JP2014081417A - Laser scanning microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser scanning microscope device which can measure or observe even a moving object, and enlarge and observe movements of a fine part of the moving object as well.SOLUTION: A laser light source 1 emits laser beam, and an acousto-optical element 3 modulates the laser beam to light of two frequencies respectively and emits them in mutually different directions. The light is two-dimensionally scanned by a two-dimensional scanning device 6, a part of the light is partially separated by a beam splitter 7, photoelectric conversion is performed by a light receiving element 8, and respective beat signals are sent out. Light from the laser light source 1 is emitted by an objective lens 11 to a sample S while it is brought closer by pupil transmission lens systems 5, 10, photoelectric conversion of transmission light from the sample S is performed by a light receiving element 9, and respective beat signals are sent out. A phase difference obtained on the basis of the beat signals from the light receiving elements 8, 9 is detected by a signal comparator 15, and a data processing control unit 16 performs data processing to obtain information about the sample S and matches a view point to an object in the sample S on the basis of the data processing.

Description

  The present invention relates to a laser scanning microscope apparatus that performs high-speed measurement and observation of a surface shape profile of a measurement object and a surface state of a cell that is the measurement object by scanning with a laser beam.

  Optical heterodyne interferometry is well known as a method for measuring and observing minute objects with high accuracy. In this method, two laser beams having different frequencies are caused to interfere with each other, a beat signal having a frequency difference between them is generated, and a change in the phase of the beat signal is detected to measure or observe the object to be measured.

  In Japanese Patent Application Laid-Open No. 59-214706 of Patent Document 1 below, two beams having different wavelengths are generated adjacent to each other using an acousto-optic device, and a phase change between these two beams is detected. A method for accumulating phase changes to obtain a surface profile is disclosed. However, in the technique of Patent Document 1, information in the beam profile cannot be extracted, and the resolution in the beam profile that is in the plane cannot be increased.

  On the other hand, a technique called a DPC (Differential Phase Contrast) method has been conventionally known. This is a technique that was first applied to an electron microscope by Dekkers and de Lang, and was later extended to an optical microscope by Sheppard and Wilson et al. That is, the DPC method detects a differential signal between detectors arranged in a far field with respect to an electromagnetic wave irradiated to an object to be measured and symmetrical with respect to an irradiation axis of the electromagnetic wave. Thus, the profile information of the object is obtained.

  On the other hand, using the acousto-optic device, the present inventors scan two beams that have slightly different frequencies and cause a slight misalignment between each other, and place them in the far field. A system has been devised for heterodyne detection of the differential output from the phase change between these two beams obtained by a plurality of light receiving elements. That is, this method can be said to be a method using a fusion of the DPC method and the heterodyne method, and the surface profile of the object is obtained by this method.

  These methods using the DPC method and methods combining the DPC method and the heterodyne method can acquire three-dimensional profile information in one scan, so that very high-speed processing can be performed, and transparent cells and plankton lived. In addition, it can be observed without using means such as staining, and nanoparticles can be observed, which can be said to be a very useful measurement and observation means.

JP 59-214706 A

  However, plankton, cells, etc. move spontaneously in three dimensions, and even in the measurement of nanoparticles etc., the measurement object to be measured and observed by Brownian motion etc. moves, and sufficient measurement and observation I couldn't. Further, along with this, it is extremely difficult to see the detailed movement of the object in a particularly enlarged manner even with the conventional method.

  The present invention has been made in view of the above-described background, and enables a laser scanning microscope that enables measurement and observation of a moving object, and further enables observation of an enlarged movement of details of the moving object. An object is to provide an apparatus.

In order to achieve the above object, the present invention provides a laser light source that emits laser light,
A scanning optical element for two-dimensionally scanning laser light;
An objective lens that emits laser light to a sample having a pupil position and a symmetrical object that can move three-dimensionally;
A light receiving element for receiving reflected light or transmitted light from the sample;
A data processing control unit that obtains information on an output difference from the light receiving element and performs data processing, and matches a viewpoint to an object in the sample based on the data processing,
It is set as the laser scanning microscope apparatus characterized by including.

Furthermore, the present invention provides a laser light source that emits laser light,
An optical modulator that emits light in different directions while modulating the laser light into two lights having different frequencies;
A scanning optical element for two-dimensionally scanning two lights;
An objective lens that emits two lights to a sample having a pupil position and a symmetrical object that can move three-dimensionally;
A pupil transfer lens system that is positioned between the light modulator and the objective lens and causes the light emitted in the mutually different directions by the light modulator to approach each other on the sample;
A light receiving element that receives reflected light or transmitted light from the sample and photoelectrically converts it to generate a beat signal; and
A signal comparator for detecting a phase difference between two lights obtained based on a beat signal of the light receiving element and an amplitude of the beat signal itself;
A data processing control unit that obtains phase information based on the phase difference detected by the signal comparator and intensity information based on the amplitude to perform data processing, and matches a viewpoint to an object in the sample based on the data processing,
It is set as the laser scanning microscope apparatus characterized by including.

