JP2008233227A - Microscope device and control method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably make an observation by precisely correcting a position shift of the optical axis of laser light even when the observation is temporally made for a long time. <P>SOLUTION: The microscope device 1 comprises: a light source 7; an intensity modulator 8, a scanner 14 which makes a scan with intensity-modulated laser light L; an objective lens 15 which converges the laser light L on the sample A and converges fluorescent light generated by a sample A; an optical detector 17 which detects the converged fluorescent light; a laser position detector 11 which detects a position shift of the laser light L from the light source 7 and a laser position corrector 10 which corrects the position shift on the basis of a detection result thereof between the intensity modulator 8 and objective 15; and a control unit 4 which controls them. The control unit 4 controls the intensity modulator 8 to operate before time tracked back by the sum of a time needed to stabilize the intensity modulator 8 from fluorescent light detection start time when the optical detector 17 and scanner 14 are placed in operation and a time needed for the position detection and position shift correction of the laser light L. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microscope apparatus and a control method thereof.

  Conventionally, an illumination optical system for introducing laser light from a light source into a microscope is provided with an intensity modulator composed of an acousto-optic element or an electro-optic element, and a laser beam system is provided between the light source and the intensity modulator. There is known a microscope apparatus in which a beam expander for adjustment and a beam shifter for correcting the optical axis position of a laser are arranged (see, for example, Patent Document 1).

  In the microscope apparatus disclosed in Patent Document 1, a laser beam incident on a narrow opening of an intensity modulator is shattered to cause a power loss, or an optical axis misalignment or an angle change occurs when the wavelength of the laser beam is switched. In order to prevent this, a beam expander or beam shifter is used for correction.

JP 2005-345614 A

  However, even if the optical axis position shift or angle change is corrected once by the beam expander or beam shifter, a laser light source or an intensity modulator is required as time elapses when observing over time for a long time. As a result, the optical characteristics of the optical element change and the optical axis of the laser beam fluctuates. In particular, there is a problem in that stable observation cannot be performed when observation is performed after a long pause time from the previous observation.

  The present invention has been made in view of the above-described circumstances, and can perform stable observation by accurately correcting the positional deviation of the optical axis of laser light even when performing observation over time for a long time. It is an object of the present invention to provide a microscope apparatus and a control method thereof.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source that emits laser light, an intensity modulator that modulates the intensity of laser light from the light source, a scanner that scans laser light that has been intensity-modulated by the intensity modulator, and a scanner that scans the laser light. An objective lens for condensing the fluorescence generated in the specimen, a photodetector for detecting the fluorescence condensed by the objective lens, the intensity modulator, and the objective lens A laser position detector that detects a positional deviation of the laser beam from the light source, a laser position correction unit that corrects a positional deviation of the laser beam based on a detection result by the laser position detection mechanism, and And a control unit for controlling the time required to stabilize the intensity modulator from the fluorescence detection start time when the photodetector and the scanner are operated, and the level of the laser light. Providing a microscope apparatus for controlling so as to actuate the intensity modulator before time going back by the sum of the time required for the displacement detection and the positional deviation correction.

  According to the present invention, when fluorescence detection is started, the laser light emitted from the light source is intensity-modulated by the intensity modulator, scanned by the scanner, and condensed on the sample by the objective lens. In the specimen, the fluorescent material is excited at the position where the laser beam is irradiated to generate fluorescence, and the generated fluorescence is collected by the objective lens and then detected by the photodetector. A fluorescence image can be constructed based on the scanning position by the scanner and the fluorescence intensity detected by the photodetector.

  The laser beam emitted from the intensity modulator is detected by the laser position detection unit for the positional deviation of the optical axis, and the laser position correction unit corrects the optical axis deviation based on the detection result. Therefore, even if the laser beam is misaligned for some reason, it can be corrected and the sample can be accurately irradiated.

  In this case, when a plurality of microscopic observations are performed over a long period of time, even if the positional deviation has been eliminated by correction once, the positional deviation is further caused by a change in optical characteristics over time. It is thought that it will occur. According to the present invention, the control unit operates the intensity modulator prior to the start of fluorescence detection, and performs laser beam position shift detection and position shift correction after the intensity modulator is stabilized. Light misalignment has been eliminated. In this case, according to the present invention, the positional deviation detection of the laser beam can be performed after the intensity modulator is stabilized, and the positional deviation of the laser beam can be accurately corrected.

