JP2006308709A - Microscope stage, and focal position measuring instrument and confocal microscope system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably displace the focal position of an optical microscope under an optical microscope at a high speed without causing overshooting and vibration. <P>SOLUTION: A microscopic sample is displaced at high speed in the optical axis direction of the microscope by extending/shortening the piezoelectric element using a calibration table of the voltage-extension characteristics for a piezoelectric element without using a feedback circuit etc. Also, the extension axis of the piezoelectric element is freely adjustable in order to strictly parallel the extension axis of the piezoelectric element and the optical axis for the optical microscope. The focal position, i.e. the position of the microscope sample with respect to an objective lens is measured by using an objective lens type reflective optical system of a numerical aperture ≥1.3 under the optical microscope. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microscope stage for moving a focal position and such an apparatus / system for obtaining three-dimensional information by an optical microscope.

  By installing a video rate camera on the imaging plane of the optical microscope, the microscopic specimen can be observed in real time. A confocal microscope can acquire a cross-sectional image of a microscopic sample specimen with a thin optical section, so that a three-dimensional image can be acquired by scanning the objective lens in the optical axis direction and arranging the acquired optical sections in the optical axis direction. Can do. In order to obtain an image with little optical blur, a technique is disclosed in which the objective lens is moved stepwise outside the camera shooting time, the focus movement is completed, and the focus is fixed during the shooting time. (For example, refer to Patent Document 1) In Patent Document 1, the focal position of the objective lens is controlled by a piezoelectric actuator in synchronization with a vertical synchronization signal of a video rate camera. Therefore, a confocal image at the required focal position can be acquired in accordance with the video frame.

  Further, when the objective lens is driven by a piezo actuator, the response of the piezo actuator to the voltage is not linear, and has a hysteresis characteristic that the response varies depending on the increase or decrease of the voltage. Therefore, there is a technique for correcting the hysteresis characteristics in a piezo driver that includes a piezo actuator that includes a sensor that knows the degree of distortion of the piezo element, and that controls the driving of the piezo actuator. When the objective lens is driven stepwise, the objective lens always vibrates due to its weight. As a method of suppressing the vibration, there is a method of giving an electrical braking signal by adding a feedback circuit or the like to the piezo driver.

As a method for measuring the focal position of a microscope, a method of calculating the focal position using reflected light from a microscopic sample specimen obtained during microscopic observation and return light as scattered light as an index is often used. . (For example, refer to Patent Document 2) This technique irradiates a microscopic sample specimen using a low coherence light source, detects the interference light generated by the light source and its return light, and detects the light intensity. This is a technique that adjusts the focus so that it is maximized or maximized. In this technique, in order to generate interference, it is necessary to make the optical path length variable, and the optical system becomes complicated. Since it is difficult to acquire the absolute position of the focal position, these techniques are mainly used to correct the deviation of the focal position during microscopic observation.
JP 2004-184699 A JP 2004-321696 A

  In the method of acquiring a three-dimensional image as described above, there is a problem that when the focus is changed stepwise, the objective lens causes overshoot due to the weight of the objective lens and vibrates. In order to solve the above problem, if the physical resistance is increased physically or an improvement such as adding a feedback circuit is added, overshoot and vibration can be suppressed to some extent, but time delay occurs in the staircase drive. The staircase will have at least a few ms to rise. Further, when a piezo driver that controls the driving of the piezo actuator is provided with a sensor that knows the degree of distortion of the piezo as described above, the objective lens can be accurately driven with an error of several nm. Similarly, a time delay occurs in the staircase drive. Furthermore, when the focus is moved, the above-described means for acquiring the focus position has a problem that the optical system becomes complicated. In addition, there is a method to calculate the focal position by image processing etc. from the reflected light from the microscopic sample specimen obtained at the time of microscopic observation and the return light that is scattered light, but this method takes time to calculate. Therefore, it is impossible to acquire the focal position at high speed.

