Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0036—Scanning details, e.g. scanning stages
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
  • G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
  • G02B26/10—Scanning systems
  • G02B26/101—Scanning systems with both horizontal and vertical deflecting means, e.g. raster or XY scanners
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
  • G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
  • G02B26/10—Scanning systems
  • G02B26/105—Scanning systems with one or more pivoting mirrors or galvano-mirrors
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
  • G02B27/0025—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration
  • G02B27/0031—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration for scanning purposes

Definitions

• the present invention relates to a laser scanning device and a laser scanning microscope.
• a general laser scanning microscope includes a galvanometer mirror that scans a specimen surface in the X direction with a laser beam, and a galvanometer mirror that scans in the Y direction. If these two galvanometer mirrors are controlled in a coordinated manner, it is possible to observe the laser beam trajectory (hereinafter referred to as “scan line”) as a free curve (hereinafter referred to as “free line observation”).
• scan line the laser beam trajectory
• free line observation a free curve
• the axon of a nerve cell has a string shape, but if a free line is observed with a scanning line that traces the axon, it is possible to capture high-speed changes that occur in the axon.
• the specimen is a living body, it is susceptible to damage, and if the specimen is fluorescently stained, fading also occurs, so the number of times the specimen is irradiated with laser light must be kept to a minimum. .
• an object of the present invention is to provide a laser scanning device and a laser scanning microscope capable of surely setting conditions during laser scanning while suppressing damage to an irradiated surface.
• the laser scanning device of the present invention includes an optical deflecting unit disposed in an optical path of laser light directed to a surface to be scanned, a user interface that allows a user to specify an operation content of the optical deflecting unit, and the specified operation Generating means for generating a drive signal for the light deflection means according to the content; driving the light deflection means with the drive signal while the laser beam is turned off; and the light deflection means during the drive. And a test means for measuring the operation content of.
• the laser scanning device of the present invention includes an optical deflecting unit disposed in an optical path of laser light toward the surface to be scanned, a user interface that allows a user to specify the operation content of the optical deflecting unit, and Generating means for generating a drive signal for the light deflection means in accordance with the specified operation content; and the driving in a state where the intensity of the laser beam is weaker than the intensity of the scanning surface during the main scan.
• the optical deflection means is driven on a trial basis by a signal, and a test means for measuring the operation content of the light deflection means during the driving is provided.
• the user interface notifies the user of the measured operation content.
• the laser scanning device of the present invention further includes correction means for comparing the measured operation content with the designated operation content and correcting the drive signal so that the former approaches the latter. Is desirable.
• the laser scanning microscope of the present invention includes the laser scanning device of the present invention and a detector that detects the intensity of light generated on the surface to be scanned. The invention's effect
• [001 1] According to the present invention, it is possible to realize a laser scanning apparatus and a laser scanning microscope capable of reliably setting conditions during laser scanning while suppressing damage to an irradiated surface.
• FIG. 1 is an overall configuration diagram of the system.
• FIG. 2 is a configuration diagram of a galvano scanner 11 and a controller 20.
• FIG. 4 is a diagram showing a setting screen.
• FIG. 5 is a diagram for explaining a method of generating a drive waveform.
• FIG. 6 is a diagram showing a setting screen at another timing.
• FIG. 7 is a diagram showing a display example of observation information.
• FIG. 8 is an operation flowchart (first half) of the controller 20 and the computer 21 in the second embodiment.
• FIG. 9 is an operation flowchart (second half) of the controller 20 and the computer 21 in the second embodiment.
• FIG. 10 is a diagram showing a setting screen according to the second embodiment.
• FIG. 11 is a diagram showing a setting screen of the second embodiment at another timing.
• This embodiment is an embodiment of a fluorescence confocal laser scanning microscope system.
• FIG. 1 is an overall configuration diagram of the present system. As shown in FIG. 1, this system includes a microscope main body 100, a controller 20, a computer 21, a monitor 22 and an input device 23 such as a mouse or a keyboard.
• the microscope main body 100 includes a laser unit 1, an optical fiber 7, a collimating lens 8, a dichroic mirror 9, a galvano scanner 1 1, a relay lens 14, an objective lens 15 and a sample 16.
• An optical lens 17, a pinhole diaphragm 18 for confocal detection, an optical detector 19, etc. are arranged.
• the specimen 16 is, for example, a fluorescent specimen supported on a stage (not shown).
• the galvano scanner 11 has two galvanometer mirrors arranged in series (galvano mirrors 1 1 1 X, which will be described later). 1 1 1 1 Y) is provided.