In the above invention, the control unit is configured such that a plurality of set fluorescence detection start times are closer to the sum of the time required for stabilizing the intensity modulator and the time required for detecting and correcting the positional deviation of the laser beam. In this case, the intensity modulator may be controlled so as not to be stopped between the fluorescence detection start times.
By doing so, prior to the second and subsequent fluorescence detections, laser beam positional deviation detection and positional deviation correction can be performed without waiting for the stability of the intensity modulator. In addition, the optical axis deviation of the laser beam can be corrected with high accuracy, and the sample can be irradiated with high accuracy.

  In the above invention, the light source can emit laser light having a plurality of wavelengths, and the controller starts fluorescence detection by operating the scanner and the photodetector when the wavelength of the laser light is switched. Control is performed so that the switching of the wavelength of the laser beam is started before the time which is backed by the sum of the time required to stabilize the light source from the time and the time required for detecting and correcting the positional deviation of the laser beam. Also good.

  In this way, the laser light wavelength is switched to stabilize the light source, and the intensity modulator is operated to stabilize the intensity modulator, and then the laser light positional deviation detection and positional deviation correction are performed with high accuracy. The observation can be started by irradiating the sample with laser light with high accuracy from a predetermined fluorescence detection start time.

  In the above invention, the light source can emit laser light of a plurality of wavelengths, and the control unit switches the wavelength with respect to the optical axis of the laser light at the head wavelength before the group of observations performed at the same time. The amount of positional deviation is stored for each wavelength with respect to other wavelengths, and when observing at the head wavelength, the time required to stabilize the light source from the fluorescence detection start time for operating the photodetector and the scanner, and laser light Fluorescence detection that starts switching of the wavelength of the laser light before a time that goes back by the sum of the time required for position shift detection and position shift correction, and activates the photodetector and the scanner during observation with the remaining wavelengths The intensity modulator is actuated before the time required to stabilize the intensity modulator from the start time, and before the time required to stabilize the light source. The switching of the wavelength of the laser light is started, may be controlled to perform positional deviation correction by position shift amount stored during these times.

  By doing so, only when observing with the first wavelength, the light source is stabilized and the intensity modulator is stabilized, and then the laser beam positional deviation detection and positional deviation correction are performed. The detection of the positional deviation of the laser beam is omitted, and the positional deviation correction is performed by the positional deviation amount based on the wavelength change stored in advance. In a group of observations performed at the same time, it is possible to detect and correct optical axis deviations due to changes in optical characteristics over time by detecting and correcting positional deviations only when observing with the first wavelength. Even in observation over time, the specimen can be accurately irradiated with laser light. In addition, when observing with a wavelength other than the first wavelength, it is possible to perform observation for different wavelengths in a short time by omitting the detection of positional deviation, and it is possible to reduce the time required for a group of observations. .

In the above invention, a shutter may be provided that blocks the optical path before the objective lens and opens the optical path between the fluorescence detection start time and the fluorescence detection end time by the photodetector.
By doing so, at the time of detecting the positional deviation of the laser light before the fluorescence detection start time and correcting the positional deviation, the optical path is blocked by the operation of the shutter, thereby preventing the laser light from being irradiated on the specimen. . Thereby, generation | occurrence | production of the fading etc. of the sample by a laser beam can be prevented beforehand, and the soundness of a sample can be improved. In addition, by opening the optical path between the fluorescence detection start time and the fluorescence detection end time, it is possible to irradiate the sample with laser light whose positional deviation has been corrected with high accuracy.

  Further, the present invention modulates the intensity of the laser beam emitted from the light source by the intensity modulator, detects and corrects the positional deviation of the optical axis of the laser beam, scans the corrected laser beam on the sample, A control method of a microscope apparatus for detecting fluorescence generated in a specimen, which is used to detect a time deviation required to stabilize the intensity modulator from a preset fluorescence detection start time, and to detect and correct a positional deviation of a laser beam. Provided is a control method of a microscope apparatus that controls to operate an intensity modulator before a time that is back by the sum of required time.

  According to the present invention, the intensity modulator is operated prior to the start of fluorescence detection, and the position deviation of the laser light is detected and corrected after the intensity modulator is stabilized. Misalignment has been resolved. In this case, according to the present invention, the positional deviation detection of the laser beam can be performed after the intensity modulator is stabilized, and the positional deviation of the laser beam can be accurately corrected.

  In the above invention, when a plurality of set fluorescence detection start times are closer to the sum of the time required to stabilize the intensity modulator and the time required for laser beam position shift detection and position shift correction. The intensity modulator may be controlled so as not to stop between these fluorescence detection start times.

  By doing so, prior to the second and subsequent fluorescence detections, laser beam positional deviation detection and positional deviation correction can be performed without waiting for the stability of the intensity modulator. In addition, the optical axis deviation of the laser beam can be corrected with high accuracy, and the sample can be irradiated with high accuracy.

