JP2011154312A - Laser scanning microscope and control method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To suppress an error involved in a change in a scanning condition of a laser beam. <P>SOLUTION: An X-axis scanning means 22 and a Y-axis scanning means 23 drive an X scanning mirror 18 and a Y scanning mirror 19 according to a driving signal which is based on a predetermined condition to thereby perform scanning with a laser beam. Angle sensors 22b and 23b output a position signal corresponding to an irradiation position with the laser beam on a sample 13. In a state where intensity in the irradiation of the sample 13 with the laser beam is suppressed (including stop) to be lower than intensity in observation by a light transmittance varying means 16 for adjusting the irradiation of the sample 13 with the laser beam, the driving signal is supplied to the X-axis scanning means 22 and the Y-axis scanning means 23 to thereby preliminarily drive the X scanning mirror 18 and the Y scanning mirror 19, and the position signal is measured at the predetermined time, then a lag time from a delay time of the position signal predicted in advance relative to the driving signal is calculated. The invention is applied to, for example, the laser scanning microscope. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a laser scanning microscope and a control method.

  Conventionally, laser light irradiated on a sample is scanned, reflected light or fluorescence emitted from the sample is introduced into a photodetector, and the intensity of the detected light is correlated with the scanning position of the laser light. There is a laser scanning microscope that acquires an image within the scanning range and observes a sample. In a laser scanning microscope, the magnification of an image (hereinafter referred to as a scanning magnification as appropriate) can be changed by changing the scanning range of the laser beam. For example, when the scanning range of the laser beam is reduced, the magnification is further increased. An image is acquired.

  For example, after acquiring an image at a predetermined reference scanning magnification, the user designates a region of interest in the image (hereinafter referred to as a region of interest as appropriate) and scans the region of interest with laser light. The laser scanning microscope is operated as follows. As a result, an image obtained by enlarging the region of interest that is a part of the image at the reference scanning magnification is acquired.

  As described above, when the region of interest is designated from the image of the reference scanning magnification and the scanning magnification is changed, it is desirable that the region of interest designated by the user coincides with the image acquired after the scanning magnification is changed. For example, when the user designates the attention area so that the target observation object in the sample is arranged at the center, the target observation object is arranged in the center of the image after changing the scanning magnification, that is, the attention area. The center position of the image and the center position of the image acquired after changing the scanning magnification are expected to match.

  However, in general, in a laser scanning microscope, for example, when a galvano motor drives a scanning mirror in accordance with a drive signal output from a control device and scans a laser beam, and the scanning range of the laser beam is changed, that is, If the amplitude of the scanning mirror is changed, the behavior of the scanning mirror with respect to the drive signal changes. Due to the error caused by the change in the behavior of the scanning mirror, the center position of the attention area designated by the user and the center position of the image acquired after changing the scanning magnification are not matched. For example, the target observation target arranged at the center in the attention area designated by the user moves from the center of the image after the change of the scanning magnification, which hinders good observation.

  Note that the behavior of the scanning mirror changes not only when the scanning magnification is changed but also when the scanning speed of the laser beam is changed. Hereinafter, the scanning magnification or the scanning speed or the combination of the scanning magnification and the scanning speed is referred to as a scanning condition as appropriate.

  In order to avoid such an error from occurring due to a change in scanning conditions, for example, in Patent Document 1, control is performed by detecting reflection on the back surface of a scanning mirror by a photodetector provided with a slit. A laser scanning microscope is disclosed.

JP 2009-45826 A

  However, the laser scanning microscope disclosed in Patent Document 1 can detect only the timing at which the scanning mirror is positioned corresponding to the center of the image (the center of the total amplitude of the scanning mirror). For this reason, when the scanning condition is changed so that the center of the region of interest is arranged at an arbitrary position other than the center in the image before changing the scanning condition, it is not possible to suppress the occurrence of an error due to the change of the scanning condition. It was.

  The present invention has been made in view of such a situation, and is intended to suppress an error associated with a change in scanning conditions of laser light.

  The laser scanning microscope of the present invention is a laser scanning microscope that scans a laser beam irradiated on a sample and observes the sample, and scans the laser beam according to a drive signal based on a predetermined scanning condition. Scanning means, position output means for outputting a position signal corresponding to the scanning position by the scanning means, irradiation intensity adjusting means for adjusting the irradiation intensity of the laser beam to the sample, and the irradiation intensity adjusting means to the sample In a state where the irradiation of the laser beam is suppressed (including the stop) from the intensity at the time of observation, a driving signal is supplied to the scanning unit to preliminarily drive the scanning unit, and the position is determined at a predetermined time. And a calculation means for measuring a signal and calculating a deviation time from a delay time of the position signal predicted in advance with respect to the drive signal.

  The control method of the present invention is a control method of a laser scanning microscope that scans a laser beam applied to a sample and observes the sample, and the irradiation intensity adjusting means applies the laser light to the sample. In a state of suppressing (including stopping) the intensity at the time of observation, the scanning means is preliminarily driven by supplying a driving signal to the scanning means, and the position signal is measured at a predetermined time, The method includes a step of calculating a deviation time from a delay time of the position signal predicted in advance with respect to the drive signal.

  In the laser scanning microscope and the control method of the present invention, the drive signal is supplied to the scanning means in a state where the irradiation intensity adjusting means suppresses the laser beam irradiation to the sample from the observation intensity (including stoppage). Then, the scanning unit is preliminarily driven, the position signal is measured at a predetermined time, and the deviation time from the delay time of the position signal predicted in advance with respect to the drive signal is calculated.

  According to the laser scanning microscope and the control method of the present invention, it is possible to suppress errors associated with changes in the scanning conditions of the laser light.

