JPH04296811A - Scanning microscope - Google Patents

Abstract

PURPOSE:To prevent variation in photographing range of a scanning microscope for scanning lighting light on a sample by retaining an optical system for radiating the lighting light on the sample at the end of a tuning fork, and by vibrating the tuning fork through a vibrating means. CONSTITUTION:Amplitude of a tuning fork 30 is detected by measuring leaking magnetic flux between the end of the tuning fork 30 and an electromagnet 31 by means of a Hall device 71, and an amplitude detection signal Sm for this is input into a control circuit 72. When the amplitude is lower than an aimed value, a control signal Se for increasing the vibrating frequency of VCO, or a control signal Se' for reducing is input into VCO by the control circuit 72, and the number of vibration of the magnetic field due to the electromagnet 31 thus follows the variation in resonance frequency of the tuning fork 30.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an optical scanning microscope, and more specifically, an optical system for irradiating a sample with illumination light is held in a tuning fork, and the tuning fork is vibrated to direct the illumination light onto the sample. The present invention relates to a scanning microscope configured to perform scanning at a location.

0002

[Prior Art] Conventionally, illumination light is converged into a minute light spot, and this light spot is scanned two-dimensionally on a sample, and at this time, the light that passes through the sample or the light that is reflected thereon, and the sample An optical scanning microscope is known in which the fluorescence generated from the sample is detected by a photodetector to obtain an electrical signal carrying an enlarged image of the sample. Furthermore, Japanese Patent Publication No. 1986-21
No. 7218 discloses an example of this scanning microscope.

In conventional optical scanning microscopes, the scanning mechanism is such that the illumination light beam is split into two by an optical deflector.
Many dimensional deflection mechanisms were used.

However, this mechanism has a drawback in that it requires an expensive optical deflector such as a galvanometer mirror or an acousto-optic optical deflector (AOD). In addition, in this mechanism, the illumination light beam is deflected by an optical deflector, so this light beam enters the objective lens of the light transmission optical system at different angles from time to time, and the resulting aberrations are corrected. It has also been recognized that this makes it difficult to design the objective lens. In particular, when an AOD is used, a special correction lens is required in addition to the objective lens because astigmatism occurs in the light beam emitted from the AOD, making the optical system more complicated.

In view of the above points, it has been conventionally considered to scan the illumination light spot without deflecting the illumination light beam. For example, patent application No. 1-2469 filed by the present applicant.
No. 46 discloses that the illumination light spot is scanned by moving the light transmitting optical system relative to the sample stage.

[0006] As one of the specific mechanisms for relatively moving the optical system and the sample stage in this way, as shown in Japanese Patent Application No. 198550/1999 filed by the present applicant, the optical system A tuning fork that is held at its tip, and an electromagnet that applies a magnetic field whose strength periodically changes to cause the tuning fork to resonate has been proposed. Furthermore, as shown in Japanese Patent Application No. 2-198550 also filed by the present applicant, a piezoelectric element is attached to the above-mentioned tuning fork,
A mechanism has also been proposed in which the voltage applied to the piezoelectric element is controlled to periodically distort the element, thereby causing the tuning fork to resonate.

[0007] Such a movement mechanism allows the relative movement width of the optical system, that is, the illumination light scanning width (this is determined by the amplitude of the tuning fork), to be large compared to a movement mechanism that uses a piezo element, an ultrasonic vibrator, or the like. This is advantageous in ensuring a larger imaging range for the scanning microscope.

[0008]

[Problems to be Solved by the Invention] However, when using an illumination light scanning mechanism that combines a tuning fork and an excitation means that makes it resonate, the imaging range in the scanning direction due to tuning fork vibration is small in the captured microscope image. However, it may fluctuate. In this case, the output image size may vary depending on how the output signal of the photodetector is processed, or the magnification may vary even if the image size is constant.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a scanning microscope in which the above-mentioned fluctuations in the imaging range do not occur when using a scanning mechanism that moves an optical system using a vibrating tuning fork. It is.

0010

[Means for Solving the Problems] As described above, a scanning microscope according to the present invention includes a tuning fork that holds an optical system at its tip, and a force whose strength changes periodically to act on the tuning fork. By moving the optical system relative to the sample stage using an optical system moving mechanism composed of an excitation means that resonates, the illumination light is caused to main and sub-scan on the sample,
In a scanning microscope that captures a sample image by detecting light from a portion of the sample scanned by the illumination light using a photodetector, ◆ means for detecting the amplitude of the tuning fork, and ◆ amplitude detection outputted by this means. The present invention is characterized in that a control circuit is provided which receives a signal and inputs a control signal corresponding to the signal to the vibration excitation means to converge the tuning fork amplitude to a target value.