  In the above invention, the light source can emit laser light having a plurality of wavelengths, and when the wavelength of the laser light is switched, the light source is stabilized from the fluorescence detection start time at which the scanner and the photodetector are operated. It is also possible to perform control so that the switching of the wavelength of the laser light is started before a time that is back by the sum of the time required for the detection and the time required for detecting and correcting the positional deviation of the laser light.

  In this way, the laser light wavelength is switched to stabilize the light source, and the intensity modulator is operated to stabilize the intensity modulator, and then the laser light positional deviation detection and positional deviation correction are performed with high accuracy. The observation can be started by irradiating the sample with laser light with high accuracy from a predetermined fluorescence detection start time.

  In the above invention, the light source can emit laser light having a plurality of wavelengths, and before the group of observations performed at the same time by switching the wavelength, the positional deviation amount of the laser light at the head wavelength with respect to the optical axis is changed. Are stored for each wavelength, and at the time of observation by the head wavelength, the time required to stabilize the light source from the fluorescence detection start time at which the photodetector and the scanner are operated, the positional deviation detection of the laser light, and Starting the switching of the wavelength of the laser light before the time that goes back by the sum required for the positional deviation correction, and when observing with the remaining wavelengths, the intensity from the fluorescence detection start time for operating the photodetector and the scanner The intensity modulator is activated before the time required to stabilize the modulator, and the wavelength of the laser light is adjusted before the time required to stabilize the light source. It was started Toggles may control to perform positional deviation correction by position shift amount stored during these times.

  By doing so, only when observing with the first wavelength, the light source is stabilized and the intensity modulator is stabilized, and then the laser beam positional deviation detection and positional deviation correction are performed. The detection of the positional deviation of the laser beam is omitted, and the positional deviation correction is performed by the positional deviation amount based on the wavelength change stored in advance. In a group of observations performed at the same time, it is possible to detect and correct optical axis deviations due to changes in optical characteristics over time by detecting and correcting positional deviations only when observing with the first wavelength. Even in observation over time, the specimen can be accurately irradiated with laser light. In addition, when observing with a wavelength other than the first wavelength, it is possible to perform observation for different wavelengths in a short time by omitting the detection of positional deviation, and it is possible to reduce the time required for a group of observations. .

Moreover, in the said invention, it is good also as opening an optical path between fluorescence detection start time and fluorescence detection end time, and interrupting | blocking an optical path in other than that.
By doing so, the laser beam is prevented from being irradiated to the specimen by blocking the optical path during the positional deviation detection and positional deviation correction of the laser light before the fluorescence detection start time. Thereby, generation | occurrence | production of the fading etc. of the sample by a laser beam can be prevented beforehand, and the soundness of a sample can be improved. In addition, by opening the optical path between the fluorescence detection start time and the fluorescence detection end time, it is possible to irradiate the sample with laser light whose positional deviation has been corrected with high accuracy.

  According to the present invention, even when observation is performed over time for a long time, the positional deviation of the optical axis of the laser light is accurately corrected, and stable observation can be performed.

A microscope apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the microscope apparatus 1 according to this embodiment includes a light source unit 2 that emits a short pulse laser light L for multiphoton excitation, and a short pulse laser light L emitted from the light source unit 2. A microscope main body 3 that irradiates the specimen A to detect fluorescence, a control unit 4 that controls these, a display unit 5 that displays an image acquired by the microscope main body 3, and an input unit 6 that inputs observation conditions. I have.

  The light source unit 2 includes a laser light source 7 that emits a short pulse laser beam L, an acoustooptic element (intensity modulator) 8 such as an AOTF that modulates the intensity of the short pulse laser beam L emitted from the light source, and A dispersion compensator 9 for imparting a negative chirp that preferentially transmits a short wavelength light component, a beam shifter 10 for adjusting the positional deviation and inclination of the optical axis of the short pulse laser light L, and the optical axis of the short pulse laser light L. A position shift detector 11 that detects position shift and inclination, and a shutter 12 that turns on and off the emission of the short pulse laser beam L from the light source unit 2 are provided.

  As shown in FIG. 2, the beam shifter 10 includes two plane mirrors 10a and 10b and a motor (not shown) for driving them, and simultaneously drives the mirrors 10a and 10b along the X and Y axes. Thus, the optical axis of the short pulse laser beam L can be translated, and the tilt of the optical axis of the short pulse laser beam L can be adjusted by swinging the mirrors 10a and 10b about the axes θx and θy. It can be done.