It is a figure which shows the structural example of one Embodiment of the laser scanning microscope to which this invention is applied. It is a figure explaining a drive signal, a position signal, and various timing signals. It is a figure explaining the center position of an image shifting before and after the change of scanning magnification. It is a figure explaining the method of correct | amending delay time. It is a block diagram which shows the structural example of a control apparatus. It is a flowchart explaining a preliminary scanning process. It is a flowchart explaining an image acquisition process.

  Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing a configuration example of an embodiment of a laser scanning microscope to which the present invention is applied.

  In the laser scanning microscope 11 of FIG. 1, the sample 13 placed on the stage 12 is observed. The laser light output from the light source 14 is converted into parallel light by the condenser lens 15 and enters the light transmittance varying means 16. The light transmittance varying means 16 adjusts the intensity of the laser light according to the control of the control device 28. Examples of the light transmittance varying means 16 include AOTF (Acousto Optic Tunable Filter) and AOM (Acousto Optic Modulator).

  The laser light that has passed through the light transmittance varying means 16 is reflected toward the sample 13 through the dichroic mirror 17 by a galvano scanner configured to include an X scanning mirror 18 and a Y scanning mirror 19, and is a condensing lens. After being imaged once by 20, it is condensed on the sample 13 by the objective lens 21. Then, the X-axis scanning unit 22 and the Y-axis scanning unit 23 drive the X-scanning mirror 18 and the Y-scanning mirror 19 according to the control of the control device 28, thereby deflecting the laser beam and forming spots on the sample 13. Are scanned two-dimensionally.

  Here, in the laser scanning microscope 11, the X scanning mirror 18 and the Y scanning mirror 19 are driven, so that the spot of the laser light can scan a predetermined range of the sample 13, but a wider range of scanning is realized. Therefore, the drive mechanism 24 is configured to drive the stage 12 in the XY direction (a direction orthogonal to the Z direction, where the optical axis of the laser beam irradiated on the sample 13 is the Z direction). The drive mechanism 24 is configured by a manual mechanism that is manually driven by a user, an electric mechanism that is electrically controlled and driven, or the like.

  When the fluorescent material included in the sample 13 is irradiated with laser light, fluorescence is emitted from the fluorescent material, and the laser light path is reversed through the objective lens 21 and the condensing lens 20, and the dichroic mirror 17. Head for. The fluorescence that has entered the dichroic mirror 17 has a wavelength longer than that of the laser light, and is reflected by the dichroic mirror 17, collected by the condenser lens 25, and incident on the photodetector 26. The photodetector 26 converts the incident light into an electrical signal corresponding to the intensity and outputs it to the intensity signal imaging circuit 27.

  The intensity signal imaging circuit 27 emits light at a timing according to the control of the control device 28, that is, at a timing associated with the angles of the X scanning mirror 18 and the Y scanning mirror 19 according to the position of the spot on the sample 13. The electrical signal output from the detector 26 is sampled, and the intensity signal obtained as a result is converted into an intensity signal sequence corresponding to the scanning surface on the sample 13 to form an image. Then, the intensity signal imaging circuit 27 outputs the image signal to a display device (not shown) for display, or outputs the image signal to a storage device (not shown) for storage.

  The control device 28 controls each part of the laser scanning microscope 11 according to scanning conditions supplied from a host device (not shown).

  For example, the control device 28 supplies a light transmittance control signal indicating the transmittance of laser light by the light transmittance varying means 16 to the light transmittance varying means 16 and controls the intensity of the laser light irradiated on the sample 13. . In addition, as will be described later with reference to FIG. 2, for example, the control device 28 outputs various timing signals such as a horizontal synchronization signal, a horizontal sampling effective signal, an intensity signal sampling synchronization signal, and a vertical synchronization signal to an intensity signal imaging circuit. The intensity signal sampling circuit 27 controls the intensity signal sampling by the intensity signal imaging circuit 27.

  Further, the control device 28 supplies a saw-shaped X scanning axis drive signal (FIG. 2) to the X axis scanning means 22 and controls the rotation angle of the X scanning mirror 18 driven by the X axis scanning means 22.

  The X-axis scanning means 22 incorporates an angle sensor 22b that detects the angle of the scanning axis 22a to which the X-scanning mirror 18 is fixed, and outputs the angle sensor 22b (that is, indicates the angle of the X-scanning mirror 18). Information) is supplied to the control device 28. Here, the angle of the X scanning mirror 18 corresponds to the position in the X direction of the spot of the laser beam formed on the sample 13, and the control device 28 outputs from the angle sensor 22 b of the X axis scanning means 22. Is controlled as an X-axis position signal indicating the position of the spot of the laser beam formed on the sample 13 in the X direction. For example, a galvano motor can be used as the X-axis scanning unit 22, and the galvano motor includes an angle sensor in order to realize a stable behavior with respect to the drive signal, and outputs the angle sensor to the control device 28. By providing the servo system with feedback, the stabilization when the galvano motor is driven can be achieved.

  Similarly to the X-axis scanning unit 22, the control device 28 supplies a Y-scanning axis drive signal to the Y-axis scanning unit 23 to drive the Y-scanning mirror 19. The output of the angle sensor 23b that detects the angle of the scanning axis 23a to which the Y scanning mirror 19 is fixed is supplied to the control device 28, and the control device 28 outputs the output to the laser formed on the sample 13. Control is performed as a Y-axis position signal indicating the position of the light spot in the Y direction.

