquiring an observation image.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  In the present embodiment, the confocal microscope system 1 shown in FIG. 1 will be described as an example.

  As shown in FIG. 1, the confocal microscope system 1 includes a light source unit 10, a confocal microscope 20, and a computer 50.

  The light source unit 10 includes a light source that emits illumination light to the confocal microscope 20. As the light source, in addition to the laser, white light such as an ultra-high pressure mercury lamp, a xenon lamp, a metal halide lamp, or the like is used. When white light is used as the light source, an excitation filter is installed between the light source and a beam shaping optical system described later. Hereinafter, a case where a laser light source is provided will be described as an example.

  The confocal microscope 20 includes a beam shaping optical system 21, a confocal slit diaphragm 22, an illumination collimating lens 23, a beam splitter 24, a scanner 25, a scanner lens 26, a second objective lens 27, an objective lens 28, a stage 29, and an image. Each unit includes a system collimating lens 30, a condenser lens 31, an imaging unit 32, reflection mirrors 33 and 34, and a filter 35. The beam shaping optical system 21 is, for example, a cylindrical microlens, and the beam splitter 24 is, for example, a dichroic mirror. The filter 35 is, for example, a barrier filter. The scanner 25 includes a scan mirror 25a whose both surfaces are reflection surfaces. The scan mirror 25a is rotatable in the direction of arrow A in FIG. 1, and scans the irradiation light in the direction of arrow B by rotating (details will be described later). For example, a specimen T such as a biological sample is placed on the stage 29. The stage 29 can be moved in a three-dimensional direction (xyz direction in FIG. 1) by a driving unit (not shown). The imaging unit 32 includes, for example, an imaging element such as a CCD, a light receiving element, and the like, and generates two-dimensional luminance distribution data (observation image) from imaging light described later.

  The computer 50 includes an operation unit 51 such as a mouse and a keyboard, a display unit 52 including an LCD, etc., and a control unit 53 that detects an operation on the operation unit 51 and displays an image or the like on the display unit 52. Further, the control unit 53 controls the light source unit 10, the scanner 25, and the imaging unit 32, and acquires a two-dimensional observation image from the imaging unit 32 (details will be described later).

  Next, with reference to FIG. 1, the light guide in the confocal microscope system 1 is demonstrated easily.

  Illumination light emitted from the light source unit 10 is formed into a linear light beam that is long in one direction by the beam shaping optical system 21. This linear light beam (hereinafter referred to as “light beam S”) is collected at a position optically conjugate with the observation surface Ta of the sample T. At this position, a confocal slit diaphragm 22 having a plurality of slit-like openings is installed.

  The light beam S formed in a line shape by the beam shaping optical system 21 enters the opening of the confocal slit diaphragm 22. The light beam S is formed into a line-shaped light beam having a predetermined width by the opening.

  The light beam S that has passed through the confocal slit diaphragm 22 is converted into parallel light by the illumination system collimating lens 23, and is reflected in the direction of the scanner 25 by the reflection mirror 33. Then, the light beam S reflected by the first surface 25a-s of the scan mirror 25a is reflected in the direction of the scanner lens 26. The light beam S that has passed through the scanner lens 26 passes through the second objective lens 27 and the objective lens 28 in order, and is condensed on the observation surface Ta of the sample T as a linear illumination region.

  By irradiating the sample T with the light beam S, the sample T emits fluorescence or reflects the light beam S. This fluorescent or reflected light (hereinafter referred to as “light beam K”) is guided to the confocal slit diaphragm 22 along a path opposite to that of the light beam S as illumination light. That is, the light beam K is guided to the scanner 25 through the objective lens 28, the second objective lens 27, and the scanner lens 26 in order. The light beam K reflected by the first surface 25 a-s of the scan mirror 25 a is reflected in the direction of the reflection mirror 33, and further reflected by the reflection mirror 33 in the direction of the illumination system collimating lens 23. The light beam K collected by the illumination system collimating lens 23 enters the confocal slit stop 22. At this time, the confocal slit diaphragm 22 functions as a confocal diaphragm of the imaging optical system, and cuts off excess light out of focus.