  The position shift detector 11 includes, for example, a beam splitter 11a that branches a part of the short pulse laser light L from the optical path downstream of the beam shifter 10, and a different optical path for the short pulse laser light L branched from the optical path by the beam splitter 11a. Two sensors 11b and 11c that detect the length are provided. Reference numeral 11d is a beam splitter, and reference numeral 11e is a mirror.

  Each of the sensors 11b and 11c is, for example, a four-divided photodiode, and the original balance of the short pulse laser light L is determined by the output balance of four sensor portions (not shown) corresponding to the spot position of the received short pulse laser light L. The offset amount along the direction perpendicular to the optical axis with respect to the optical axis can be detected. In addition, the amount of inclination of the short pulse laser beam L with respect to the original optical axis can be detected according to the difference in offset amount between the two sensors 11b and 11c via different optical path lengths.

  The microscope main body 3 includes a stage 13 on which the specimen A is mounted, a scanner 14 that two-dimensionally scans the short pulse laser light L output from the light source unit 2, and the short pulse laser light L scanned by the scanner 14. An objective lens 15 that condenses and irradiates the specimen A while condensing the fluorescent light generated in the specimen A, and a dichroic mirror 16 that branches the fluorescent light collected by the objective lens 15 from the optical path of the short pulse laser beam L. And a photodetector 17 for detecting the fluorescence branched by the dichroic mirror 16. Reference numeral 18 denotes a condenser lens.

The scanner 14 includes, for example, two galvanometer mirrors 14a and 14b that are supported so as to be swingable about mutually perpendicular axes. According to the scanner 14, the two pulsed galvanometer mirrors 14a and 14b are swung in synchronization, whereby the short pulse laser light L can be scanned two-dimensionally, for example, in a raster scan method. Yes.
The photodetector 17 is, for example, a photomultiplier tube.

  The control unit 4 includes a storage unit 19 that stores the maximum correction processing time Tmax necessary for correcting the positional deviation of the optical axis of the short pulse laser beam L. The storage unit 19 stores other temporary data.

The maximum correction processing time Tmax is expressed by the sum of the time required for position shift detection and the total time Ta i required for position shift correction and the time Tao required for stabilizing the acoustooptic device 8.
The control unit 4 performs the following processing according to the observation conditions input from the input unit 6.

As the observation conditions, for example, the number of image acquisitions, the image acquisition start time, the interframe interval, the scan speed, and the image size are input. Based on the input observation conditions, the control unit 4 determines a waiting time Trest between a plurality of observation operations with a time interval and a scan start time Tnext for operating the scanner 14 and the photodetector 17 in each observation operation. The correction processing start time Tstart in each observation operation is calculated by the following formula.
Tstart = Tnext-Tmax

  Next, the control unit 4 waits until the correction processing start time Tstart, and then shuts off the optical path by the shutter 12, while operating the acoustooptic device 8 in a state where the optical path is blocked by the shutter 12, An internal shutter (not shown) is opened so that the pulse laser beam L is emitted. At this time, the dimming value of the acoustooptic device 8 may be equal to that at the time of fluorescence detection.

At this time, the blanking operation by the acoustooptic device 8, that is, the process for turning on and off the short pulse laser light L to limit the irradiation range of the short pulse laser light L irradiated to the specimen A is stopped. It is like that.
Further, the control unit 4 activates the position shift detector 11 when the time Tao required to stabilize the acoustooptic element 8 from the start of the operation of the acoustooptic element 8 to activate the optical axis of the short pulse laser beam L. The position shift of the optical axis is detected and the beam shifter 10 is operated to correct the position shift of the optical axis.

Then, the control unit 4 opens the shutter 12 at the stage where the optical axis positional deviation correction by the beam shifter 10 is completed.
Thereafter, a blanking operation by the acousto-optic element 8 is started, and the scanner 14 and the photodetector 17 are operated to acquire two-dimensional fluorescence luminance information in a predetermined range of the specimen A. This completes one observation operation, so that the above steps are repeated as many times as the number of image acquisitions input under the observation conditions.
The luminance information of the specimen A detected by the photodetector 17 is stored in the storage unit 19 in association with the scanning position information of the scanner 14, and a fluorescent image is created by the control unit 4 and displayed on the display unit 5. It has become.

A control method of the microscope apparatus 1 according to the present embodiment configured as described above will be described below with reference to FIGS. 3 and 4.
In order to perform long-time observation (long-time time-lapse observation) of the specimen A using the microscope apparatus 1 according to the present embodiment, first, imaging conditions are input from the input unit 6 (step S1).

The control unit 4 calculates the standby time Trest, the scan start time Tnext, and the correction process start time Tstart based on the input imaging conditions (step S2).
Then, it is determined whether or not the time T has reached the correction processing start time Tstart (step S3). If the time T has been reached, the shutter 12 is actuated to block the optical path to the microscope body 3 (step S4). .