[0011]

[Operation] According to the research conducted by the present inventors, it has been found that the above-mentioned variation in the imaging range is caused by variation in the tuning fork amplitude. That is, the resonance frequency of the tuning fork may shift due to disturbances such as temperature. Although this deviation is slight, the resonance curve of the tuning fork has a sharp peak, so even a slight deviation in the resonance frequency causes a large change in its amplitude.

In the device of the present invention having the above configuration, even if the resonant frequency of the tuning fork shifts due to a disturbance and the amplitude changes, the driving of the excitation means is controlled in response to the change, and the amplitude of the tuning fork is kept at the target value. Since it is made to converge, the tuning fork amplitude can be kept constant.

[0013]

Effects of the Invention: If the amplitude of the tuning fork can be controlled to be constant as described above, the movement width of the optical system moved by this tuning fork,
In other words, the scanning width of the illumination light can be kept constant. Therefore, since the image capturing range is kept constant, the size or magnification of the output image will not fluctuate.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be explained in detail below based on embodiments shown in the drawings.

FIG. 2 shows a monochrome reflection type confocal scanning microscope according to a first embodiment of the present invention, and FIG. 1 shows the planar shape of its scanning mechanism in detail. As shown in FIG. 2, the monochromatic laser 10 emits illumination light 11 of a single wavelength. This linearly polarized illumination light 11 enters the film surface 25a of the polarizing beam splitter 25 in a P-polarized state and is transmitted therethrough. The illumination light 11 that has passed through the polarization beam splitter 25 has a wavelength of λ/2 for polarization plane adjustment.
The light passes through the plate 12, is focused by the entrance lens 13, and is made to enter the polarization-maintaining optical fiber 14.

As shown in the cross-sectional shape of FIG. 4, this polarization-maintaining optical fiber 14 includes a core 14b disposed within a cladding 14a, and stress applying portions 14c, 14c formed on both sides of the core 14b. So-called PANDA
type is used. By appropriately rotating the λ/2 plate 12, the linearly polarized illumination light 11 is brought into a state in which the direction of the polarization plane is aligned with the direction in which the stress applying portions 14c and 14c are arranged, or with the direction perpendicular thereto (this example In the latter direction, that is, the direction of arrow U in FIG. 4), the light is made to enter the optical fiber 14.

One end of this optical fiber 14 is connected to the probe 1
5, and the illumination light 11 propagated within the optical fiber 14 is emitted from this one end. At this time, one end of the optical fiber 14 emits illumination light 11 in the form of a point light source. A light transmitting optical system (also serving as a light receiving optical system) 18 consisting of a collimator lens 16 and an objective lens 17 is fixed to the probe 15 . Note that a λ/4 plate 19 is disposed between the collimator lens 16 and the objective lens 17.

The above illumination light 11 is transmitted through the collimator lens 1.
6, the light is made into parallel light, passes through the λ/4 plate 19, becomes circularly polarized light, and is then focused by the objective lens 17. ) Image is formed into a minute light spot P. The reflected light 11'' reflected by the sample 23 becomes circularly polarized light with the opposite direction of rotation, and λ/
The light passes through the four plates 19 and becomes linearly polarized light whose plane of polarization is perpendicular to that of the illumination light 11 . The beam of this reflected light 11'' is condensed by the collimator lens 16 and made to enter the polarization maintaining optical fiber 14.At this time, the direction of the polarization plane of the reflected light 11'' is in the direction of arrow V in FIG. becomes. The reflected light 11'' propagated through the optical fiber 14 is emitted from one end thereof, and is converted into parallel light by the lens 13.

After passing through the λ/2 plate 12, this reflected light 11'' enters the film surface 25a of the polarizing beam splitter 25 in the S polarization state.
incident on and reflected there. This reflected light 11'' is focused by a condensing lens 26 and detected by a photodetector 28 through an aperture pinhole 27.
Reference numeral 8 includes, for example, a photomultiplier (photomultiplier tube) or the like, from which a continuous signal S indicating the brightness of the illumination light irradiated portion of the sample 23 is output.

As described above, by providing the optical isolator composed of the λ/4 plate 19 and the polarizing beam splitter 25, the reflected light 11'' is prevented from returning to the laser 10 side, and a larger amount of light is reflected. The light 11" is detected by the photodetector 28
Become guided by. Further, the illumination light 11 reflected by the entrance lens 13 or the end face of the optical fiber 14 is prevented from entering the photodetector 28, and the signal S with a high S/N is prevented.
will be obtained.