  An object of the present invention is to provide an apparatus capable of scanning a focal position at high speed under an optical microscope and a technique capable of acquiring the focal position at high speed with a simple optical system.

  According to the present invention, in an optical microscope, a glass chamber for putting a microscopic sample specimen, and a piezo element for directly controlling the focal position by directly attaching the glass chamber and specifying a depth position in a focal direction for obtaining an image. And a calibration table that measures the displacement response to the voltage of the piezo element in advance, and has a tilt screw that adjusts the expansion / contraction direction of the piezo element in parallel with the optical axis of the optical microscope. By driving the piezo element, a microscope stage can be obtained which scans the focal position at high speed without using a feedback circuit.

  Further, according to the present invention, the glass chamber contains two glass rectangular plates having a size of 9 mm × 9 mm or less and a thickness of 150 to 400 μm, in which an observation microscope sample is accommodated, and a 9 mm × 9 mm or less surface is 50 μm or less A microscope stage characterized by a structure in which they are bonded to each other so as to have a gap is obtained.

  According to the present invention, in the optical microscope in which the glass chamber, an objective lens having a numerical aperture of 1.3 or more, and a mirror is disposed on the back focal plane of the objective lens, the interface between the solution in the glass chamber and the glass The optical path of the total reflected light changes due to the movement of the interface, which is the focal point, and the change in the optical path becomes an angular change at the position of the mirror on the rear focal plane of the objective lens. Thus, a focal position measuring apparatus is obtained, wherein the angle change is detected by a two-divided or four-divided photodiode, and the focal position at the time of microscopic observation is detected and adjusted.

  In addition, according to the present invention, there is obtained a focal position measuring apparatus characterized in that, in the focal position measuring apparatus, the microscopic focal point is moved on the microscope stage of the present invention while confirming the focal position.

  Further, according to the present invention, a laser for irradiating a microscopic sample specimen, a video camera for converting a two-dimensional optical section represented by x and y axis coordinates into a video signal, and a memory for recording the video signal as digital data 2. A medium, a microscope stage according to claim 1, a waveform output device for voltage-controlling the piezoelectric element of the stage, and a high-voltage amplifier, and taking a video frame for each vertical synchronizing signal of the video camera. By shifting in a stepped manner outside the time and fixing the focus within the video frame shooting time, it is possible to obtain a clear optical section, and the acquired optical section is arranged in accordance with the z-axis coordinate. A confocal microscope system characterized by continuously acquiring original digital image data in real time is obtained.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to scan a focus position at high speed under an optical microscope, and to acquire the focus position at high speed with a simple optical system.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 shows a configuration of a microscope stage and a confocal microscope system according to the present embodiment. As shown in FIG. 1, the microscope stage includes a piezo element 3 for scanning the glass chamber 1 containing a microscopic specimen in the optical axis direction, a base 4 for fixing the piezo element, and a piezo element. A tilt screw 5 for finely adjusting the scanning axis, and a base 6 and a base 7 for fixing these to the upper part of the objective lens. A waveform output device 9 provided with a calibration table of voltage-displacement characteristics of the piezo element 3 in advance sends an analog voltage signal V1 within 10V to the high voltage amplifier 8. The voltage amplifier 8 gives an analog voltage signal V2 obtained by amplifying the voltage signal V1 sent from the waveform output device 9 to the piezo element.

  In addition to the microscope stage, the confocal microscope system includes an objective lens 2 for observing a microscopic specimen in the glass chamber 1, a confocal scanner unit 12, an image intensifier 13, a video camera 14, and a video camera. 14 includes a recording medium 10 for accumulating the video signal VIDEO sent from 14 and performing image processing. The confocal scanner unit 12 houses a microlens array, a pinhole array, a beam splitter, and a rotation control unit that rotationally drives the microlens array and the pinhole array. The waveform output device 9 receives the synchronization signal SYNC from the video rate camera 14 and sends an analog voltage signal V 1 synchronized with the SYNC to the high voltage amplifier 8. FIG. 2 shows an example in which the focal position is scanned by synchronizing the synchronization signal SYNC and the displacement of the piezo element 3.

