WO2004059244A1

WO2004059244A1 - High-powered microscope observation device

Info

Publication number: WO2004059244A1
Application number: PCT/JP2003/016203
Authority: WO
Inventors: Katsumi Yoshino; Jyunya Kobayashi
Original assignee: Japan Science And Technology Agency
Priority date: 2002-12-25
Filing date: 2003-12-17
Publication date: 2004-07-15
Also published as: JP2004205366A; JP3754958B2

Abstract

A highly visible marker or the like is attached to the slide glass, stage or he like of a microscope and pattern image recognition is utilized, whereby a more accurate full closed controller which does not require the conventional expensive length measuring scale, and work prepared by using the same are realized. To solve the above problem, the invention provides a high-powered microscope observation device comprising a predetermined pattern formed on a microscope sample holding member or stage, a recognition means for recognizing the predetermined pattern, and a moving means for moving the microscope sample holding member to a predetermined position on the basis of positional information recognized by the recognition means.

Description

Description High-magnification microscope

TECHNICAL FIELD The present invention relates to a high-magnification microscopic observation device. More specifically, a high-magnification (400 to 20000 times) digital micro microscope, long-time Timelabs observation microscope, nanomaterial alignment device, UV laser microcutter (cell trimming) Suitable for various fields, such as chromosome force, microscopic laser traversing (capture and movement) equipment, egg cell injection equipment, DNA chip spotting equipment, and others (ink jet EL manufacturing equipment, elibuso, near field) The present invention relates to a high-magnification microscopic observation device that can be used for a microscope. Background art

For example, when controlling a sub-micro stage such as a microscope, a length measuring device using a magnetic scale, optical scale, laser interference, etc. is attached to the stage to directly grasp the movement amount of the stage and the current position. Position control is almost impossible unless full-closed control, which feeds the signal from the long tool to the motion control unit, is adopted.

However, even if the stage is controlled precisely, if there is an error due to a difference from the thermal expansion of the work, for example, it is not possible to say that full-closed control is required to constantly hold the work at a fixed position. Therefore, measures such as using a material with a similar thermal expansion coefficient or separately providing an environmental sensor for temperature compensation have been taken, but this is complicated as a symptomatic treatment and has restrictions.

In addition, since individual differences between workpieces naturally occur, it is necessary to set the control position of the stage in accordance with individual differences between workpieces, and this is the biggest problem in terms of yield by automation. 。 It is not realistic, for example, the same accuracy is required for the perforated structure itself. Furthermore, in a system that incorporates a length measuring device on the stage, even if the stage can be precisely positioned, if the observation system moves, the error will Could not be corrected.

In addition, if a single-axis drive motor is used, a two-axis motor of XY is naturally required, and a plurality of (for example, XY®) length measuring devices are inevitably required. As a result, errors are inevitably accumulated, so that the alignment accuracy becomes stricter and higher costs cannot be avoided. Therefore, ideally, it is ideal to directly observe the work itself and adjust it to the desired position of the work, and if the position information is given to the control system, the work itself should be precisely adjusted. Is possible. Therefore, improvement in yield can be expected.

Furthermore, when fabricating highly integrated products such as semiconductors and biochips, it is the most cost-effective method to capture the work placed on the stage in two dimensions. The method is being actively adopted. The method of illuminating the target object has been devised and the target has been devised to make it easier to confirm, but there was no solution with high visibility. In addition, the database of coding and managing patterns had to be newly considered.

An object of the present invention is to attach a pattern with high visibility to a slide glass, a stage, or the like of a microscope to use pattern image recognition. That is, the disclosure of the invention aims at realizing a more accurate full-closed control device which does not require a conventional expensive measuring scale at all, and a work manufactured using the same.

According to the present invention, in order to solve the above problems, a predetermined pattern formed on a microscope sample holding member stage, a recognition unit for recognizing the predetermined pattern, and a recognition unit for recognizing the predetermined pattern. A high-magnification microscopic observation apparatus is provided, comprising: moving means for moving the sample holding material for a microscope to a predetermined position based on position information. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a configuration diagram of a high magnification microscopic observation apparatus according to the present invention. Fig. 2 (a) is a top view of the positioning XY stage, and (b) is a side view of the positioning XY stage.

FIG. 3 is a diagram showing a positioning marker.

FIG. 4 is a diagram showing a position detection method.

FIG. 5 is a diagram showing a method of scanning the liquid crystal display and specifying a position from FIGS. (A) to (g).