  The light beam K that has passed through the confocal slit stop 22 is reflected by the beam splitter 24 toward the imaging system collimating lens 30. The light beam K converted into parallel light by the imaging system collimator lens 30 is reflected by the reflecting mirror 34 toward the scanner 25. The light beam K is reflected in the direction of the condensing lens 31 by the second surface 25a-r of the scan mirror 25a, and is imaged on the detection surface of the imaging unit 32 by the condensing lens 31 as a two-dimensional luminance distribution. The Note that the luminance distribution imaged on the detection surface of the imaging unit 32 corresponds to the luminance distribution on the observation surface Ta.

  In the confocal microscope system 1 having the above-described configuration, a basic operation of the control unit 53 when acquiring an observation image of the specimen T will be described with reference to FIG. When acquisition of an observation image is instructed via the operation unit 51, the control unit 53 detects this and controls the light source unit 10 to start irradiating the specimen T with illumination light. The control unit 53 controls the scanner 25 to rotate the scan mirror 25a. Then, by changing the amplitude angle of the scan mirror 25a, the irradiation region of the light beam S on the sample T is moved in the minor axis direction (direction of arrow B). The direction of the arrow B corresponds to the scanning direction by the scanner 25, and the scanner 25 scans the sample T by scanning the light beam S. Further, the scanner 25 descans the light beam K reflected by the sample T and guides it to the imaging unit 32.

  Next, details of the operation of the control unit 53 when acquiring the observation image of the specimen T will be described with reference to the flowchart of FIG. 2 and the timing chart of FIG.

  Prior to the observation start instruction, the user operates the operation unit 51 to move the stage 29, and arranges the portion to be observed in the sample T at the center of the visual field of the objective lens 28. Further, the stage 29 is moved also in the Z direction, and approximate focusing is performed on a portion to be observed. Note that the user can make these adjustments by visually observing an observation image acquired by the imaging unit 32 and displayed on the display unit 52. Further, when the confocal microscope system 1 has a configuration capable of observing the phase difference, the user may perform the above-described adjustment by visually observing the phase difference image.

  In step S1, the control unit 53 determines whether or not an observation start instruction has been issued. The control unit 53 waits until it is determined that an observation start instruction has been issued, and proceeds to step S2 if it is determined that an observation start instruction has been issued. The observation start instruction is performed by a user operation via the operation unit 51. Further, when the confocal microscope system 1 has a timer or the like, the control unit 53 itself may issue an observation start instruction based on the timer.

  In step S2, the control unit 53 controls each unit and performs pre-scanning. This pre-scan is a scan intended to detect a Top slice and a Bottom slice in step S3 described later. The control unit 53 performs imaging by the imaging unit 29 while moving the stage 29 in the Z direction, and acquires a plurality of pre-scan images.

  Note that the pre-scan is performed under predetermined illumination conditions and imaging conditions. For example, the imaging conditions may be lower than when performing actual observation (hereinafter referred to as “main observation”). Also, the start point and end point in the Z direction in the pre-scan are, for example, positions that are moved up and down by a predetermined width with reference to the position in the Z direction when the observation start instruction is given. Further, the pre-scan interval (the moving speed of the stage 29 and the imaging interval by the imaging unit 32) may be specified in advance by a user operation via the operation unit 51, or may be automatically specified by the control unit 53. good.

  In step S3, the control unit 53 detects a Top slice and a Bottom slice based on the plurality of prescan images obtained by the prescan performed in step S2.

  First, the control unit 53 obtains a histogram of the luminance distribution for each of the plurality of prescan images obtained in step S2, and determines the presence or absence of an object (for example, a cell). Presence / absence of the object can be determined by comparing a threshold value determined in advance based on the illumination condition and the imaging condition at the time of executing the pre-scan and the luminance value of each pre-scan image. The threshold value described above may be specified by a user operation via the operation unit 51.

  For example, the control unit 53 determines the presence / absence of an object in the Z direction in order from the side closer to the objective lens 28, and sets a pre-scan image that is initially determined to have “the object” as a Top slice. Further, the control unit 53 determines the presence / absence of an object along the Z direction, and sets a pre-scan image that has changed from “the object is present” to “the object is not present” as a Bottom slice.