  In the laser scanning microscope 11 configured as described above, when the X scanning axis driving signal is supplied from the control device 28 to the X axis scanning means 22, the X scanning mirror 18 causes the X scanning mirror 18 to perform a predetermined reciprocating motion. The fluorescence from the sample 13 is detected by the intensity signal imaging circuit 27 as an intensity signal according to the angle of the X scanning mirror 18, that is, as an intensity signal according to the position of the spot formed on the sample 13. Therefore, an image is constructed by associating the spot position with the intensity signal.

  Incidentally, since the X-axis scanning means 22 drives the X-scanning mirror 18 in accordance with the X-scanning axis drive signal from the control device 28, the waveform of the X-axis position signal has substantially the same shape as the X-scanning axis drive signal. Since the X-axis scanning means 22 is a mechanism for driving the scanning shaft 22a by electromagnetic force such as a galvano motor, the X-axis position signal follows the change of the X scanning axis drive signal with a certain delay time. It will be. Similarly, the Y-axis position signal follows the change of the Y scanning axis drive signal with a certain delay time. That is, as shown in FIG. 2, the position signal is delayed with respect to the drive signal, and the control device 28 needs to output various timing signals to the intensity signal imaging circuit 27 in consideration of this delay time. There is.

  With reference to FIG. 2, the drive signal output from the control device 28, the position signal input to the control device 28, and various timing signals will be described. Note that FIG. 2 describes the X-axis direction (horizontal direction), but the same applies to the Y-axis direction (vertical direction).

  On the upper side of FIG. 2, a sawtooth drive signal and a position signal are shown. In the drive signal (solid line) and the position signal (broken line), the vertical axis represents the signal voltage value (V) and the horizontal axis represents time (T).

  Below the waveforms of the drive signal and the position signal, a horizontal synchronization signal, a light transmittance control signal, a horizontal sampling effective signal, an intensity signal sampling synchronization signal, and a readout clock signal are shown in order from the top.

  The horizontal synchronization signal is a signal indicating the timing at which one line of horizontal scanning is started. The light transmittance control signal is a light transmittance variable means 16 so that the sample 13 is irradiated with laser light in a section corresponding to an image acquisition section which is a section in which an image is acquired by laser light emitted from the light source 14. This is a signal for controlling (ON / OFF) the laser beam passing through.

  The horizontal sampling effective signal is a signal indicating a section in which sampling by the intensity signal imaging circuit 27 is effective in the horizontal scanning of the laser light. The intensity signal sampling synchronization signal is a signal indicating the timing at which the intensity signal imaging circuit 27 samples the intensity signal. The read clock signal is a signal indicating a timing at which a D / A (Digital / Analog) converter included in the control device 28 reads numerical data when generating a drive signal that is an analog signal from the numerical data string.

  The drive signal is a signal for controlling the rotation angle of the X-scanning mirror 18 driven by the X-axis scanning unit 22, and is driven so that the X-scanning mirror 18 reciprocates according to the drive signal. The drive signal is inverted after scanning so that the spot moves at a constant speed in a certain range, and immediately after the inversion, the drive signal has a sawtooth shape that is driven non-linearly to minimize the time for inversion.

  When the X scanning mirror 18 is driven by such a drive signal, the locus of the spot on the sample 13 becomes a constant speed in the section in which the X scanning mirror 18 is driven at a constant speed, and the intensity signal sampling synchronization at equal intervals is performed. Since the intensity signal sampled by the signal changes linearly, this interval is suitable for the intensity signal imaging circuit 27 to sample and image the intensity signal.

  Here, assuming that there is no delay in the response of the X scanning mirror 18 to the drive signal, as shown in FIG. 2, the horizontal synchronization signal, the light transmittance control signal, the horizontal sampling valid signal, and the intensity signal sampling synchronization signal are , And output from the control device 28 to the intensity signal imaging circuit 27.

  That is, a certain time Ts (a certain time Ts is a mechanical factor of the scanning means) until Low is output to the horizontal synchronization signal at the start time of one cycle of the drive signal and the rotation speed of the X scanning mirror 18 is stabilized. After the elapse of (determined by the electrical characteristics of the drive circuit), the horizontal sampling valid signal is switched from invalid to valid, and a certain image acquisition interval and horizontal sampling valid signal are validated. In addition, the light transmittance control signal is switched from OFF to ON before the horizontal sampling effective signal is effectively switched at the timing corresponding to the rise time of the light output of the light source 14, and this time difference causes At the time when the horizontal sampling valid signal is effectively switched, the output of the laser beam has reached a stable state.

  Then, in the image acquisition period in which the horizontal sampling valid signal is valid, the intensity signal sampling synchronization signal is output with the number of pulses corresponding to the horizontal resolution. The intensity signal imaging circuit 27 samples the output signal of the photodetector 26 in synchronization with the intensity signal sampling synchronization signal, thereby obtaining an intensity signal string corresponding to the scanning position of the X scanning mirror 18, and according to the intensity. By performing the display, one line of scanning can be imaged. Then, every time one line is scanned in the horizontal direction by the X scanning mirror 18, the Y scanning mirror 19 is repeatedly moved by one line in the vertical direction, whereby the intensities in the horizontal direction differing in the vertical direction by one line. A signal sequence is accumulated, and a two-dimensional image can be obtained.

  As described above, the X-axis scanning unit 22 drives the X-scanning mirror 18 in accordance with the drive signal, and an image is acquired. However, as shown in FIG. 2, the position signal output from the X-axis scanning unit 22 is driven. There is a delay with respect to the signal. This delay varies depending on the scanning conditions. Therefore, when the timing at which the horizontal sampling effective signal is turned on is the same before and after the change of the scanning condition, an error occurs in the imaged region due to the change in the delay time. Due to such an error, a deviation occurs between the center position of the image before changing the scanning condition and the center position of the image after changing the scanning condition.