  In this state, the acoustooptic device 8 is operated (step S5), and the incidence of the short pulse laser beam L on the dispersion compensator 9 is started. At this time, it is determined whether or not the time Tao required for the acoustooptic device 8 to stabilize has elapsed (step S6). After the time Tao has elapsed, the positional deviation of the optical axis by the positional deviation detector 11 is detected. Detection is performed (step S7), and the positional shift is corrected by the beam shifter 10 (step S8).

As a result, the positional deviation of the optical axis of the short pulse laser beam L is corrected, so that the short pulse laser beam L is accurately incident on the microscope body 3 by opening the shutter 12 (step S9).
The short pulse laser beam L incident on the microscope main body 3 is two-dimensionally scanned by the scanner 14, condensed on the specimen A by the objective lens 15, and generates fluorescence by the multiphoton excitation effect at the collection position. The generated fluorescence is condensed by the objective lens 15 and then branched from the optical path of the short pulse laser beam L by the dichroic mirror 16 and detected by the photodetector 17 (step S11).

  At this time, the blanking operation is performed by the operation of the acoustooptic device 8, and the irradiation range of the short pulse laser light L to the specimen A is limited (step S10). Therefore, irradiation of the short pulse laser beam L to the specimen A during the blanking period is stopped, and the short pulse laser beam L having a uniform photon density is irradiated within a predetermined range. Thereby, the fluorescence image information for one sheet is acquired, and one observation operation is completed. At this time, it is determined whether or not the number of times of image acquisition set as the observation condition has been reached. If not, the steps from Step S2 are repeated (Step S12).

  After the set number of pieces of fluorescent image information are acquired, a fluorescent image is generated based on the fluorescent image information from the photodetector and the scanning position information of the scanner 14 (step S13), and is displayed on the display unit. It is displayed (step S14).

  As described above, according to the microscope apparatus 1 and the control method thereof according to the present embodiment, since the acoustooptic element 8 waits for stability, the optical axis position deviation detection and the position deviation correction of the short pulse laser beam L are performed. The optical axis of the short pulse laser beam L can be accurately corrected. Since the sample A is irradiated with the short pulse laser light L with the optical axis corrected with accuracy as described above, the sample A can be prevented from being damaged by the optical components and power loss, and the stable uniform short pulse laser light L can be applied to the sample A. The specimen A can be accurately observed by irradiation.

  In addition, since the optical axis is corrected every time a fluorescent image is acquired, the optical axis position shifts over time due to the temperature rise of the laser light source 7 and other optical components in time-lapse observation over a long period of time. However, there is an advantage that this can be corrected accurately and stable observation can be performed.

  In the present embodiment, the correction processing start time Tstart is calculated and the operation of the acoustooptic device 8 is started for each of a plurality of observation operations performed at intervals. When the interval is short and the correction processing start time Tstart cannot be calculated, as shown in FIG. 5, the acoustooptic element 8 is not turned on and off between observation operations, and the acoustooptic element 8 is always in an operating state. Deviation detection and position deviation correction may be performed. In this case, it is only necessary to cause the acoustooptic device 8 to perform a blanking operation and to open the shutter 12 only during the time when the scanner 14 and the photodetector 17 are operated to acquire a fluorescent image.

Next, a microscope apparatus 1 and a control method thereof according to the second embodiment of the present invention will be described with reference to FIGS.
In the description of the present embodiment, portions having the same configuration as those of the microscope apparatus 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

The microscope apparatus 1 according to the present embodiment includes a variable laser light source that can selectively emit an arbitrary wavelength in a plurality of wavelengths, for example, a wavelength range of 700 nm to 1000 nm, as the laser light source 7.
Further, the storage unit 19 includes a time Tali required for position shift correction, a time Texm required for position shift detection, a time Tao required for the acoustooptic device 8 to be stabilized, and a time required for the laser light source 7 to be stabilized after wavelength switching. Tlaser is stored. Here, it is known that the standby time Tlaser at the time of wavelength switching increases as the wavelength change width Δλ increases. For example, the relationship between Δλ and Tlaser shows a distribution as shown in FIG. Therefore, it is preferable to store the system-specific distribution in the storage unit 19 in advance.

  First, the control unit 4 performs a pre-scan without irradiating the sample A with the short pulse laser beam L, thereby obtaining a positional deviation correction value for each wavelength and storing it in the storage unit 19. Specifically, as shown in FIG. 6, the emission wavelength of the laser light source 7 is adjusted to the first wavelength λA, the acoustooptic device 8 is simultaneously operated, and the shutter 12 is closed.