  In addition, when detecting the Top slice and the Bottom slice more accurately, the time point when “the object is present” is changed to “the object is present”, and the case where the “object is present” is changed to “the object is not present”. At this point, the determination of the presence / absence of the object related to the previous pre-scan image may be performed again with the condition regarding the threshold changed narrowly. With such a configuration, it is possible to detect the Top slice and the Bottom slice more accurately like hill-climbing AF.

  Further, the detected Top slice and Bottom slice are reflected in the movement range in the Z direction during the main observation described later. Therefore, in order to ensure that this moving range includes the portion where the object exists, the Top slice may be positioned closer to the objective lens 28 and the Bottom slice may be positioned farther from the objective lens 28. For example, for the Top slice, the pre-scan image closer to the objective lens 28 than the pre-scan image initially determined as “the object is present” is the Top slice, and the Bottom slice is “the object is present”. A pre-scan image farther from the objective lens 28 than the pre-scan image changed to “no object” may be used as a Bottom slice.

  Note that the prescan described in step S2 and the detection of the Top slice and Bottom slice described in step S3 may be performed simultaneously. That is, the control unit 53 determines the presence or absence of an object every time a pre-scan image is acquired. By adopting such a configuration, the pre-scan interval is narrowed until the Top slice is detected, and the pre-scan interval is increased after the Top slice is detected. It is also possible.

  In step S4, the control unit 53 selects a reference slice based on the Top slice and the Bottom slice detected in step S3. The reference slice is a slice that is a target for adjusting the brightness adjustment parameter in step S5 described later. Which slice is selected as the reference slice based on the Top slice and the Bottom slice may be determined in advance, or may be designated by a user operation via the operation unit 51. Here, a description will be given assuming that, in addition to the Top slice and Bottom slice, a total of four slices corresponding to 1/3 and 2/3 of the Top slice and Bottom slice are selected as reference slices.

  In step S5, the control unit 53 adjusts the brightness adjustment parameter for the reference slice selected in step S4. Here, the laser intensity of the laser light source in the light source unit 10 will be described as an example of the brightness adjustment parameter.

  The control unit 53 performs imaging by the imaging unit 32 while adjusting the laser intensity for each of the reference slices selected in step S4. Then, an index value is obtained from the image obtained by imaging, and an optimal brightness adjustment parameter is determined by comparing with an threshold value. The index value is a luminance histogram, the number of luminance saturation pixels, or the like. For example, when the luminance value is 0 to 4095 and the ratio of the pixels having the luminance value of 4095 (luminance saturation pixel) is 0.05 to 0.06%, the laser intensity at that time is the optimum luminance adjustment. It can be determined that it is a parameter.

  It is assumed that the brightness adjustment parameter has been linearly corrected in advance. Further, an image obtained by performing imaging by the imaging unit 32 may be displayed on the display unit 52 while adjusting the laser intensity.

  Further, when the optimum brightness adjustment parameter cannot be obtained even after a predetermined number of adjustments, the optimum brightness adjustment parameter may be obtained again by changing the threshold value to be compared with the index value and relaxing the condition. Further, the user may be notified that the optimum brightness adjustment parameter cannot be obtained using the display unit 52 or the like.

  The brightness adjustment parameter for each reference slice obtained in this way has a different value for each slice. When the brightness adjustment parameter is laser intensity, the value of the laser intensity increases as the slice is farther from the objective lens 28.

  In step S6, the control unit 53 determines the luminance adjustment parameter for each slice of the main observation based on the adjustment result of the luminance adjustment parameter performed in step S5.

  The control unit 53 obtains a luminance adjustment parameter for each slice of the main observation in step S7 described later. The slice to be subjected to the main observation may be determined in advance, or may be designated by a user operation via the operation unit 51. Here, slices at the same interval as the pre-scan performed in Step S2 between the Top slice detected in Step S3 and the Bottom slice are targeted for the main observation.

  For example, the control unit 53 performs spline interpolation using the brightness adjustment parameter of the reference slice obtained in step S5, and determines the brightness adjustment parameter of each slice. An interpolation method other than spline interpolation, such as linear interpolation, may be used. Further, the interpolation method may be determined according to the number of reference slices selected in step S4.