  For example, the amplitude between the drive voltage Vs at the start of the image acquisition period and the drive voltage Ve at the end of the image acquisition period corresponds to the scanning magnification. When the amplitude of the driving voltage is reduced, the scanning magnification is increased. Get higher. When the scanning magnification is changed in this way and the timing at which the horizontal sampling valid signal is turned on is the same, a shift occurs in the center position of the image before and after that.

  Next, with reference to FIG. 3, the shift of the center position of the image before and after the change of the scanning magnification will be described.

  On the left side of FIG. 3, a drive signal (solid line) for acquiring an image at a reference scan magnification which is a reference scan magnification in the laser scanning microscope 11 and an output from the X-axis scanning means 22 driven according to the drive signal. The position signal (dashed line) is shown. Further, on the right side of FIG. 3, a drive signal (solid line) and a position signal (broken line) when an image is acquired at a scanning magnification of n times the reference scanning magnification are shown.

  The amplitude Vn of the drive voltage corresponding to the image acquisition interval in the drive signal at the scanning magnification of n is 1 / n with respect to the amplitude VR of the drive voltage corresponding to the image acquisition interval in the drive signal at the reference scanning magnification. is there. The position signal at the reference scanning magnification is measured as a waveform having substantially the same shape as the drive signal as an output corresponding to the drive signal after a predetermined delay time DR. Note that the position signal does not have the same shape at a portion where the drive signal changes sharply due to a mechanical response limit.

  For example, when it is desired to acquire an image centered on a position corresponding to the center voltage Vc of the amplitude VR of the drive signal at the reference scanning magnification, the reference time at which the cycle of the drive signal starts (timing at which the horizontal synchronization signal Low is output) Thus, after an appropriate fixed time Ts has passed so that the center voltage Vc becomes the central time of the image acquisition interval, the horizontal sampling effective signal is enabled only in the image acquisition interval, thereby corresponding to the center voltage Vc of the drive signal. An image centered on the position can be acquired.

  When it is desired to enlarge the vicinity of the center of the image at the reference scanning magnification by a factor of n, a spot is formed in a narrow range on the sample 13 with the same number of samplings (number of pixels) by changing the driving voltage to 1 / n. As a result, an optical n-fold image can be obtained.

  However, as described above, the delay time DR with respect to the drive signal having the amplitude VR when the image is acquired at the reference scanning magnification, and the delay time Dn with respect to the drive signal having the amplitude Vn when the image is acquired at the scanning magnification of n times. In general, since the time is different, if only the amplitude of the drive signal is changed while maintaining the section in which the horizontal sampling effective signal is enabled, the range of the acquired image (corresponding to the visual field) is shifted. Will occur.

  At this time, it is considered that the occurrence of deviation in the image range can be corrected by changing the timing at which the horizontal sampling effective signal is activated in accordance with the change in the scanning magnification, but the scanning magnification is changed. The change in the delay time with respect to the change in the amplitude of the drive signal at this time is the mass of the scanning mirror, the torque of the drive mechanism, the dynamic friction coefficient of the drive mechanism, the deviation between the center of gravity of the entire movable part and the movable center, Since various factors such as current supply capability are related and do not have a linear relationship, the shift time of the delay time from the change of the scanning magnification (the shift time from the delay time at the time of the reference scanning condition) (hereinafter referred to as the delay appropriately) It is difficult to calculate (referred to as time). For example, when the scanning speed is 500 Hz or 1 kHz, the delay time is around 160 μsec, and a shift time of about several μsec occurs in the delay time as the scanning magnification is changed.

  Therefore, the laser scanning microscope 11 performs a preliminary scan without irradiating the sample 13 with the laser beam before acquiring the image of the sample 13, and measures and corrects the delay time so that the scanning condition is satisfied. Generation of errors due to changes can be suppressed.

  Next, a method for correcting the delay time will be described with reference to FIG.

  FIG. 4A shows the vicinity of the center voltage Vc corresponding to the center of the image acquisition interval for the drive signal (solid line) and the position signal (broken line) at the reference scanning magnification.

  With respect to the drive signal supplied to the X-axis scanning means 22, the X scanning mirror 18 reaches the position indicated by the drive signal after a delay time DR. Therefore, the intensity at the intended position is sampled by controlling the intensity signal imaging circuit 27 to sample the intensity signal with a time difference corresponding to the delay time DR from the drive signal.

  When the vicinity of the center of the image acquired at the reference scanning magnification is designated as the attention area, and the scanning magnification is changed so as to acquire the image of the attention area, the drive signal and the position signal are as shown in FIG. 4B. It becomes a slant.

  When the scanning condition is changed and the X-axis scanning means 22 drives the X-scanning mirror 18 with a drive signal having an inclination as shown in FIG. 4B, the position signal has a delay time ΔD rather than the delay time DR at the reference scanning magnification. Will be late. At this time, if the intensity signal is sampled at the same timing as that at the reference scanning magnification, the intensity signals at different positions are sampled according to the error voltage ΔV.

  Therefore, it is considered that the deviation corresponding to the error voltage ΔV can be eliminated by changing the timing by the delay time ΔD and sampling the intensity signal, but the intensity signal output from the control device 28 is considered. Since the sampling synchronization signal is generated based on a predetermined reference clock, it is difficult to change the intensity signal sampling synchronization signal at an arbitrary time as opposed to changing the intensity signal sampling synchronization signal in units of reference clocks. is there.

  Therefore, for example, after changing the intensity signal sampling synchronization signal in units of reference clocks in a direction in which the delay time ΔD is eliminated, a minute time difference that cannot be changed in units of reference clocks is determined according to the error voltage ΔD. By offsetting, an effect equivalent to the time difference can be obtained with high accuracy.