  In the case of FIG. 6, the time Tlaser required for the laser light source 7 to stabilize is longer than the time Tao required for the acoustooptic device 8 to stabilize, so the time Tlaser required for the laser light source 7 to stabilize is awaited. Then, position deviation detection and position deviation correction are performed, and the position deviation correction value is stored in the storage unit 19. Thereafter, the positional deviation correction value is similarly obtained for the second wavelength λB and stored in the storage unit 19. When observing other wavelengths as well, a positional deviation correction value is obtained in the same manner and stored in the storage unit 19.

Next, long time lapse observation is performed.
In this embodiment, the long time lapse observation is a group of observation operations for detecting fluorescence from the specimen A while switching the wavelength of the short pulse laser light L at substantially the same time (in FIG. 7, according to the first wavelength λA). This is an observation method in which the observation operation and the observation operation using the second wavelength λB) are performed a plurality of times over a long period of time with a time interval, and the change over time of the specimen A is observed.

  Specifically, as shown in FIG. 9, first, an observation condition is input from the input unit 6 (step S21). Then, the wavelength λA of the short pulse laser beam L emitted from the laser light source 7 is selected (step S22), and an observation operation based on this is performed (step S23). Next, the wavelength λB is selected (step S24), and an observation operation based on this is performed (step S25).

  Then, it is determined whether or not the group of observation operations has been performed a predetermined number of times with a time interval in accordance with the input observation conditions (step S26), and after the set number of observations has been performed. Based on the fluorescence image information from the photodetector and the scanning position information of the scanner 14, a fluorescence image is generated (step S27) and displayed on the display unit (step S28).

Here, each observation operation (steps S23 and S25) will be described with reference to FIG. 10 and FIG.
First, based on the wavelength λ of the selected short pulse laser beam L, a positional deviation correction value is read from the storage unit 19 (step S31). Thereafter, the position shift correction depending on the wavelength of the short pulse laser beam L is performed by the operation of the beam shifter 10 based on the position shift correction value obtained in the pre-scan (step S32).

In addition, the control unit 4 determines the standby time Trest between a plurality of observation operations with a time interval based on the input observation conditions, and the scan start time Tnext for operating the scanner 14 and the photodetector 17 in each observation operation. Is calculated, the time Tali + Texm + Tlaser required for the correction process is read, and the correction process start time Tstart is calculated by the following equation (step S33).
Tstart = Tnext- (Tali + Texm + Tlaser)

  When the correction processing start time Tstart is reached (step S34), the shutter 12 is closed (step S35), the wavelength of the laser light source 7 is switched to the first wavelength λA (step S36), and the acoustooptic device 8 is activated. (Step S37). By waiting for the elapse of time Tlaser (step S38), the laser light source 7 is stabilized and the acoustooptic device 8 is also stabilized, so that the position deviation is detected (step S39), and the position deviation correction value obtained in the pre-scan. It is determined whether or not a further positional deviation has occurred (step S40).

  If a positional shift has occurred, the positional shift correction by the beam shifter 10 is performed (step S41), and a new positional shift correction value is stored in the storage unit in association with the wavelength of the short pulse laser beam. (Step S42). On the other hand, when there is no positional deviation, no positional deviation correction is performed.

Thereafter, waiting for the scan start time Tnext (step S43), the shutter 12 is opened (step S44), the acoustooptic device 8 is blanked (step S45), and the scanner 14 and the photodetector 17 are activated. (Step S46).
Thereby, the observation operation ends.

  According to the microscope apparatus 1 and the control method thereof according to the present embodiment, the setting of the acousto-optic element 8 and the setting of the dispersion compensator 9 are performed by switching the wavelength of the short pulse laser light L emitted from the laser light source 7. Although the optical axis fluctuates due to switching, the positional deviation detection and the positional deviation correction are performed after waiting for the time required for the laser light source 7 to stabilize, so the optical axis positional deviation is always accurately corrected and the microscope main body is corrected. 3 can be made incident.

  In addition, pre-scanning is performed in advance to store a positional deviation correction value for each wavelength, and whenever the wavelength is switched, the stored positional deviation correction value is read and positional deviation correction is performed. The deviation can be corrected quickly. In addition, since the position shift is detected every time the fluorescence detection operation is performed by operating the scanner 14 and the light detector 17, in an observation mode in which the position shift of the optical axis is likely to occur with time like the long time-lapse observation. In addition, the optical axis deviation can be corrected with high accuracy.