• the laser light emitted from the laser unit 1 is incident on one end of the optical fiber 7, then propagates inside the optical fiber 7, and is emitted from the other end of the optical fiber 7. After collimated light is collimated by the collimating lens 8, the light enters the dichroic mirror 9.
• the laser beam passes through the dichroic mirror 9, is sequentially reflected by the two galvanometer mirrors of the galvano scanner 1 1, and then passes through the relay lens 1 4 and the objective lens 1 5, and is collected at one point on the sample 1 6. .
• the galvano scanner 11 is driven in this state, the laser beam scans the sample 16.
• Fluorescence generated at the condensing position of the laser light on the specimen 16 is directed to the dichroic mirror 9 through the same optical path as the laser light.
• the fluorescence is reflected by the dichroic mirror 9, collected by the condenser lens 17, passes through the pinhole diaphragm 18, removes excess rays, and then enters the photodetector 19 to fluoresce. Converted to a signal.
• the controller 20 of this system includes a laser unit 1 and a galvano scanner.
• the photodetector 19 is controlled synchronously, and the fluorescence signal is repeatedly acquired while scanning the sample 16 with the laser beam.
• the fluorescence signal captured at this time is sent to the computer 21 as observation information, and is output to the monitor 22 or stored by the computer 20 as necessary. Using this observation information, the user observes sample 16.
• FIG. 2 is a configuration diagram of the galvano scanner 11 and the controller 20.
• the galvano scanner 11 is provided with two galvanometer mirrors 1 1 1 1 X and 1 1 1 Y.
• the galvanometer mirror 1 1 1 X is driven while the laser beam is projected onto the galvano scanner 1 1, the laser beam scans the specimen 1 6 in a predetermined direction (X direction), and the galvanometer mirror 1 1 1 Y
• the laser beam scans the sample 16 in a direction perpendicular to the X direction (Y direction).
• the driver 2 0 4 X which is the driving circuit is connected to the galvano mirror 1 1 1 X
• the driver 2 0 4 Y which is the driving circuit is connected to the galvano mirror 1 1 1 Y. Is connected.
• the galvanometer mirror 1 1 1 X is provided with a position sensor 1 1 2 X that detects the mirror position.
• 1 1 1 Y is equipped with a position sensor 1 1 2 ⁇ that detects its mirror position.
• the controller 20 includes a scanner control unit 202 that is a dedicated control circuit for the galvano scanner 11 1, a laser control unit 207 that is a dedicated control circuit for the laser unit 1, and a control circuit dedicated to the photodetector 19.
• reference numerals 205 X and 205 Y indicate AZD converters that perform AZD conversion on signals output from the galvano scanner 11, and are indicated by reference numerals 203 X and 203 Y. Is a DZA converter that DZA converts the signal output from the scanner control unit 202.
• the computer 21 prompts the user to operate the input device 23 prior to observation, and inputs scanning conditions.
• This scanning condition includes at least the scanning line and scanning speed desired by the user, and usually includes the laser intensity desired by the user.
• the input scanning condition is transmitted from the computer 21 to the controller 20.
• the CPU 201 of the controller 20 recognizes the scanning condition via the interface circuit 209.
• the CPU 201 records necessary information in the laser control unit 207, the detector control unit 208, and the scanner control unit 202 according to the recognized scanning conditions, so that the laser unit 1 and the photodetector 1 are recorded.
• the CPU 201 determines the waveform of the drive signal to be given to the driver 204 X of the galvano scanner 1 1 based on the setting line and the setting speed information included in the scanning conditions (hereinafter referred to as the following). "X drive waveform” . ) And the waveform of the drive signal to be given to the driver 20 4 Y (hereinafter referred to as “ ⁇ drive waveform”), and store the waveform information in the memory 2 0 2 ⁇ of the scanner control unit 2 0 2 To do.
• CPU 2 0 1 gives an instruction to laser controller 2 07, detector controller 2 0 8 and scanner controller 2 0 2 under the above settings.
• the laser unit 1, the photodetector 19 and the galvano scanner 11 are driven synchronously.
• the scanner control unit 20 2 generates a drive signal according to the information of the X drive waveform stored in the memory 2 0 2 A, and sends it to the DZ A converter 2 0 3 X.
• Driver 2 0 4 Send to X sequentially.
• the scanner control unit 202 generates a drive signal in accordance with the information of the Y drive waveform stored in the memory 202 A and sends it to the driver 20 04 via the DZA converter 20 03 Y. Send sequentially to Y.