  That is, as shown in FIG. 4C, by offsetting the drive signal, as a result, sampling is performed at a desired position at the same timing as the reference scanning magnification (that is, timing delayed by the delay time DR). be able to.

  As described above, in the laser scanning microscope 11, when the scanning condition is changed, an error occurs with the change of the scanning condition by adjusting the timing of the intensity signal sampling synchronization signal and offsetting the drive signal. Can be prevented.

  Here, a method for obtaining the timing adjustment value and the offset value for adjusting the timing will be further described.

  As described above, the laser scanning microscope 11 can turn on / off the irradiation of the laser beam to the sample 13 by the light transmittance varying unit 16, and before performing image acquisition by irradiating the laser beam. By preliminarily driving the X scanning mirror 18 and the Y scanning mirror 19 under the same conditions as the scanning conditions for acquiring an image with the laser light irradiation turned off, and analyzing the position signal, The delay time ΔD is accurately measured. As described above, when the preliminary driving is performed, it is possible to avoid damaging the sample 13 by preventing the sample 13 from being irradiated with the laser beam.

  The position delay with respect to the drive signal is measured as a response delay time with respect to the drive signal from the position at that time by measuring the position signal with a sampling signal having a fixed time relationship from the drive signal. Specifically, since it is known that the mechanical response to the drive signal is delayed, it is clear that if the position measurement is performed at the start time of the image acquisition section, the position that has not reached the start position is measured. It is. Therefore, for example, if the position signal is measured at the central time of the image acquisition section of the drive signal in consideration of the approximate response delay time (a constant time Ts in FIG. 3), the image acquisition is actually performed. It can be expected that the position can be measured almost at the central time, and that the estimated position is insufficient or excessively deviated from the image acquisition section even if the estimated position is insufficient. For this reason, the measurement near the center of the image acquisition section is the most reasonable measurement timing.

  Here, as shown in FIG. 2, from the drive voltage Vs when the image acquisition period starts and the drive voltage Ve when the image acquisition period ends, a predicted delay time (a response delay time predicted in advance). Is accurate, the voltage Vr of the position signal at the central time is measured with a value of Vr = (Vs + Ve) / 2. However, since the predicted delay time is not accurate, for example, a voltage Vf different from the voltage Vr is measured. From the difference between the voltage Vr and the voltage Vf and the voltage change amount Sc per unit time in the image acquisition effective section, the delay time ΔD can be calculated by ΔD = (Vr−Vf) / Sc.

  By calculating the delay time ΔD in this way, a correction value for correcting the delay can be obtained. Since the delay time ΔD is an analog quantity, it is desirable to correct within the effective digit range of the correction calculation for precise correction. However, since the timing signal for image acquisition needs to be synchronized with the readout clock of the D / A converter that generates the scanning signal, it is usually generated based on a signal derived from a common reference clock. Therefore, the period unit of the reference clock is the minimum unit for time adjustment.

  Usually, the clock of a digital circuit needs to be kept at a frequency as low as possible in order to reduce noise, and cannot be set to a high frequency for the purpose of improving time resolution without darkness. For example, when scanning is performed at a horizontal scanning speed of 1 kHz, one scanning period is 1 msec, and assuming that the image acquisition section is about 50% of one scanning period, the image acquisition section is 500 μsec. Also, assuming that the resolution at the time of imaging is 512, the time per pixel is about 1 μsec. Therefore, the reference clock that is the basis for generating the timing is usually a fraction of that. If the reference clock for generating timing is 1/8 of the image acquisition timing signal, the reference clock is about 125 nsec. That is, the image acquisition timing cannot be adjusted unless the time unit is about 125 nsec.

  In addition, assuming that the image acquisition time per pixel is about 1 μsec, if the adjustment time unit is 125 nsec, it is 1/8 of 1 pixel, so the image position adjustment accuracy can be paraphrased as 1/8 pixel. . That is, while the image is acquired with the same scanning magnification, it can be said that the adjustment accuracy is sufficient, but assuming that the center of the first acquired image is enlarged 50 times with the scanning magnification, the original position adjustment is performed. Since the accuracy includes an error of 1/8 pixel, an error after enlargement of 50 times results in an error of about 6 pixels. Therefore, when enlarging with the scanning magnification, it is desirable to keep the position accuracy of the original image as high as possible.

  Furthermore, when the delay time ΔD is adjusted by the timing signal based on the reference clock, the frequency of the original reference clock becomes very high if it is to be adjusted precisely, which is not realistic. For example, if an adjustment is made in units of 10 nsec, a reference clock of 100 MHz is required, and the frequency cannot be handled by a general programmable logic array or the like.

  Therefore, in the laser scanning microscope 11, paying attention to the fact that the drive signal in the image acquisition section increases linearly, instead of adjusting the delay time ΔD, the fine adjustment of time offsets the drive signal in the voltage direction. As a result, as described with reference to FIG. 4C, what is equivalent to the time adjustment is realized by adjusting the analog offset voltage.

  If the offset voltage is an analog signal, the resolution of the D / A converter becomes the limit of adjustment. If a 16-bit DA converter is used, it becomes 1/65536 of the total output amplitude, but about half of the total amplitude. Assuming that is used for the image acquisition interval, the resolution is 1/32768. Assuming that the pixel resolution at the maximum amplitude is 512, 512/32768 ≒ 0.016, which is less than about 2/100 pixels, and performs higher-precision adjustment than the adjustment by the timing signal based on the reference clock. be able to.

  Next, FIG. 5 is a block diagram illustrating a configuration example of the control device 28.