  In the present embodiment, the acoustooptic device 8 is operated simultaneously with the start of wavelength switching of the short pulse laser light L emitted from the laser light source 7, but instead, as shown in FIG. The acousto-optic element 8 starts to operate at a time Texm + Tali required for position deviation detection and position deviation correction from the scan start time Tnext and at a time that goes back by the time Tao necessary for the acousto-optic element 8 to be stabilized. Also good.

  In addition, depending on the observation conditions, when a sufficient time can be secured for a group of observation operations, the laser light source 7 is stabilized as the wavelength of the short pulse laser beam l is switched as shown in FIG. The acoustooptic device 8 may be operated after the time required for the operation has elapsed, that is, after the laser light source 7 is stabilized.

  Further, in the present embodiment, the variable laser light source 7 that can arbitrarily change the wavelength of the short pulse laser light L in the range of 700 nm to 1000 nm is employed, but instead, a single wavelength laser light can be emitted. You may employ | adopt the laser light source unit which combined the some laser light source. In this case, as compared with the variable laser light source, the time required for the laser light source to become stable can be reduced to a negligible level. Therefore, it is only necessary to set the correction processing start time Tstart according to the time required for the waiting time when switching the wavelength of the short pulse laser light L to be greatly reduced and the acoustooptic device 8 to be stabilized.

  Further, in the present embodiment, in each group of observation operations performed almost at the same time, the optical axis position deviation is detected every time the wavelength of the short pulse laser beam L is switched. Instead, in the prescan, the difference value of the positional deviation of the optical axis between the first wavelength (leading wavelength) and the other wavelengths in each group of observation operations is detected and stored, and FIG. As shown in FIG. 4, position shift detection is performed only when the first wavelength is set in each group of observation operations, and only position shift correction based on the stored position shift difference value is performed at other wavelengths. You may decide.

The difference value of the positional deviation of the optical axis generated by switching the wavelength does not change even when the temperature of the optical component rises, so this is memorized, and the positional deviation of the optical axis due to the temperature rise of the optical component By detecting and correcting at the first wavelength of a group of observation operations, it is possible to perform position shift correction accompanying temperature rise for each wavelength only by correcting position shift for the other wavelengths based on the difference value.
By doing in this way, it is not necessary to detect the positional deviation of the optical axis for switching the wavelength, and therefore it is possible to shorten the standby time associated with the wavelength switching.

That is, if only the optical axis positional deviation correction based on the stored difference value is performed, it can be performed in an open loop, and there is no need to wait for the stability of the laser light source 7 and the acousto-optic element 8. Therefore, the positional deviation correction can be performed simultaneously with the start of the correction process started by closing the shutter 12, and the interval of the fluorescence detection operation performed by operating the scanner 14 and the photodetector 17 can be made stable for the laser light source 7. It can be shortened only to the longer one of the time required or the time required for stabilization of the acoustooptic device 8.
Thereby, a plurality of observation operations performed by switching a plurality of wavelengths can be performed as close as possible to each other, and the simultaneity in a group of observation operations can be enhanced.

1 is an overall configuration diagram schematically showing a microscope apparatus according to a first embodiment of the present invention. It is a figure which shows an example of the position shift correction mechanism with which the microscope apparatus of FIG. 1 is equipped. It is a flowchart which shows the control method of the microscope apparatus of FIG. It is a time chart explaining the control method of FIG. It is a time chart which shows the modification of the control method of FIG. It is a flowchart which shows the control method of the microscope apparatus which concerns on the 2nd Embodiment of this invention. It is a time chart explaining the control method of FIG. It is a graph which shows the relationship between the wavelength interval of the laser light source in the microscope apparatus of FIG. 6, and waiting time. 7 is a flowchart showing a main flow of a control method of the microscope of FIG. 6. 10 is a flowchart for explaining an observation operation routine in the flowchart of FIG. 9. It is a time chart which shows the 1st modification of the control method of FIG. It is a time chart which shows the 2nd modification of the control method of FIG. It is a time chart which shows the 3rd modification of the control method of FIG. It is a time chart which shows the 4th modification of the control method of FIG.

Explanation of symbols

A Specimen L Short pulse laser beam (Laser beam)
Tao Time required to stabilize the intensity modulator Tali, Tali + Texm Time required to detect and correct laser misalignment Tnext Fluorescence detection start time 1 Microscope device 4 Control unit 7 Laser light source (light source)
8 Acousto-optic elements (intensity modulator)
10 Beam shifter (laser position correction unit)
11 Laser Position Detection Unit 12 Shutter 14 Scanner 15 Objective Lens 17 Photodetector

Claims (10)