FIG. 6 is an enlarged view of the circle in FIG. 5 (f) in FIG. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the sample holding material for microscope is, for example, microscope slide glass, on which a predetermined pattern such as a code or a marker is formed. This predetermined pattern is, for example, a mark obtained by irradiating a slide glass with a laser or the like during observation under a microscope, a chromium vapor-deposited film, a screen-printed film, a photosensitive material, or the like marked with a UV or YAG laser. An electronic paper that is engraved or laser-irradiated on an EL material so that it can be erased and rewritten; a material that displays such a code II shape by applying such a luminous body; Examples thereof include pressing a probe pen with pressure to mark the portion, but the present invention is not limited thereto. Further, the pattern itself can be formed by a light emitting element.

The light-emitting element is a liquid crystal that generates an electro-optical effect when excited electrically, chemically, optically or magnetically, colloid particles used for an electronic vapor, and an electroluminescent element that emits fluorescence. Examples include, but are not limited to, an element that emits phosphorescent light, an element that displays a light spot by a projector element such as a laser spot light shirt array, or the like.

The shape of the pattern itself is not particularly limited, but is preferably, for example, a lattice shape, a staggered shape, or the like. Further, by forming a predetermined pattern on a slide glass or the like and placing it on a light emitting element such as a liquid crystal panel, it is possible to use the liquid crystal panel or the like as a light source and the slide glass or the like as an aperture array.

When a liquid crystal is used as the light emitting element, a plurality of pixels are arranged, and the width of the pixel is 5

Replacement form (Rule 26) 3/1

Use a liquid crystal display panel of up to 500 urn, preferably 100 to 100 / zm. Replacement paper (Rule 26) 4 can be. If the distance is less than 5 m, the influence of interference of the diffracted light cannot be ignored by the Fraunhofer rule as in the case of the diffraction grating, and this becomes disturbance light, and the precise position cannot be specified. If the width is larger than 0 0 ^ m, the linearity characteristic of the moving position versus the output will be distorted. Further, the pitch is 5 to 500 τη, preferably 10 to 10 ~ μπι. This is for the same reason as the pixel width described above.

In the case of a liquid crystal display panel, a pair of transparent substrates are opposed to each other, and liquid crystal is sealed between them. Examples of the transparent substrate include, but are not limited to, a glass substrate and a polyacryl substrate. In addition, for example, an ITO film is preferably used as the transparent electrode film, but is not limited thereto. The transparent electrode film is formed by vapor deposition using a CVD method or the like.

Furthermore, liquid crystals include, for example, birefringent liquid crystal elements, transmission scattering liquid crystal elements, TN (twisted nematic) liquid crystals, STN (super TN) liquid crystals, ferroelectric liquid crystal elements, antiferroelectric liquid crystals, and polymers. It can be composed of any of the dispersion type liquid crystals, but is not limited thereto. The distance (cell gap) between the opposing substrates is 1 to: L00 m, and preferably 1 to 20 m.

As a recognition means, a known photodetector, for example, a high-speed imaging device such as a charge-coupled device (CCD) camera, a discharge device (CID) camera, a video camera, a photomultiplier tube, and a parallel vision chip can be used. Yes, but not limited to. In addition to the direct reading by the recognition means, for example, in the same manner as the read head of a DVD, the reading is performed by applying a material using an optical read head and a phase change that can be read and written. Alternatively, a method of reading a pit formed thereon may be used. In a similar method, a magnetic head may be used as the detection unit, and a magnetic substance may be applied on the slide glass and the pit may be read. In addition, an IC chip such as an IC flash memory may be mounted and formed on glass and read by an electronic reading device.

As the moving means, for example, a combination with other actuators such as a linear motor, a stepping motor, and a piezo ultrasonic motor may be used. 5 You can. In addition, the microscopes to which the present invention is applied include all microscopes such as an optical microscope, an AFM.STM microscope, and an electron microscope.

According to the present invention, a pattern such as a code or a force is engraved on a slide glass or the like of a microscope, and the pattern is detected by pattern matching with a CCD or the like, and is fed back to the actuator for positioning. In addition, any point of the slide glass can be specified to improve the workability of sample observation. Since the code of the slide glass is detected at an optical magnification of 10 to 50 times, the position in the slide glass can be specified with a resolution of 100 nano to 1 micron.

This code may have a pattern already created on the slide glass as an off-the-shelf product. When observing the microscope image, the printed shape and characters may be displayed on the slide glass. Not only positioning patterns but also past observation data can be stored in memory. Alternatively, a method of performing ink jet printing on slide glass may be used.