• the galvano scanner 11 is driven.
• the scanner control unit 20 while driving the galvano scanner 11 1, outputs a signal output from the position sensor 1 1 2 X (hereinafter referred to as “X position signal”) and the position sensor 1 1 2 Y.
• the signal output from the signal (hereinafter referred to as ⁇ position signal) is sampled via the AZ D converters 2 0 5 X and 2 0 5 Y and stored in the memory 2 0 2 A. This sampling rate is sufficiently high and is higher than the data sampling signal frequency in the controller 20.
• the X position signal and Y position signal acquired in this way are used effectively during freeline observation as described below.
• FIG. 3 shows the controller 20 and the computer at the time of free line observation.
• Step S 21 is an operation flowchart of 21.
• the operation program (control program) of the controller 20 is stored in advance in the ROM 2 0 1 A of the controller 20, and the operation program (operation program) of the computer 21 is stored in the hard disk of the computer 21. Stored in advance. [0034] (Step S 21)
• the computer 21 displays a setting screen such as that shown in FIG. 4 on the monitor 22 in order to allow the user to input scanning conditions such as a scanning line, a scanning speed, and laser intensity in the GUI environment.
• the setting screen displays an image I of the observation range of the specimen 16 (within the field of view of the objective lens 15).
• This image I is acquired by normal observation by this system, for example. Normal observation is to obtain observation information by setting the laser intensity at a low intensity and the scanning line in a stripe shape.
• the user operates the input device 23 to draw the scanning line L 1 on this setting screen and input characters such as the scanning speed B 1 and the laser intensity B 0.
• test button B2 main scan button B3, and the like are also arranged on this setting screen, and the user selects these buttons at a desired timing to perform a test input instruction or main scan. Instructions can also be entered into the computer 21.
• the computer 21 displays information on the scanning line L 1 and the scanning speed B 1 displayed at that time together with a test input as information on the setting line and the setting speed by the user, respectively. Send to controller 20.
• the CPU 201 of the controller 20 When the CPU 201 of the controller 20 receives the setting line and setting speed information and the test instruction, it sets the galvano scanner 11 according to the information.
• the CPU 201 disassembles the setting line into a plurality of unit vectors.
• the unit vector size is an increasing function of the set speed.
• the CPU 201 generates an X drive waveform (Fig. 5 (b)) by converting the X component of the disassembled setting line into a voltage value using a predetermined conversion formula, and converts the Y component of the disassembled setting line to By converting to voltage value with predetermined conversion characteristics, A dynamic waveform (Fig. 5 (c)) is generated.
• the predetermined conversion characteristics are galvanometer mirror 1 1
• the CPU 201 stores the generated X drive waveform and Y drive waveform information in the memory 202 A of the scanner control unit 202.
• the CPU 201 of the controller 20 gives an instruction to the scanner control unit 202 and drives the galvano scanner 11. However, at this time, the CPU 201 does not drive the laser unit 1 and the photodetector 19 at all, so that the laser beam does not enter the sample 16.
• the X position signal and the Y position signal output from the galvano scanner 11 are sampled by the scanner control unit 202 and sequentially stored in the memory 202 A.
• the actual scanning line measured in this way is called the “measured line” to distinguish it from the set line.
• the CPU 201 of the controller 20 reads the X position signal and the Y position signal stored in the memory 202 A, converts them into coordinates on the image I, calculates a measured line, and information on the measured line Is sent to the computer 21.
• the computer 21 When the computer 21 receives the information on the measured line, the computer 21 displays the measured line L 2 together with the scanning line L 1 on the setting screen as shown in FIG. By this display, the user can intuitively recognize the deviation between the scanning line L1 input by the user and the actual measurement line L2.
• test button B2 If the user is dissatisfied with the actual measurement line L2, redraw the scanning line L1 to a gentle curve or change the scanning speed B1 to the lower speed side, and then select the test button B2 again. That's fine.
• the test button B 2 is selected, the above-described test ⁇ is repeated.
• this scan button B3 may be selected.
• the computer 21 displays information on the scan line L 1, scan speed B 1, and laser intensity BO displayed at that time, the user set line, set speed, and set intensity. This information is sent to the controller 20 together with the main scanning instruction.
• the CPU 201 of the controller 20 receives the setting line, setting speed, setting intensity information, and main scanning instruction, the settings of the laser unit 1, the galvano scanner 1 1, and the light detector 19 are set according to the information. Do. Incidentally, when the setting line and the setting speed are the same as the previous value, the setting of the galvano scanner 11 is omitted.