A light source that emits laser light;
An intensity modulator for modulating the intensity of laser light from the light source;
A scanner that scans laser light intensity-modulated by the intensity modulator;
An objective lens for condensing the fluorescence generated in the specimen while condensing the laser beam scanned by the scanner on the specimen;
A photodetector for detecting fluorescence collected by the objective lens;
A laser position detector that is disposed between the intensity modulator and the objective lens and detects a positional deviation of the laser light from the light source;
A laser position correction unit that corrects a positional deviation of the laser beam based on a detection result by the laser position detection mechanism;
A control unit for controlling these,
The control unit goes back by the sum of the time required to stabilize the intensity modulator and the time required to detect and correct the positional deviation of the laser light from the fluorescence detection start time at which the photodetector and the scanner are operated. Microscope device that controls the intensity modulator to operate before the specified time.   When the plurality of set fluorescence detection start times are closer to the sum of the time required for stabilizing the intensity modulator and the time required for laser beam position shift detection and position shift correction, The microscope apparatus according to claim 1, wherein control is performed so that the intensity modulator is not stopped between these fluorescence detection start times. The light source is capable of emitting laser light having a plurality of wavelengths;
When the wavelength of the laser beam is switched, the control unit detects the time required to stabilize the light source from the fluorescence detection start time at which the scanner and the photodetector are operated, and detects and corrects the positional deviation of the laser light. The microscope apparatus according to claim 1, wherein control is performed so as to start switching of the wavelength of the laser light before a time traced back by the sum of the time required for the operation. The light source is capable of emitting laser light having a plurality of wavelengths;
Before the group of observations performed at the same time by switching the wavelength, the control unit stores the positional deviation amount with respect to the optical axis of the laser light at the head wavelength for each wavelength,
At the time of observation by the head wavelength, the time required for stabilizing the light source and the time required for detecting and correcting the positional deviation of the laser light are traced back from the fluorescence detection start time at which the photodetector and the scanner are operated. Start switching the wavelength of the laser light before the time
When observing with the remaining wavelengths, the intensity modulator is operated before the time required to stabilize the intensity modulator from the fluorescence detection start time at which the photodetector and the scanner are operated to stabilize the light source. 2. The microscope apparatus according to claim 1, wherein switching of the wavelength of the laser beam is started before the time required, and control is performed so as to perform positional deviation correction by a positional deviation amount stored during these times.   The microscope according to any one of claims 1 to 4, further comprising a shutter that blocks an optical path in a front stage of the objective lens and opens the optical path between a fluorescence detection start time and a fluorescence detection end time by the photodetector. apparatus. The intensity of the laser beam emitted from the light source is modulated by the intensity modulator, the positional deviation of the optical axis of the laser beam is detected and corrected, the corrected laser beam is scanned over the sample, and the fluorescence generated in the sample is detected. A control method of a microscope apparatus to detect,
Operates the intensity modulator before the time that is the sum of the time required to stabilize the intensity modulator from the preset fluorescence detection start time and the time required to detect and correct the positional deviation of the laser beam. A control method of a microscope apparatus for controlling to perform.   When a plurality of set fluorescence detection start times are closer than the sum of the time required to stabilize the intensity modulator and the time required for laser beam position shift detection and position shift correction, these fluorescence detections The method for controlling a microscope apparatus according to claim 6, wherein the intensity modulator is controlled not to be stopped between the start times. The light source is capable of emitting laser light having a plurality of wavelengths;
When the wavelength of the laser beam is switched, a time required for stabilizing the light source from a fluorescence detection start time for operating the scanner and the photodetector, and a time required for detecting the positional deviation of the laser light and correcting the positional deviation. The method for controlling a microscope apparatus according to claim 6, wherein control is performed so as to start switching of the wavelength of the laser beam before a time that is back by the sum. The light source is capable of emitting laser light having a plurality of wavelengths;
Before the group of observations that are performed at the same time by switching the wavelength, the positional deviation amount with respect to the optical axis of the laser beam at the leading wavelength is stored for each wavelength for the other wavelengths,
At the time of observation by the head wavelength, the time required for stabilizing the light source and the time required for detecting and correcting the positional deviation of the laser light are traced back from the fluorescence detection start time at which the photodetector and the scanner are operated. Start switching the wavelength of the laser light before the time
When observing with the remaining wavelengths, the intensity modulator is operated before the time required to stabilize the intensity modulator from the fluorescence detection start time at which the photodetector and the scanner are operated to stabilize the light source. 7. The method of controlling a microscope apparatus according to claim 6, wherein switching of the wavelength of the laser beam is started before the time required, and control is performed so that the positional deviation is corrected by the amount of positional deviation stored during these times.   The method for controlling a microscope apparatus according to any one of claims 6 to 9, wherein an optical path is opened between a fluorescence detection start time and a fluorescence detection end time, and the optical path is blocked at other times.

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