Further, according to the present invention, a predetermined pattern formed on the microscope sample holding material, means for recognizing the predetermined pattern, and a processing apparatus for processing the sample held on the microscope sample holding material are provided. Further, a high-magnification microscopic observation apparatus is provided which includes a moving means for moving the sample holding material for microscope or the processing apparatus to a predetermined position based on the position information recognized by the recognition means. This makes it possible to perform high-speed positioning between the sample processing apparatus and the sample.

Here, examples of the processing apparatus for processing a sample include a manipulator, a laser cutter, a laser marker, and the like, but are not limited thereto. Any processing machine represented by microfactory can be used. It is preferable that the processing device is installed at a location for recognizing the above-mentioned predetermined pattern.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a configuration of a high-magnification microscopic observation apparatus according to the present invention. Reference numeral 1 denotes a positioning XY stage, which is moved in an XY direction by an actuator such as a piezoelectric motor (not shown). Can be. Slide glass 2 is placed on XY stage 1, 6 Place the sample sample S (see Fig. 2) on the glass slide 2 and cover it with a force glass (not shown). A microscope lens barrel 3 is arranged at an approximate position of the specimen sample, and the microscope lens barrel 3 accommodates an optical system such as an objective lens and a condensing lens, a photoelectric element for imaging, and the like. The image observed by the microscope is displayed on the monitor 4.

Further, the microscope lens barrel 3 is provided with a detector 5 which is branched and made of CCD, and this detector 5 is arranged on an approximate position of a positioning marker 1 to be described later. The image of the detector 5 is displayed on the monitor 14 as a multi-window, and the signal is input to the positioning controller (CPU) 6, where the position is recognized. A predetermined movement amount is calculated by the position recognition processing, and the positioning XY stage 1 moves in a work (not shown).

Fig. 2 shows the state of the slide glass 2 on the positioning XY stage 1. Figure (a) is a top view and (b) is a side view. The same members as those in FIG. 1 are denoted by the same reference numerals. The slide glass 2 is disposed in contact with three positioning pins 9 provided on the positioning XY stage 1, and the sample S is covered with a cover glass (not shown) by the vacuum chuck 10.

A positioning marker 7 is formed on the inner surface of the slide glass 2 with a chrome evaporated film 12 (see FIG. 3) and the like. The positioning marker 7 forms a pattern as shown in FIG. 3, and has an opening 11 of 15 im square. A liquid crystal display module 8 is adhered to the positioning marker 7, and a specific lighting light of the liquid crystal display module 8 is transmitted through the opening 11. As a result, an arbitrary position of the positioning marker 7 can be turned on.

The liquid crystal display module 8 has a structure in which liquid crystal is sandwiched between an upper substrate and a lower substrate. For example, the entire size is 20 mm × 20 mm, and is 105 dots vertically and 7 dots horizontally. It consists of 4-dot pixels. Note that the size of the liquid crystal display dot is 200 to 300 microns square, since one dot is allocated to one opening of each positioning marker. Even if the opening 15 is displaced during mounting, the object can be achieved if it is within the range of 200 to 300 microns, and positioning can be tolerated. Each pixel is connected by a lead 7 Selective energization allows the liquid crystal sandwiched between the upper and lower substrates to be driven by the energization, allowing light from a backlight (not shown) installed on the bottom of the substrate to transmit and light. Thus, the pattern can be turned on. When specifying the position of the sample on the slide glass 2, marking on the slide glass 2 makes it easy to install the slide glass 2, and even if high accuracy is required, Ease remains the same. Any position between the openings on the slide glass 2 where the absolute coordinates can be specified can be divided by the number of pixels by the detector 5 which is a CCD.