  In the control device 28, the arithmetic processing circuit 31 performs setting for each block of the control device 28 in accordance with the scanning conditions supplied from an external device. The arithmetic processing circuit 31 receives outputs from the angle sensor 22b of the X-axis scanning means 22 and the angle sensor 23b of the Y-axis scanning means 23 via the position signal detection circuit 32, and the X-axis position signal and the Y-axis position signal. The arithmetic processing circuit 31 calculates the X offset value, the Y offset value, and the timing adjustment value from the delay time ΔD of the X axis position signal and the Y axis position signal with respect to the X scan axis drive signal and the Y scan axis drive signal. calculate.

  The position signal detection circuit 32 has a timing according to the X position signal detection timing signal and the Y position signal detection timing signal supplied from the timing generation circuit 33, and the angle sensor 22b of the X axis scanning means 22 and the Y axis scanning means 23. The output from the angle sensor 23b is sampled and supplied to the arithmetic processing circuit 31 as an X-axis position signal and a Y-axis position signal.

  The arithmetic processing circuit 31 supplies a scanning condition setting signal to the timing generation circuit 33 to set the scanning condition in the timing generation circuit 33. The timing generation circuit 33 generates various timing signals from a predetermined reference clock serving as a reference according to the setting by the arithmetic processing circuit 31, and supplies the timing signals to each block.

  For example, the timing generation circuit 33 supplies the light transmittance control signal to the light transmittance varying means 16 and outputs the horizontal synchronization signal, the vertical synchronization signal, the horizontal sampling effective signal, and the intensity signal sampling synchronization signal to the intensity signal imaging circuit 27. To supply. Further, the timing generation circuit 33 supplies the X scan axis drive waveform generation pixel address signal and the read clock signal to the X scan axis drive waveform generation circuit 34, and Y scans the Y scan axis drive waveform generation line address signal and the read clock signal. This is supplied to the shaft drive waveform generation circuit 35.

  The arithmetic processing circuit 31 holds the calculated timing adjustment value in the timing adjustment value holding circuit 36 and supplies the timing adjustment value to the timing generation circuit 33 via the timing adjustment value holding circuit 36. Thereby, the timing generation circuit 33 adjusts the timing signal to be output according to the timing adjustment value.

  In addition, the arithmetic processing circuit 31 generates X scan axis drive waveform data for driving the X scan mirror 18 in accordance with a predetermined control program according to the scanning conditions, and sets the generated data in the X scan axis drive waveform generation circuit 34. The calculated X offset value (drive center) is held in the X offset value holding circuit 37. The arithmetic processing circuit 31 can generate waveforms corresponding to various scanning conditions by generating the X scanning axis drive waveform data according to the control program.

  The X scan axis drive waveform generation circuit 34 has a rewritable memory (for example, RAM (Random Access Memory)), and stores X scan axis drive waveform data (numerical data string) in the memory. Then, the X scan axis drive waveform generation circuit 34 reads the numerical value stored at the address corresponding to the X scan axis drive waveform generation pixel address signal supplied from the timing generation circuit 33 according to the read clock signal (FIG. 2). By sequentially reading out at a predetermined timing, a signal indicating a waveform that changes in time series is generated. Then, the X scan axis drive waveform generation circuit 34 generates an X scan axis drive waveform signal by offsetting the signal with the X offset value held in the X offset value holding circuit 37, and an X scan axis drive circuit 39. To supply.

  The X scan axis drive circuit 39 D / A converts the X scan axis drive waveform signal to generate an X scan axis drive signal (drive signal in FIG. 2) that is an analog voltage, and supplies it to the X axis scanning means 22. . As a result, the X-axis scanning unit 22 drives the X-scanning mirror 18 in accordance with the X-scanning axis drive signal, and X-axis scanning is performed.

  Here, the arithmetic processing circuit 31 separates the X scan axis drive waveform data and the X offset value for generating the X scan axis drive waveform signal and supplies them to the X scan axis drive waveform generation circuit 34, that is, By supplying the X offset value to the X scanning axis driving waveform generation circuit 34 via the X offset value holding circuit 37, the driving waveform is based on the center voltage of the entire amplitude voltage width based on the amplitude and the scanning speed. Generalized as amplitude and period changes, the offset voltage can statically give the position of the amplitude center.

  As the X-axis scanning unit 22 is driven in this way, an X-axis position signal indicating the actual position of the X-scanning mirror 18 is supplied from the X-axis scanning unit 22 to the position signal detection circuit 32, and the arithmetic processing circuit 31. Can measure the actual position of the X scanning mirror 18 at an arbitrary timing.

  Similarly to the X-axis scanning, the arithmetic processing circuit 31 sets the Y-scanning-axis driving signal in the Y-scanning-axis driving waveform generation circuit 35 and holds the calculated Y offset value in the Y offset value holding circuit 38. In accordance with the Y scanning axis driving signal output from the X scanning axis driving circuit 40, the Y scanning mirror 19 is driven by the Y axis scanning means 23 to perform Y axis scanning. Then, a Y-axis position signal indicating the actual position of the Y-scanning mirror 19 is supplied from the Y-axis scanning unit 23 to the position signal detection circuit 32, and the arithmetic processing circuit 31 can measure the actual position of the Y-scanning mirror 19. .

  Next, FIG. 6 is a flowchart for explaining the preliminary scanning process performed by the laser scanning microscope 11.

  In step S11, the control device 28 controls, for example, the X-axis scanning unit 22 and the Y-axis scanning unit 23 so as to perform scanning at the reference scanning magnification, and the intensity signal imaging circuit 27 outputs the image signal at the reference scanning magnification. The data is output and displayed on a display device (not shown). When the user designates a region of interest with respect to the image at the reference scanning magnification, scanning conditions (scanning speed, scanning magnification, center position, etc.) for scanning the region of interest are determined, and the arithmetic processing circuit 31 If supplied, the process proceeds to step S12.