    When the recording medium 10 starts acquiring the video signal VIDEO, the waveform output device 9 receives the synchronization signal SYNC from the video rate camera 14 and sends the analog voltage signal V1 synchronized with the SYNC to the high voltage amplifier 8. By arranging digital video images stored in the recording medium 10 in the z-axis direction in the order of frames as shown, a three-dimensional image can be constructed and acquired in real time. FIG. 3 shows a frame configuration.

  FIG. 4 shows the voltage-displacement characteristics of the piezo element 3 obtained by vibrating the voltage 50 times with a triangular wave of 1 Hz having an amplitude of 96 V and obtaining the extension of the piezo element 3 at that time. In FIG. 4, all the data which were vibrated 50 times are plotted. The displacement response was different when the voltage was increased from 0V to 96V and when the voltage was decreased from 96V to 0V. However, the reproduction error due to repetition is as small as 10 nm at the maximum when the applied voltage is 0V and 16 nm at the maximum when 96V. Therefore, by sending a voltage signal to the piezo element 3 using FIG. 4 as a calibration table, the piezo element can be accurately expanded and contracted without using a strain sensor or a piezo driver.

  FIG. 5 shows an embodiment of a glass chamber in which a microscopic sample specimen is placed. In the present invention, since the glass chamber 1 is scanned by the piezo element 3, the weight of the glass chamber 1 causes overshoot and vibration of focal movement. The glass chamber 1 should be made as small as possible. In this embodiment, two glass rectangular plates having a size of 9 mm × 9 mm and a thickness of 170 μm were prepared, and an enamel resin was sandwiched between them to produce a chamber. When an oil immersion lens is selected as the objective lens 2, when the glass chamber 1 is displaced by a piezo element, a glass using a 170 μm thick glass is used due to the viscosity of the immersion oil filling between the oil immersion lens and the glass chamber. In the chamber, the observation surface in the glass chamber is curved, and the microscopic sample specimen bonded to the glass surface vibrates. Therefore, when an oil immersion lens is selected as the objective lens 2, if a glass chamber is used using glass having a thickness of 340 μm, the vibration of the microscopic sample specimen due to the curvature is reduced. In addition, the viscosity of the immersion oil also causes a time delay or vibration of the driving of the glass chamber 1. Instead of the immersion oil, a silicone oil having a refractive index of 1.50 having a viscosity lower than that of the immersion oil may be used.

  FIG. 5 also shows a three-dimensional schematic diagram of the embodiment of the microscope stage shown in FIG. The reference numerals correspond to those in FIG. The glass chamber 1 and the piezo element 3 are directly bonded by an adhesive. The type of the adhesive is not particularly limited as long as it is non-fluorescent, but is preferably an organic solvent-soluble one in order to facilitate adhesion and peeling between the glass chamber 1 and the piezoelectric element 3. In this embodiment, an acetone-soluble polymer resin MMP (Methyl Methacrylate Polymer) is used, and a 10% MMP solution using acetone as a solvent is used as an adhesive. By extending and shortening the piezo element 3, the glass chamber 1 moves and scans the focal position. This scanning axis needs to be strictly parallel to the optical axis. When the focal point is scanned while observing the microscopic specimen, if the drive axis and the optical axis are not parallel, the observed microscope image moves in the direction perpendicular to the optical axis simultaneously with the scanning of the focal position. If the drive shaft is adjusted so that the observed image does not move left and right or up and down within the microscopic field even when the focal position is scanned using the tilt screw 5, the drive shaft is Become parallel. Also, when the focal position is desired to be displaced stepwise, the voltage applied to the piezo element is not a simple rectangular wave, but the rising edge of the rectangular wave is a sine wave. Thereby, overshoot and vibration at the time of displacement of the focal position are reduced.