As for the positioning method, the shape of the opening 11 is stored in advance as a pattern by a pattern matching method, and the position is specified at every pixel number depending on which coordinates on the image position the center of gravity of the shape. It becomes possible. In other words, the pattern of the opening 11 represented on the image (CCD image) composed of 64 × 480 pixels captured by the detector 5 and the portion appearing at the position of the center of gravity are recorded as observation points, When specifying the observation position again, the actuator is controlled to reproduce the image. Instead of forming such an opening in the slide glass 2, it may be formed directly on the glass panel upper surface of the liquid crystal display module 8 below the slide glass 2 by chromium vapor deposition and etching. Fig. 4 shows the method of position detection. 5 (a) to 5 (g) each show a liquid crystal display module, and show a method of scanning and position determination of the liquid crystal display. As described above, for example, the entire liquid crystal display module has a size of 20 mm × 20 mm, and is composed of 105 dots vertically and 74 dots horizontally. In Fig. 5, an enlarged view of the circle in Fig. (F) is shown in the circle in Fig. 6. This circle corresponds to the liquid crystal display unit dot. This is, for example, divided into 64 × 60 (270 °) in the horizontal direction and 480 × (190 / im) in the vertical direction. The resolution is 500 nm each. 13 is the position of the center of gravity of the pattern on the image, and 14 is the positioning marker opening (15 μιη angle). In FIG. 5 (g), reference numeral 15 denotes image information (640 pixels × 480 pixels) obtained from the image sensor, and 17 denotes an opening.

Two sheets of clean paper (Rule 26) 8 The method of specifying the position is performed based on the flow shown in Fig. 4. The slide glass 2 is set on the XY stage 1 for positioning, and the sample, that is, the sample S, is observed by the microscope column 3, and the measurement point is set. In the detector 5 integrated with the lens barrel 3, the specific opening 11 of the positioning marker 7 on the slide glass 2 comes into view. At that time, the liquid crystal display module 8 is sequentially turned on as shown in FIGS. 5 (a) to (g). That is, first, the pixels in one column direction are displayed, and the columns are sequentially scanned (FIGS. 5A to 5C). The position of the detector is indicated by a circle in the figure. When it enters the field of view of the detector 5, the columns are turned on one by one pixel (liquid crystal display unit dot) row by row (Figures (d) to (f) in Figure 5). As a result, the position of an opening in the field of view of the detector 5 is specified from the address of the liquid crystal display. The aperture in the field of view of the detector 5 stores a pattern in advance as shown in Fig. 5 (g), and the location of the center of gravity in the field of view as coordinates on the image. Find out.

The pattern memory allows the size and shape of the opening to be recognized as a pattern, so as shown in Fig. 5 (g), the CPU selects the periphery of the opening and the opening in advance as a figure to the CPU. Recognize. For example, if one liquid crystal dot is further divided into 64 × 480 pixels to determine the position, the area selected to register as pattern information should be about 30% of 64 × 480 pixels. Choose from X300 pixels to the size of 300 X300 pixels. The size of the area to be selected is determined by the size of the opening. If it is less than 60 × 60, the patterning accuracy will be reduced, resulting in poor positioning accuracy. If the number of pixels is more than 300 × 300 pixels, the detectable effective area that can be actually detected on the image is reduced, which causes a non-measurable area in the entire liquid crystal panel. The most effective size is on the order of 60 × 60 to 100 × 100 pixels.

This makes it possible to narrow down a wide range of positions on the slide glass. Since the position can be specified from the address of the liquid crystal and the coordinates on the image, if the address and the coordinates on the image are stored in memory, the position can be surely reproduced.

The high magnification microscope according to the present invention has many effects and advantages as described below.

Replacement paper (Rule 2β) Has 9 points. That is, according to the high-magnification microscopic observation apparatus of the present invention, even if the slide glass is once detached, the observation position can be easily determined again at the 100-nanometer level. In addition, even if the slide glass or lens barrel of the stage shifts due to temperature change or vibration, the position can be quickly identified. In addition, if the image is constantly recognized and observed after pattern matching has been performed, the correction is performed autonomously in real time, so that long-time Timelabs observation and the like can be easily performed. If a high-speed parallel processing image sensor is used, an active anti-vibration function of about 1 to 10 kHz can be realized.

In addition, the present invention is applicable not only to slide glass but also to various positioning works. When finely positioning the work, if such markings are printed in advance and the marking is placed on the liquid crystal display, the same positioning is possible. That is, even when moving from one processing apparatus to another, by transmitting the address of the liquid crystal and the coordinate information on the image, the positioning can be easily and accurately performed.

Further, in a system in which a length measuring device is incorporated on a conventional stage, even if the stage can be precisely positioned, the error cannot be corrected if the observation system or the like moves, but according to the present invention, Since the stage is captured as an image from the observation unit, the entire optical system is very stable. In particular, if a manipulator for processing the sample is installed at the observation section, it is possible to perform live positioning of the manipulator and the sample.