Next, two-dimensional scanning of the light spot P of the illumination light 11 will be explained with reference to FIG. The probe 15 is fixed to one tip of a horizontally arranged tuning fork 30 with the optical axis of the optical system 18 being vertical. This tuning fork 30
The base 30a is fixed to a pedestal 32, and is capable of vibrating at a predetermined natural frequency. Then, on the inside of the tuning fork 30, there is a slight gap between both ends of the tuning fork.
An electromagnet 31 is provided. This electromagnet 31 is fixed to a pedestal 32 via a mounting member 34.

A rectangular pulse voltage Vd having a frequency equal to the natural frequency of the tuning fork 30 is applied to the electromagnet 31 from a drive circuit 33 which also constitutes an excitation means. As a result of the magnetic field acting intermittently on both ends of the tuning fork 30, the tuning fork 30 resonates at its natural frequency. Therefore, the probe 15 fixed to this tuning fork 30 is shown in FIG.
, moves back and forth at high speed in the X direction (horizontal direction) in FIG. 2, and main scanning of the light spot P is performed.

The sample stage 22 also has a Z movement stage 24Z that can reciprocate in the Z direction (optical axis direction of the optical system 18) with respect to the mount 32, and a Z movement stage 24Z that can reciprocate in the Y direction perpendicular to both the X and Z directions. It is attached via a Y-transformable stage 24Y. Therefore, when the light spot P is main scanned as described above, if the Y moving stage 24Y is simultaneously driven back and forth, the light spot P is sub-scanned.

Each time the light spot P is scanned two-dimensionally, the Z movement stage 24Z is moved to create an image in focus on all surfaces within the range in which the sample 23 is moved in the Z direction. It becomes possible to obtain the signal S that carries the signal.

In this embodiment, as shown in FIG.
A dummy probe 15' having the same configuration as the probe 15 is attached to the other tip of the probe 15. Thereby,
It becomes possible to maintain good mechanical balance between one end and the other end of the tuning fork 30, and to configure a nearly ideal resonance system. Furthermore, in this embodiment, the electromagnet 31 is placed inside the tuning fork 30 to apply a magnetic field to both ends of the tuning fork 30, so the electromagnet is placed only outside one end of the tuning fork 30. Compared to the case, the magnetic flux density acting on the tuning fork 30,
In other words, the force acting can be made larger.

Next, the electrical configuration will be explained with reference to FIG. The continuous analog signal S output from the photodetector 28 mentioned above is amplified by an amplifier 40 and then sent to an A/D
The signal is input to the converter 41, where the period and timing are defined based on the sampling clock C from the sampling clock generation circuit 70, and the signal is sampled, quantized, and converted into a digital image signal Sd. This image signal Sd is subjected to image processing such as gradation processing in an image processing device 42, and then input to an image reproduction device 43 such as a CRT display device. In this image reproducing device 43, the image carried by the image signal Sd,
That is, the microscopic image of the sample 23 is reproduced.

A computer 44, such as a personal computer, is connected to the image reproducing device 43 via an image processing device 42, and receives image processing commands and basic operation commands of the scanning microscope, that is, images for searching the field of view. All imaging commands such as the imaging command for the observation image and the imaging command for the observation image are given using an input operation unit such as a keyboard of the computer 44.

Further, the aforementioned Y moving stage 24Y is driven by a driver 46 which receives a signal of a predetermined frequency from an oscillator 45 so as to reciprocate at that frequency. Further, the Z movement stage 24Z is driven by a driver 48 so as to be at a predetermined Z position based on a Z-axis control signal Fs output from the image processing device 42 and converted into an analog signal by the D/A converter 47. . and oscillator 45
, D/A converter 47 are each operated and controlled based on a vertical synchronization signal Vs and a focus direction signal Fs issued from the image processing device 42, and the movements of the stages 24Y and 24Z are synchronized.

On the other hand, the electromagnet drive circuit 33 is composed of a voltage controlled oscillator (VCO) 60, a 1/n frequency divider 61, and a driver 50 at the subsequent stage. The driver 50 includes an open collector buffer 51, a photocoupler 52,
It consists of a power MOS-FET 53, a diode 54, a capacitor 55, etc., and applies a rectangular pulse voltage Vd of the same frequency as the pulse signal output from the 1/n frequency divider 61 to the electromagnet 31. Note that the 1/n frequency divider 61 is the VCO 6
It outputs 1/n times the number of pulses as compared to the pulse signal Sf outputted by 0. In addition, the VCO 60 is connected to the horizontal synchronization signal H as described above.
The operation is controlled based on s, and stages 24Y and 24
The Z movement and the reciprocating movement of the probe 15 are synchronized.