  FIG. 6 shows the configuration of the focal position measuring apparatus according to the present embodiment. As shown in the figure, the focal position measuring device includes a glass chamber 21, an infinite objective lens 22, an imaging lens 23 for imaging a microscopic sample specimen, and a mirror placed at the focal position of the imaging lens. 24 and two or four divided photodiodes 25. The objective lens 22 and the imaging lens 23 are handled as one finite objective lens. Instead of the objective lens 22 and the imaging lens 23, one finite objective lens may be used. In order to totally reflect the laser beam at the bottom surface of the glass chamber 21, the objective lens 22 is an oil immersion lens having a numerical aperture of 1.3 or more. The refractive index of the oil filling the glass chamber 21 and the objective lens 22 is made equal to the refractive index of the glass. The mirror 24 is adjusted so that the incident laser is totally reflected at the interface between the solution in the glass chamber 21 and the glass. As the position of the microscopic sample 21 changes as indicated by the dotted line, the position where total reflection occurs also changes. The change Ad is approximately an angle change Ae on a mirror placed on the back focal plane of the optical system constituted by the infinity objective lens 22 and the imaging lens 23, and is divided into two or four-division photo. It is projected on the diode 25 and detected as a position change Ax. The two-part or four-part photodiode 25 outputs the position change Ax as a voltage. FIG. 7 shows the correlation between the focal position and the voltage of the two-part photodiode measured by the focal position measurement apparatus. The relationship between Ad and Ax shows almost linearity in theory, but in reality, it does not show linearity due to the effects of aberrations of the optical system, stray light, and the like. The focal position was moved slowly by 100 nm per second so as not to cause overshoot or vibration. With reference to FIG. 7 as a calibration calibration table, the movement of the focal position when the piezo element 3 is driven at high speed can be measured from the output of the two-part or four-part photodiode 25. In the focal position measurement apparatus according to the present embodiment, since the laser is totally reflected at the interface between the solution and glass in the glass chamber 21, the laser applies the microscopic sample specimen in the glass chamber 21 to about 200 nm in the vicinity of the interface. No other light is irradiated and no extra background light is generated during microscopic observation.

  FIG. 8 shows a focal position when the piezo element 3 is extended stepwise, which is measured using the microscope stage and the focal position measuring device. The voltage signal from the two-divided photodiode 25 used in the focal position measuring device was measured at a sampling rate of 24 kHz. There is almost no overshoot or vibration of the focal position due to high-speed driving. The rise time of the focal position was 280 is. For comparison, FIG. 9 shows the focal position when the objective lens is driven stepwise in the optical axis direction using a piezo actuator, as in the conventional focal point movement method. When the feedback circuit is not used for the piezo driver that applies voltage to the piezo actuator (broken line), a severe overshoot is observed immediately after the objective lens is step-driven, and the vibration is not yet attenuated even after 30 ms. When a feedback circuit was used for the piezo driver (solid line), the overshoot and vibration amplitude decreased compared to when the feedback circuit was not used, but the vibration was attenuated for about 20 ms. Therefore, the microscope stage of the present invention is significantly more effective than the conventional method as a focal point moving method.

  FIG. 10 shows an embodiment in which on / off of camera shooting is output as a synchronization signal SYNC from the video camera 14, and the focus is moved by the microscope stage in synchronization with the SYNC. The shooting time of the video camera 14 is 4 ms, and the image signal is transmitted to the recording medium 10 in 1 ms outside the shooting time. The upper panel of FIG. 10 shows the voltage V <b> 2 applied to the piezo element 3. By sending the V2 signal in synchronization with the SYNC, the movement of the focal point is completed without overshoot or vibration within 1 ms outside the imaging time. Therefore, according to the present invention, an image with less optical blur can be acquired at a higher speed than in the case of using the conventional focus movement method.

  The microscope stage, the focal position measurement device, and the confocal microscope system manufactured according to the present invention can be used for various industrial devices related to an optical microscope provided with a vertical fine movement mechanism.