• the CPU 201 of the controller 20 gives an instruction to the laser controller 20 7, the detector controller 208, and the scanner controller 202, and the laser unit 1, the photodetector 19, and the galvano Drive the scanner 1 1 synchronously to obtain observation information.
• This observation information acquisition is repeated, for example, a plurality of times.
• the above steps S 1 6 and 17 are the main scan.
• the CPU 201 of the controller 20 transmits the observation information acquired in the main scanning to the computer 21 together with information on the scanning conditions at the time of the main scanning. [0052] (Step S 2 8 YES ⁇ S 2 9)
• FIG. 7 is a diagram in which each scanning line information acquired by laser scanning a plurality of times from the start point P 1 to the end point P 2 of the measurement line L 2 from time t 0 to t n is arranged in chronological order. According to such a display, it becomes clear that the part (blacked part) where the reaction is detected is gradually moving from P 1 to P 2 (step S 29).
• step S 1 2 and 1 3 only the galvano scanner 11 is driven without irradiating the laser beam under the scanning conditions specified by the user, and the actual measurement at that time is performed. Measure the scanning line (measured line). Therefore, in this test, it is possible to obtain information on the measured line while preventing discoloration and damage of the specimen 16.
• the user can judge whether the scanning conditions he / she has set are good or not, and can give the system the desired instructions such as changing scanning conditions, actual scanning, and retesting.
• the scanning line L1 set by the user is displayed together with the actual measurement line L2 (see Fig. 6), so the user can intuitively recognize the deviation between the two. it can.
• This embodiment is an embodiment of a fluorescence confocal laser scanning microscope system. Here, only differences from the first embodiment will be described. The difference is that an optimization function that automatically corrects the details of the scanning conditions is installed.
• FIGS. 8 and 9 are added to the operations of the controller 20 and the computer 21 of this system, and the optimization button B 4 is arranged on the setting screen as shown in FIG.
• the setting screen is also provided with an area for inputting the optimization margin B 5 desired by the user.
• the optimization margin is the maximum It is the allowable range of deviation between the actual measurement line and the setting line after optimization, and is expressed by the number of pixels on the image I, for example.
• the computer 21 displays the information of the scanning line L 1, the scanning speed B 1, and the optimization margin B 5 displayed at that time, the setting line by the user, the setting speed, The setting margin information is sent to the controller 20 along with the optimization instruction.
• step S33 the CPU 201 of the controller 20 skips step S33 and proceeds to step S34.
• the CPU 201 of the controller 20 reads the X position signal and the Y position signal stored in the memory 202A at this time, and converts them into coordinates on the image I, thereby calculating an actual measurement line.
• the CPU 201 of the controller 20 subtracts the actual measurement line from the setting line by the user, and calculates the difference between the two. At this time, CPU 20
• the unit vector size is an increasing function of the set speed and is the same as that used when generating the drive waveform for the galvano scanner 11.
• the CPU 201 of the controller 20 determines whether or not the calculated difference is within the set margin by the user.
• the CPU 201 of the controller 20 converts the X-direction difference into a voltage value using a predetermined conversion formula, thereby correcting the X drive waveform correction amount AV X (t ) Further, the CPU 201 obtains the correction amount AV X (t) of the Y drive waveform by converting the difference in the Y direction into a voltage value using a predetermined conversion formula.
• the predetermined conversion formula is the same as that used when generating the drive waveform of the galvano scanner 11.
• the CPU 201 corrects the X drive waveform by adding the correction amount AV X (t) to the X drive waveform stored in the memory 202 A at that time. Further, the CPU 201 corrects the Y drive waveform by adding the correction amount AV Y (t) to the Y drive waveform stored in the memory 202A at that time.
• the CPU 201 of the controller 20 determines whether or not the frequency changes of the corrected X drive waveform and Y drive waveform fall within the limit frequency of the galvano scanner 11.
• the limit frequency is determined by the speed set by the user and the response characteristics of the galvanometer mirrors 1 1 1 X and 1 1 1 Y. The higher the set speed, the lower the limit frequency.
• step S34 If the modified frequency of the X and Y drive waveforms does not fall within the limit frequency, change the setting speed one step lower, return to step S34, and calculate the difference again.
• Step S 37 Y ES ⁇ S 33
• the process returns to step S33, and retest is performed with the corrected X drive waveform and ⁇ drive waveform.
• the above steps S33 to S38 are optimization.