Next, the point of controlling the amplitude of the tuning fork 30 to be constant will be explained. As shown in FIG.
A Hall element 71 as an amplitude detecting means is disposed close to the tip of the tuning fork holding the tuning fork. This Hall element 71 detects the magnitude of magnetic flux leaking laterally from the electromagnet 31. The magnitude of this leakage magnetic flux changes as the gap between the tuning fork 30 and the electromagnet 31 changes as the tuning fork 30 vibrates. In other words, as shown in (1) and (2) of FIG. 8, the larger the gap, the larger the leakage magnetic flux. Therefore, the output Sm of the Hall element 71 changes periodically as the tuning fork 30 vibrates. This output Sm is input to the control circuit 72.

The control circuit 72, for example, outputs this output Sm
Find the maximum value of. If the amplitude of the tuning fork 30 is constant, the gap between it and the electromagnet 31 changes with constant characteristics as the tuning fork vibrates. However, if the resonant frequency of the tuning fork 30 changes due to the reasons mentioned above and its amplitude becomes smaller than the predetermined target value, the minimum value of the gap becomes larger as shown by the broken line in (1) of FIG. , the maximum value will be smaller. The control circuit 72 sets this maximum value to a predetermined first reference value R.
1 (that is, when the amplitude of the tuning fork 30 is less than the target value), the fundamental frequency f of the pulse signal Sf is reduced by a small predetermined amount Δ
A control signal Se that increases by f is input to the VCO 60. If the resonance frequency of the tuning fork 30 has changed to a higher frequency side and its amplitude has decreased, then the control signal Se
is input to the VCO 60, the amplitude changes in an increasing direction. When the amplitude changes in this way, the control circuit 72 continues to input the control signal Se to the VCO 60. As a result, the tuning fork amplitude reaches the target value, and the maximum value of the output Sm increases to the second reference value R2, which is slightly larger than the reference value R1. At this point, the control circuit 72 stops outputting the control signal Se.

On the other hand, if the amplitude has decreased because the resonant frequency of the tuning fork 30 has changed to a lower frequency side, the amplitude will further decrease by inputting the control signal Se to the VCO 60. When the amplitude changes in this way, the control circuit 72 continues to input to the VCO 60 a control signal Se' that lowers the fundamental frequency f of the pulse signal Sf by a small predetermined amount Δf instead of the control signal Se. As a result, the tuning fork amplitude reaches the target value, and the maximum value of the output Sm increases to the second reference value R2. At this point, the control circuit 72 stops outputting the control signal Se'.

By carrying out the above processing, the frequency of the magnetic field generated by the electromagnet 31 changes to follow the fluctuation of the resonant frequency of the tuning fork 30, and the amplitude of the tuning fork is always kept constant.

Note that in this embodiment, the pulse signal Sf
is also input to the sampling clock generation circuit 70 and the horizontal synchronization signal generation circuit 73, and is used to define the generation timing of the sampling clock C and the horizontal synchronization signal Hs, respectively. The above horizontal synchronization signal Hs
is input to the image processing device 42.

The means for detecting the amplitude of the tuning fork 30 is as follows:
In addition to the Hall element 71 in the above embodiment, a thin piezoelectric sheet 74 attached to the tuning fork 30 can also be used as in the second embodiment shown in FIG. That is, this piezoelectric sheet 74 receives stress due to the vibration of the tuning fork 30 and generates an electromotive force E1, but the value becomes smaller as the amplitude of the tuning fork 30 decreases, so the tuning fork amplitude can be detected from this electromotive force E1. . Note that in FIG. 5, elements equivalent to those already explained are given the same numbers, and a redundant explanation of them will be omitted (hereinafter,
similar).

In the third embodiment shown in FIG. 6, the amplitude detection means includes a semiconductor laser 75 and a condenser lens that converges the laser beam 76 emitted from the semiconductor laser 75 onto the side surface of the mirror-finished tuning fork tip. 77, a condenser lens 78 that condenses the laser beam 76 reflected from the side surface, and a photodetector 79 such as a photodiode that detects the condensed laser beam 76. The semiconductor laser 75 and the condensing lens 77 are arranged so that the laser beam 76 is converged on the side surface of the tuning fork when the tuning fork 30 is not vibrating.

Therefore, when the tuning fork 30 vibrates, the side surface of the tuning fork periodically shifts forward and backward from the point of convergence. In this way, no matter which direction the tuning fork side surface deviates from the convergence point, the photodetector 79
Since the amount of light reaching S decreases, the output S of the photodetector 79 decreases.
g decreases. The degree of decrease in the amount of detected light is determined by the tuning fork 30.
decreases as the amplitude decreases. Therefore, if the minimum value of the output Sg exceeds a predetermined value, it can be determined that the tuning fork amplitude is below a predetermined target value.