In addition, an autonomous correction stage can be created by feeding back the image information of the aperture array using the light-emitting elements, so that if there is temperature fluctuation or vibration, it is possible to actively correct the position shift. Yes, this eliminates the need to install the microscope on a vibration-isolating facility such as a vibration isolation table, or to perform observation or production processing in an environment-controlled room where wind and temperature changes are suppressed, as in the past. By using this, if a high-magnification ultra-depth microscope or a long-term fluorescence observation limelabs microscope is constructed, it will be possible to obtain clearer images. Furthermore, if a high-speed imaging device such as a parallel vision chip is used, the visual feedback response speed will be much faster, and disturbances such as fluctuation, vibration, and sound can be completely removed. 10 In the future, the use of high-speed response imaging devices such as parallel vision devices will enable the removal of higher frequencies.

In particular, in the assembly process, the load on micro and nano working environments is reduced, so prototype equipment for research and development using microscopy technology (equipped with a laser cutter, laser power, manipulator, etc.), and production It can also be applied to inspection equipment and processing machines typified by micro factories. When these features are combined, according to the present invention, a disturbance prevention function, a length measuring microscope function, a high-precision positioning function, and the like can be realized. In particular, a conventional microscope has a function of performing distance measurement in the field of view of the microscope by image processing or the like, but it can also perform distance measurement including outside the field of view of the microscope.

Also, while observing a moving object with a microscope using the image recognition or pattern matching of the present invention, the moving distance and the trajectory can be simultaneously measured and analyzed using a liquid crystal panel. In other words, when the measurement object moves in long-term fluorescence observation, etc., the stage distance is moved in the process of autonomously moving the stage position according to the moving object by pattern recognition so that the object remains in the field of view. By reading the absolute coordinates in a time series in the same way on a liquid crystal panel, even if the object moves over a wide range, the movement distance can be measured in a time series with high resolution.

Furthermore, it can be replaced with a slide glass DNA chip or the like. In such a case, such a code is embedded in the side of a DNA array engraved on a slide glass. As a result, it can be used as a precision positioning chip when a method that replaces the conventional image processing has emerged in order to increase reading accuracy or throughput in the case of fluorescence identification. Industrial Applicability The high-magnification microscopic observation device according to the present invention is a high-magnification (400 to 2000 × magnification) digital micro microscope, long-time Timelabs observation microscope, nanomaterial alignment device, UV laser micro Yuichi Katsu (cell trimming chromosome cut), microscopic laser trapping (capture and movement) device, egg cell injection device, D 11

It can be suitably used in various fields such as an NA tip spotting device.

Claims

12 Scope of Claim

1. Based on a predetermined pattern formed on the microscope sample holder, a recognition unit for recognizing the predetermined pattern, and the position information recognized by the recognition unit, the microscope sample holder is positioned at a predetermined position. A high-magnification microscopic observation device, comprising: a moving means for moving.

2. The high magnification microscopic observation device according to claim 1, wherein the predetermined pattern is formed by a light emitting element.

3. The high-magnification microscopic observation apparatus according to claim 1, wherein the predetermined pattern is formed by a light-emitting element and an aperture array.

4. Colloid particles, electroluminescent devices, and organic electroluminescent devices used in liquid crystals and electronic vapors that generate electro-optical effects when the light-emitting device is electrically, chemically, optically, or magnetically excited. An element combined with a light source consisting of a luminescence element or a light emitting diode, or a colloid particle used for an electronic paper, a light source consisting of an electroluminescence element, an organic electroluminescence element or a light emitting diode, or a laser spot or 4. The high-magnification microscopic observation device according to claim 2, wherein the device is configured to display a spot of light by a projector device including an optical shutter array.

5. A predetermined pattern formed on the sample holding material for microscope, a recognizing means for recognizing the predetermined pattern, a processing device for processing the sample held on the sample holding material for microscope, and a recognizing means by the recognizing means. A high-magnification microscopic observation apparatus, comprising: a moving means for moving the sample holding material for a microscope or the processing apparatus to a predetermined position based on the obtained position information.

6. The processing equipment is manipulator, laser cutter, laser cutter, spotter or The high magnification microscopic observation device according to claim 5, wherein 13 is an STN · A FM probe.

7. The high-magnification microscope according to any one of claims 1 to 6, wherein the recognition means is a charge-coupled device (CCD) camera, a discharge device (CID) camera, a video camera, a photomultiplier tube, or a high-speed response imaging device. .

8. The high-magnification microscope according to claim 1, wherein the moving means comprises a linear motor, a stepping motor, or a combination of the linear motor and the stepping motor, and a piezo or ultrasonic motor.