• step S34 when the magnitude of the difference calculated in step S34 falls within the setting margin, the CPU 201 of the controller 20 finishes the optimization and sends the set speed and the measured line information after the optimization to the computer 21. To do.
• the method for calculating the measurement line is as described above.
• the computer 21 When the computer 21 receives information about the set speed and the measured line after optimization, the computer 21 reflects the information on the setting screen, for example, as shown in FIG. In Fig. 11, the reference speed B1 represents the set speed after optimization, and the reference line L2 'represents the measurement line after optimization. This allows the user to check the optimization results.
• the computer 21 obtains the information of the scan line L 1, the scan speed B 1 ′, the laser intensity BO, and the optimization margin B5 that are displayed at that time.
• Information on the setting line, setting speed, setting intensity, and setting margin is sent to the controller 20 along with the main scanning instruction.
• the CPU 20 1 of the controller 20 sets the laser unit 1, the light detector 19 and the galvano scanner 11 according to the setting line, setting speed and setting intensity.
• Step S 302 Y ES On the other hand, if optimization has been performed, the CPU 201 of the controller 20 sets only the laser unit 1 and the optical detector 1 9 according to the setting line, setting speed, and setting intensity, and the galvano scanner 1 1 The setting contents (driving waveform) are saved in the optimized state.
• the CPU 201 of the controller 20 gives an instruction to the laser controller 20 7, the detector controller 208, and the scanner controller 202, and the laser unit 1, the photodetector 19, and the galvano Drive the scanner 1 1 synchronously to obtain observation information.
• This acquisition of observation information is repeated continuously, for example, a plurality of times.
• the above steps S303, 304, and 305 are the main scan.
• the CPU 201 of the controller 20 transmits the observation information acquired in the main scanning to the computer 21 together with the scanning conditions at the time of the main scanning.
• the computer 21 When the computer 21 receives the observation information, the computer 21 displays the observation information on the monitor 22.
• the display method at this time is as shown in FIG. 7 (step S 50).
• step S33 to S38 in the optimization of this system (steps S33 to S38), until the difference between the measured line and the set line falls within the set margin (until YES in step S35), the test and drive waveforms The correction is repeated (step S36). By repeating this, the drive waveform gradually approaches the optimum value. In this optimization, the set speed is corrected to the low speed side as necessary to obtain the optimum value. Therefore, according to the optimization of this system, details of scanning conditions are automatically optimized.
• step S34 of this system the difference between the measured line and the set line is calculated and then converted to a voltage value. However, the measured line and the set line are converted to a voltage value. Then, the difference between the two may be calculated. However, in that case, the criterion (setting margin) in step S35 is converted to a voltage value. As the need arises, the conversion in step S 36 is not necessary.
• step S 3 5 the test and the correction of the drive waveform are performed until the difference between the measured line and the set line is within the set margin (step S 3 5 Repeated until YES, but may be repeated a predetermined number of times. Further, the number of repetitions may be specified by the user.
• step S 3 0 3 to S 3 0 5 if optimization has been performed, the drive waveform after optimization is automatically adopted. The user may select whether to use the optimized drive waveform or the newly generated drive waveform, and follow the selection result.
• controller 20 and the computer 21 of this system may operate as follows after optimization.
• the CPU 20 of the controller 20 transmits the optimized drive waveform information to the computer 21.
• the computer 21 stores the received drive waveform information in accordance with an instruction from the user. At this time, the drive waveform information is associated with the image I information of the sample 16. Thereafter, when there is a drive waveform call instruction from the user, the computer 21 reads out the stored drive waveform information and transmits it to the controller 20. In this case, the controller 20 sets the galvano scanner 11 by writing the received drive waveform to the memory 20 2 A. According to such an operation, the number of executions of the process related to optimization can be minimized. Also, the user can call and use the optimized drive waveform at a desired timing.
• the galvano scanner 11 may be driven while irradiating the laser from the laser unit 1 with an intensity lower than the intensity of the laser light irradiated in the main scanning. In this way, fading and damage to the specimen 16 can be suppressed even by making the laser light intensity weaker than the intensity during the main scan.
• the microscope main body 100 described above is a laser scanning microscope equipped with both a fluorescence detection function and a confocal detection function. However, one or both of the fluorescence detection function and the confocal detection function are provided.
• the present invention is also applicable to a laser scanning microscope that is not mounted.
• the present invention can also be applied to a laser scanning device that does not have a detection function.

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• Optics & Photonics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Microscoopes, Condenser (AREA)
• Laser Beam Processing (AREA)

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• 2007
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