  In step S12, the arithmetic processing circuit 31 performs X scanning axis drive waveform data and Y scanning for driving the X scanning mirror 18 and the Y scanning mirror 19 in accordance with a predetermined control program according to the scanning conditions determined in step S11. Axis drive waveform data is generated and set in the X scan axis drive waveform generation circuit 34 and the Y scan axis drive waveform generation circuit 35.

  In step S13, the X-scan axis drive waveform generation circuit 34 reads out the X-scan axis drive waveform data according to the read clock signal in a state where the laser beam irradiation to the sample 13 is turned off by the light transmittance varying unit 16. An X scan axis drive waveform signal is generated and supplied to the X scan axis drive circuit 39. The X scan axis drive circuit 39 D / A converts the X scan axis drive waveform signal to generate an X scan axis drive signal, and the X axis scan means 22 preliminarily moves the X scan mirror 18 in accordance with the X scan axis drive signal. To drive.

  In accordance with this driving, the position signal output from the angle sensor 22b of the X-axis scanning means 22 is supplied to the arithmetic processing circuit 31 via the position signal detection circuit 32, and the arithmetic processing circuit 31 corresponds to the central time of the image acquisition section. The position signal to be measured is measured. At this time, the position signal is measured in consideration of a predetermined time Ts measured in advance as a rough delay time. Similarly, preliminary driving is performed for the Y axis.

  In step S14, the arithmetic processing circuit 31 obtains a difference between the voltage value Vf of the position signal measured in step S13 and the voltage value Vr that should be originally. Then, the delay time ΔD is calculated from the difference between the voltage values and the voltage change amount Sc per unit time of the drive signal.

  In step S15, the arithmetic processing circuit 31 calculates a time that is an integral multiple of the time adjustment unit in the delay time ΔD as a timing adjustment value, and causes the timing adjustment value holding circuit 36 to hold it.

  In step S16, the arithmetic processing circuit 31 converts the remaining time of the delay time ΔD that could not be adjusted by an integral multiple of the time adjustment unit into an offset voltage to calculate an X offset value and a Y offset value, and retains the X offset value. The preliminary scanning process is completed after the circuit 37 and the Y offset value holding circuit 38 hold it.

  As described above, by performing the pre-scan process with the laser beam irradiation turned off, the timing adjustment for correcting the error while avoiding the influence on the sample 13 (deterioration, fading, etc.) is performed. A value, an X offset value, and a Y offset value can be calculated. In the description of this embodiment, an example is described in which the preliminary scanning is performed with the laser beam irradiation turned off. However, the present invention is not limited to this, and the laser beam intensity is observed to the extent that the sample is not affected. The preliminary scanning may be performed in a state sufficiently weaker than the intensity of time.

  Next, FIG. 7 is a flowchart for explaining image acquisition processing performed by the laser scanning microscope 11.

  In step S21, the arithmetic processing circuit 31 controls the X scan axis drive waveform generation circuit 34 and the Y scan axis drive waveform generation circuit 35 so as to use the drive signal generated in step S12 of FIG.

  In step S22, the timing generation circuit 33 is a time obtained by adding the timing adjustment value held in the timing adjustment value holding circuit 36 to the predetermined time Ts, and is delayed from the reference time at which the cycle of the drive signal starts. Is set so as to generate a timing signal so as to be effective.

  In step S <b> 23, the X scan axis drive waveform generation circuit 34 generates an X scan axis drive waveform signal by offsetting the X scan axis drive waveform data with the X offset value held in the X offset value holding circuit 37. Set as follows. The Y scan axis drive waveform generation circuit 35 generates the Y scan axis drive waveform signal by offsetting the Y scan axis drive waveform data with the Y offset value held in the Y offset value holding circuit 38. Set up.

  In step S24, the X scan axis drive waveform generation circuit 34 and the Y scan axis drive waveform generation circuit 35 perform the X scan axis drive waveform signal and the Y scan axis drive at a timing according to the read clock signal supplied from the timing generation circuit 33. The laser beam is scanned by generating and outputting the waveform signal. Also, sampling by the intensity signal imaging circuit 27 is started in accordance with the horizontal sampling effective signal output from the timing generation circuit 33. Thereby, the scanning of the laser beam and the acquisition of the image of the sample 13 are performed, and the image acquisition process is ended.

  As described above, since an image is obtained by performing correction based on the timing adjustment value, X offset value, and Y offset value calculated by the preliminary scanning process, errors due to changes in the laser light scanning conditions are suppressed. be able to. Thereby, for example, it is avoided that the center position of the image before and after the change of the scanning magnification is shifted, and the sample 13 can be observed more smoothly. In addition, since the delay time ΔD for scanning under the same scanning condition is stable in a short time, by measuring the delay time ΔD and accurately adjusting the correction amount before acquiring an image, The scanning position can be stabilized.

  Further, for example, a response delay time with respect to a representative scanning condition is measured in advance and a correction value is stored, and for a scanning condition different from the representative scanning condition, correction is performed from the representative scanning condition close to the scanning condition. The laser scanning microscope 11 calculates the correction value under the same conditions as the actual measurement conditions, rather than a technique that suppresses the error by estimating the value, so that the error can be suppressed more precisely. it can.

  In this embodiment, the timing adjustment value and the offset value are used together to correct the error. However, the error correction may be performed using only the timing adjustment value or the offset value. Good. For example, from the balance between the time adjustment system and the linearity of the D / A conversion, it is possible to select the one expected to suppress the error with high accuracy. Further, by correcting the error only with the offset value, the processing can be simplified, and the occurrence of the error can be suppressed more than the conventional case by adjusting only the offset value.