  In step S7, the control unit 53 controls each unit and performs the main observation (Z stack observation). The control unit 53 performs imaging by the imaging unit 29 while moving the stage 29 in the Z direction, and acquires a plurality of observation images.

  Note that this observation is performed using the luminance adjustment parameter for each slice determined in step S6. That is, every time the stage 29 is moved in the Z direction, the control unit 53 performs imaging by the imaging unit 29 using the luminance adjustment parameter of the slice. In the main observation, a three-dimensional image may be generated from a plurality of observation images obtained by imaging.

  In step S8, the control unit 53 records a plurality of observation images generated by the main observation performed in step S7 in a recording unit (not shown), and ends a series of processes. Note that when the three-dimensional image is generated in step S7, the control unit 53 may record only the three-dimensional image, or may record a plurality of observation images together with the three-dimensional image. Furthermore, when recording an image, the brightness adjustment parameter for each slice determined in step S6 may be recorded in association with the image.

<Addition of embodiment>
Instead of performing steps S2 and S3, the reference slice described in step S4 may be selected according to a predetermined condition. For example, the position in the Z direction at the time when the observation start instruction is given is used as a reference, and the position moved up and down by a predetermined width may be used as the reference slice.

  In step S5 and subsequent steps, the laser intensity of the laser light source in the light source unit 10 has been described as an example of the brightness adjustment parameter. However, the intensity (luminance) of incident light finally incident on the imaging unit 32 can be adjusted. A parameter may be used. For example, the scan speed (such as the time for irradiating laser light to one line) may be used as the brightness adjustment parameter, or the gain of the light source unit 10 by voltage adjustment may be used as the brightness adjustment parameter. Also, two or more parameters may be used in combination. Each parameter differs in the intensity of illumination light, the time required for observation, the image quality of the image obtained by the imaging unit 32, the influence on the sample T, and the like. Therefore, a configuration in which the brightness adjustment parameter can be selected as appropriate in accordance with the type of specimen T, the purpose of observation, and the like may be adopted. Furthermore, brightness adjustment parameters other than those described above may be used.

  In the example of FIG. 3, the case where any of the slices at the time of pre-scan is selected as the reference slice, but a slice corresponding to the middle of the slice at the time of pre-scan may be selected as the reference slice. Further, in the example of FIG. 3, the case where the interval between the slices at the time of pre-scanning and the interval between the slices at the time of the main observation are illustrated is exemplified, but these do not necessarily have to match. In either case, it can be dealt with by an interpolation calculation or the like.

  Instead of automatically performing the series of processes described in the flow of FIG. 2, the user may be authenticated and proceed to the next step. For example, a confirmation step by the user may be provided after detecting the Top slice and Bottom slice in Step S3, after selecting the reference slice in Step S4, and after determining the brightness adjustment parameter in Step S6.

  The reference slice selected in step S4, the brightness adjustment parameter determined in step S6, and the like may be recorded in a database and used for subsequent observations. For example, when performing observation with different types of specimen T (including Top slice, Bottom slice, etc.) and observation conditions (slice interval during pre-scan, slice interval during main observation, etc.), the control unit 53 Then, the reference slice and the brightness adjustment parameter recorded at the time of the previous observation are read from the database, and fine adjustment is performed by appropriately calculating according to the conditions. In this way, appropriate main observation can be performed without performing the processing described in steps S2 to S6 in the flow of FIG.

  As described above, according to the present embodiment, the objective lens, the stage on which the test object is placed, the imaging unit that captures an image of the test object, and the objective lens in the optical axis direction of the objective lens, A moving unit that changes a position relative to the stage, and obtains a brightness adjustment parameter for each of at least two surfaces in the test object having different positions in the optical axis direction, and based on the at least two brightness adjustment parameters Thus, a brightness adjustment parameter for performing imaging by the imaging unit is determined on each of the surfaces in the plurality of objects having different positions in the optical axis direction. Therefore, it is possible to acquire an image having substantially the same brightness regardless of the distance from the objective lens. At this time, the user does not need to specify illumination conditions, imaging conditions, and the like for each slice. Therefore, when generating a plurality of images with different positions of the objective lens in the optical axis direction, it is possible to suppress variations in brightness in the generated images.