1 is a block diagram of a configuration of a microscope stage capable of scanning a microscopic sample in the optical axis direction according to an embodiment of the present invention and a confocal microscope system using the stage. FIG. It is a figure which shows the scanning sequence of the microscopic sample which concerns on embodiment of this invention. It is a conceptual diagram constructed | assembled three-dimensionally from the two-dimensional optical section | slice which concerns on embodiment of this invention. It is a voltage-displacement characteristic view of the piezo element according to the embodiment of the present invention. 3D schematic diagram of an embodiment of the glass chamber to which the microscopic sample according to the embodiment of the present invention is added and the microscope stage shown in FIG. 1 shows a simple optical path diagram of a focal position measuring apparatus according to an embodiment of the present invention. The correlation between the voltage output from the two-divided photodiode and the focal position used in the focal position measuring apparatus according to the embodiment of the present invention is shown. 2 shows a focal position when a piezoelectric element is extended stepwise, which is measured using the microscope stage and the focal position measuring apparatus according to an embodiment of the present invention. Similarly to the conventional focal point moving method, the focal point position when the objective lens is driven stepwise in the optical axis direction using a piezo actuator is shown. When the focal point is moved in synchronization with the frame of the video camera using the confocal microscope system according to the embodiment of the present invention, the voltage applied to the piezo element, the synchronization signal output from the camera (exposure timing) ) And the focal position measured using the focal position measuring apparatus.

Explanation of symbols

1 Glass chamber 2 Objective lens 3 Piezo element 4 Unit 1
5 tilt screw 6 2
7 units 3
8 High Voltage Amplifier 9 Waveform Output Device 10 Storage Medium 11 Laser Light Source 12 Confocal Unit 13 Image Intensifier 14 Video Camera 15 Mirror 21 Glass Chamber 22 Objective Lens 23 Imaging Lens 24 Mirror 25 Two-Division or Four-Division Photodiode Light receiving surface

Claims (5)

  In an optical microscope, a glass chamber for placing a microscopic sample specimen, a piezoelectric element for directly adhering the glass chamber, specifying a depth position in a focal direction for obtaining an image, and controlling the focal position; and It has a tilt screw that adjusts the extension / reduction direction in parallel to the optical axis of the optical microscope, and prepares a calibration table that measures the displacement response to the voltage of the piezo element in advance. A microscope stage that is driven to scan a focal position at high speed without using a feedback circuit.   The glass chamber holds two observation glass specimens having a size of 9 mm × 9 mm or less and a thickness of 150 to 400 μm, facing each other so that the surface of 9 mm × 9 mm or less has a gap of 50 μm or less. The microscope stage according to claim 1, wherein the microscope stage has a bonded structure.   In the optical microscope in which the glass chamber, an objective lens having a numerical aperture of 1.3 or more, and a mirror is disposed on the back focal plane of the objective lens, laser optics that totally reflects the interface between the solution and the glass in the glass chamber as a focal point The optical path of the total reflected light is changed by movement of the interface that is a focal point, and the change of the optical path becomes an angle change at the position of the mirror on the rear focal plane of the objective lens, and the angle change is divided into two parts. Alternatively, a focus position measuring apparatus that detects with a quadrant photodiode and detects and adjusts the focus position during microscopic observation.   4. The focus position measuring apparatus according to claim 3, wherein the micro focus is moved on the microscope stage according to claim 1 while confirming the focus position in the focus position measuring apparatus. 2. A laser for irradiating a microscopic specimen, a video camera for converting a two-dimensional optical section represented by x and y axis coordinates into a video signal, and a storage medium for recording the video signal as digital data. Microscope stage, a waveform output device for controlling the voltage of the piezoelectric element of the stage, and a high voltage amplifier, and the focal position is displaced stepwise outside the video frame shooting time for each vertical synchronization signal of the video camera. By fixing the focal point within the video frame shooting time, it is possible to obtain a clear optical section, and to realize three-dimensional digital image data constructed by arranging the acquired optical section according to the z-axis coordinates. A confocal microscope system characterized by continuous acquisition in time.