  In addition, for example, when performing observation such as intermittently acquiring images every predetermined time for a long time (generally referred to as time lapse), the preliminary scanning process is performed again after a certain period of time has elapsed. By performing, a more accurate measurement can be performed. If it is known that the delay time with respect to a certain scanning condition is stable for a long time, the timing adjustment value and the offset value are recorded in association with the scanning condition, and the same scanning condition is set. On the other hand, the time required for observation can be shortened by using the recorded timing adjustment value and offset value.

  In addition, for example, when performing observation for irradiating a sample with a laser beam to stimulate the sample, the scanning condition is changed when performing stimulation with the laser beam. When a location to be stimulated is specified for the obtained image, a laser beam may be irradiated to a location that is shifted from the desired location. Even when performing such observations, pre-scan processing is performed under the scanning conditions that stimulate the laser beam, thereby suppressing the occurrence of errors associated with changes in the scanning conditions and irradiating the laser beam reliably to the desired location. Desired observations can be made.

  In addition to automatically correcting the error by the control device 28, for example, the measured delay time may be presented to the user, and the error may be corrected based on the user's judgment. Further, the present invention is applicable not only to observation in which an image is acquired by fluorescence emitted from the sample 13, but also to observation in which an image is acquired by reflected light when the sample 13 is irradiated with laser light. Can do.

  The control device 28 includes a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), a flash memory (for example, an EEPROM (Electronically Erasable and Programmable Read Only Memory)), and the like. Each part of the laser scanning microscope 11 is controlled by loading a program stored in the ROM or flash memory into the RAM and executing it. Note that the program executed by the CPU can be downloaded to the flash memory and updated as appropriate in addition to those stored in the ROM and the flash memory in advance.

  In addition, the processes described with reference to the flowcharts described above do not necessarily have to be processed in time series in the order described in the flowcharts, but are performed in parallel or individually (for example, parallel processes or objects). Processing). The program may be processed by one CPU, or may be distributedly processed by a plurality of CPUs.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

  11 laser scanning microscope, 12 stage, 13 sample, 14 light source, 15 condenser lens, 16 light transmittance variable means, 17 dichroic mirror, 18 X scanning mirror, 19 Y scanning mirror, 20 condenser lens, 21 objective lens, 22 X-axis scanning means, 23 Y-axis scanning means, 24 drive mechanism, 25 condenser lens, 26 photodetector, 27 intensity signal imaging circuit, 28 control device, 31 arithmetic processing circuit, 32 position signal detection circuit, 33 timing Generation circuit, 34 X scan axis drive waveform generation circuit, 35 Y scan axis drive waveform generation circuit, 36 timing adjustment value holding circuit, 37 X offset value holding circuit, 38 Y offset value holding circuit, 39 X scan axis drive circuit, 40 X scan axis drive circuit

Claims (5)

In a laser scanning microscope that observes the sample by scanning the laser beam irradiated on the sample,
Scanning means for scanning the laser beam in accordance with a driving signal based on a predetermined scanning condition;
Position output means for outputting a position signal corresponding to a scanning position by the scanning means;
Irradiation intensity adjusting means for adjusting the irradiation intensity of the laser beam to the sample;
In a state where the irradiation intensity adjusting means suppresses the irradiation of the laser beam to the sample from the intensity at the time of observation (including stoppage), a driving signal is supplied to the scanning means to preliminarily make the scanning means A laser scanning type, comprising: an operating means for driving, measuring the position signal at a predetermined time, and calculating a deviation time from the delay time of the position signal predicted in advance with respect to the drive signal. microscope. The computing means is
Calculating an offset value for offsetting the drive signal in accordance with a deviation time from the previously predicted delay time;
2. The driving signal offset by the offset value is supplied to the scanning unit in a state where the irradiation intensity adjusting unit causes the sample to be irradiated with laser light at the intensity at the time of observation. The laser scanning microscope described in 1. Further comprising an imaging means for obtaining an image signal by sampling an intensity signal corresponding to the intensity of observation light from the sample;
The computing means is
Calculating a timing adjustment value for adjusting a timing of starting sampling by the imaging unit according to a deviation time from the previously predicted delay time;
A signal instructing the start of sampling is supplied to the imaging means at a timing adjusted by the timing adjustment value in a state in which the irradiation intensity adjusting means irradiates the sample with laser light at the intensity at the time of observation. The laser scanning microscope according to claim 1. The timing for starting sampling by the imaging means can be adjusted by an integral multiple of a predetermined time adjustment unit,
The computing means is
The timing for starting sampling by the imaging means is adjusted by an integer multiple of the predetermined time adjustment unit, and the drive signal is set according to the remainder of the deviation time from the delay time equal to or less than the predetermined time adjustment unit. Calculate the offset value to be offset,
The laser scanning microscope according to claim 3, wherein the driving signal offset by the offset value is supplied to the driving unit. In a control method of a laser scanning microscope for observing the sample by scanning a laser beam irradiated on the sample,
The laser scanning microscope is
Scanning means for scanning the laser beam in accordance with a driving signal based on a predetermined scanning condition;
Position output means for outputting a position signal corresponding to a scanning position by the scanning means;
An irradiation intensity adjusting means for adjusting irradiation of the laser beam to the sample, and
In a state where the irradiation intensity adjusting means suppresses the irradiation of the laser beam to the sample from the intensity at the time of observation (including stoppage), a driving signal is supplied to the scanning means to preliminarily make the scanning means A control method comprising: driving, measuring the position signal at a predetermined time, and calculating a deviation time from a delay time of the position signal predicted in advance with respect to the drive signal.

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