  The present invention can be similarly applied to a microscope apparatus having a configuration other than that described in FIG. 1 of the present embodiment. For example, a pinhole type confocal stop which has been conventionally used in a confocal microscope may be combined. That is, the above-mentioned confocal slit diaphragm having a slit-shaped opening and a pinhole type confocal diaphragm are provided, and they are exchanged as appropriate according to the type of objective lens to be used and the type of sample to be observed. It is good also as a structure to use.

  In the present embodiment, the confocal microscope system 1 including the light source unit 10, the confocal microscope 20, and the computer 50 has been described as an example. However, the present invention is not limited to this example. For example, in the present embodiment, the example in which the scanner 25 serves as both an illumination system scanner and an imaging system scanner has been described, but an illumination system scanner and an imaging system scanner may be provided independently. Further, the present invention can be similarly applied to a confocal microscope in which an imaging unit (including a simple photodetector and an electronic camera) can be exchanged. Furthermore, the present invention can be similarly applied to a confocal microscope incorporating a light source unit and a computer described in the present embodiment.

DESCRIPTION OF SYMBOLS 1 ... Confocal microscope system, 10 ... Light source part, 20 ... Confocal microscope, 29 ... Stage, 32 ... Imaging part, 50 ... Computer, 53 ... Control part

Claims (8)

An objective lens;
A stage on which the test object is placed;
An imaging unit for imaging an image of the test object;
A moving unit that changes a relative position between the objective lens and the stage in the optical axis direction of the objective lens;
A calculation unit for obtaining a brightness adjustment parameter for each of at least two surfaces in the test object having different positions in the optical axis direction;
A determination unit configured to determine a luminance adjustment parameter for performing imaging by the imaging unit on each of a plurality of surfaces in the plurality of objects having different positions in the optical axis direction based on at least two of the luminance adjustment parameters; A microscope apparatus comprising: The microscope apparatus according to claim 1, wherein
The microscope apparatus, wherein the brightness adjustment parameter includes at least one of illumination intensity when illuminating the test object, a moving speed of the stage, and a time interval of imaging by the imaging unit. The microscope apparatus according to claim 2, wherein
An illumination unit that irradiates the test object with laser light,
The brightness adjustment parameter includes, as the illumination intensity, at least one of an intensity of the laser beam and a gain when the laser beam is generated from a light source. The microscope apparatus according to any one of claims 1 to 3,
The determination unit performs an interpolation operation based on at least two of the brightness adjustment parameters, and determines a brightness adjustment parameter for performing imaging by the imaging unit on each of a plurality of surfaces having different positions in the optical axis direction. A microscope apparatus characterized by that. In the microscope apparatus according to any one of claims 1 to 4,
A controller that performs imaging by the imaging unit at a predetermined time interval while moving the stage at a predetermined speed by the moving unit, and generates a plurality of pre-scan images,
The calculation unit estimates a reference plane in the optical axis direction of the test object based on a plurality of the prescan images, and obtains the brightness adjustment parameter for each of the plurality of planes including the reference plane. A microscope apparatus characterized by the above. The microscope apparatus according to any one of claims 1 to 5,
The control unit performs imaging by the imaging unit while moving the stage by the moving unit based on the luminance parameter determined by the determining unit, and generates a plurality of observation images. apparatus. The microscope apparatus according to claim 6, wherein
A microscope apparatus comprising: a three-dimensional image generation unit that generates a three-dimensional image of the test object based on the plurality of observation images. The objective lens, the stage on which the test object is placed, the imaging unit that captures an image of the test object, and the relative position of the objective lens and the stage are changed in the optical axis direction of the objective lens. A control program for realizing control of a microscope apparatus including a moving unit by a computer,
A calculation step for obtaining a brightness adjustment parameter for each of at least two surfaces in the test object having different positions in the optical axis direction;
A determination step for determining a luminance adjustment parameter for performing imaging by the imaging unit on each of a plurality of surfaces in the plurality of objects having different positions in the optical axis direction based on at least two of the luminance adjustment parameters; A control program comprising:

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