Furthermore, with the electrical configuration shown in FIG.
Detecting the back electromotive force E2 generated in the electromagnet 31, the tuning fork 3
It is also possible to detect an amplitude of zero. In this fourth embodiment, a back electromotive force E2 generated in the electromagnet 31 due to the vibration of the tuning fork 30 is detected by a differential amplifier 81 via a transformer 80 arranged in parallel with the electromagnet 31. When the gap between the tuning fork 30 and the electromagnet 31 changes as shown in (1) in Fig. 8, this back electromotive force E2 changes as shown in (3) in the same figure.
Changes as shown in . That is, the absolute value of the back electromotive force E2 becomes smaller as the gap becomes larger.

Therefore, when the amplitude of the tuning fork 30 becomes smaller than a predetermined target value and the gap becomes larger overall as shown by the broken line in FIG. 8 (1), the back electromotive force E2
The absolute value of is reduced by the amount shown by a and b in (3) of FIG. Therefore, if the reference voltage on the + side of the differential amplifier 81 is set to a value as shown by Th in (3) of FIG.
When the amplitude of the tuning fork 30 falls below a predetermined target value, the differential amplifier 81 can output a predetermined high level signal. At this time, the process of increasing the amplitude of the tuning fork 30 to a predetermined target value may be performed by the control circuit 72 in the same manner as in the first embodiment.

In the embodiment described above, the frequency of the magnetic field generated by the electromagnet 31 is changed to follow the resonant frequency of the tuning fork to increase the amplitude of the tuning fork. In addition to this, the voltage value of the rectangular pulse voltage Vd applied to the electromagnet 31 or its duty ratio may be increased.

The embodiments applied to a scanning microscope in which the tuning fork 30 is vibrated by the electromagnet 31 have been described above, but the present invention is also applicable to vibration of the tuning fork 30 by using other excitation means, such as a piezoelectric element attached to the tuning fork 30. It can also be applied to the case of vibration, and the effect of preventing the shift of the imaging range as described above can be achieved as well.

Furthermore, although the scanning microscope of the embodiment described above is of a monochrome reflection type, the present invention is applicable to other scanning microscopes that take color images, transmission type scanning microscopes, and even scanning microscopes. It can also be applied to fluorescence microscopes, etc.

[Brief explanation of drawings]

FIG. 1 is a plan view showing the main parts of a scanning microscope according to a first embodiment of the present invention.

[Figure 2] A partially cutaway front view showing the above scanning microscope.

[Figure 3
] Electrical circuit diagram of the above scanning microscope

[Figure 4] Cross-sectional view of the polarization-maintaining optical fiber used in the above scanning microscope

FIG. 5 is a plan view showing the main parts of a scanning microscope according to a second embodiment of the present invention.

FIG. 6 is a plan view showing the main parts of a scanning microscope according to a third embodiment of the present invention.

FIG. 7 is an electrical circuit diagram of a scanning microscope according to a fourth embodiment of the present invention.

[Figure 8] Graph showing the relationship between the distance between the electromagnet and tuning fork of a scanning microscope, leakage magnetic flux, and back electromotive force

[Explanation of symbols]

10 Monochromatic laser 11 Illumination light 11 Reflected light 14 Polarization preserving optical fiber 15 Probe 16 Collimator lens 17 Objective lens 18 Light transmission optical system 22 Sample stage 23 Samples 26, 77, 78 Condensing lens 27 Aperture pinholes 28, 79 Light Detector 30 Tuning fork 31 Electromagnet 32 Frame 33 Electromagnet drive circuit 41 A/D converter 70 Sampling clock generation circuit 71
Hall element 72 Control circuit 73 Horizontal synchronization signal generation circuit 74 Piezoelectric element 75 Semiconductor laser 76 Laser beam 80 Transformer 81 Differential amplifier

Claims (1)

[Claims] Claim 1: By moving an optical system that irradiates illumination light onto the sample relative to a sample stage on which the sample is placed, the illumination light is caused to scan in the main and sub-scans on the sample. In a scanning microscope that captures a sample image by detecting light from a portion of the sample scanned by illumination light using a photodetector, a moving mechanism for moving the optical system includes a tuning fork that holds the optical system at its tip. and excitation means for causing the tuning fork to resonate by applying a force whose strength changes periodically to the tuning fork;
A scanning microscope characterized in that it is provided with a control circuit that receives an amplitude detection signal outputted by the means and inputs a control signal corresponding to the signal to the excitation means to converge the tuning fork amplitude to a target value. .