In the present invention, a beam splitter that partially separates the light from the light modulator and reflects the reflected light from the sample,
The light receiving element is
A first light receiving element for receiving the light separated by the beam splitter;
A second light receiving element for receiving reflected light from the sample reflected by the beam splitter;
Including
It is preferable to use a signal comparator to create a beat signal between the modulation signal received by the first light receiving element and the output signal received by the second light receiving element.
Similarly, having a beam splitter that partially separates the light from the light modulator,
The light receiving element is
A first light receiving element for receiving the light separated by the beam splitter;
A second light receiving element for receiving transmitted light from the sample;
Including
It is preferable to use a signal comparator to create a beat signal between the modulation signal received by the first light receiving element and the output signal received by the second light receiving element.

  On the other hand, the signal comparator according to the present invention creates a beat signal between the modulation signal applied to the optical modulator and the output signal of the light receiving element, so that two lights obtained based on the beat signal are obtained. It is preferable to detect the phase difference between each other and the amplitude of the beat signal itself. An optical modulator according to the present invention includes an acoustooptic device that makes the laser beam emitted from the laser light source incident thereon, and a signal generator that applies a carrier AC signal (fc) and a sine wave signal (fm) to the acoustooptic device. Is preferably included. Further, the scanning optical element according to the present invention includes a one-dimensional scanning element of a galvano mirror and a resonant mirror, a two-dimensional scanning optical system comprising two one-dimensional scanning devices and a pupil transfer lens system, or a one-dimensional or two-dimensional microscopic element. It is preferable to be a mirror device.

  On the other hand, the light receiving element according to the present invention or the second light receiving element may be a divided light receiving element that is divided into at least two parts in a direction perpendicular to a separation direction of light emitted in different directions by the light modulator. Is preferred. The output of the light receiving element according to the present invention is the sum signal of all the divided light receiving elements of the divided light receiving elements divided into two or more of the light receiving elements, or the division at the corresponding position of the divided light receiving elements divided into two or more. It is preferable to obtain from a difference signal between the light receiving elements.

Furthermore, the present invention is a laser scanning microscope apparatus in which the sample is placed on a mounting table,
A first step of identifying a target area where the target object exists;
A second step of binarizing the intensity information of the target region to detect the center of gravity;
A third step of evaluating the degree of variation in the histogram of the intensity information;
A fourth process for identifying the direction in which the variation is large;
Each process consisting of
By moving the mounting table, the object in the sample is moved to the center of gravity calculated from the second process,
It is preferable to move the objective lens in the direction of large variation by the fourth process.
In the fourth process according to the present invention, it is preferable that in the fourth process, the objective lens is vibrated slightly to move the objective lens in a direction in which variation is large.

In addition, the present invention provides a near-infrared laser light source that emits infrared light;
A collimator lens that collimates infrared light from the near-infrared laser light source;
A sub-beam splitter disposed on the optical axis between the pupil transmission lens system and the objective lens or between the pupil transmission lens system and the two-dimensional scanning device and reflecting infrared light to the sample side When,
Is preferably included.
In addition, it is preferable that the infrared cut filter according to the present invention is disposed between the light receiving element and an infrared light introducing optical path.

The operation of the claimed invention will be described below.
According to the DPC method and the fusion method of the DPC method and the heterodyne method, three-dimensional profile information can be acquired in one scan, and the following calculation is performed based on the information.
Specifically, images from a sample having a symmetrical object that can move three-dimensionally are acquired continuously over time to obtain image data for each frame, and the calculation area of this object is specified. The differential signal of the information obtained from the observation of the object is binarized, and the barycentric coordinates X and Y are detected.
X = Σxnnx / Σnx
Y = Σynny / Σny
Here, xn, yn are the x coordinate and y coordinate corresponding to the binarized pixel, and nx, ny are the number of pixels occupying the x, y coordinate.
Based on the center-of-gravity coordinates X and Y calculated by this calculation, the x and y values of the moving stage on which the objective lens and the sample are placed are changed to move the object with respect to the optical axis so that the object is at the center of the field of view. Control so that it is always located.

On the other hand, since objects such as cells and plankton move three-dimensionally, a blurred image is obtained unless the objective lens or moving stage is moved in the focus direction (Z-axis direction) in addition to the two-dimensional tracking. .
At this time, when the histogram of the contrast of the signal intensity detected from the object is created based on the data of the barycentric coordinates X and Y, the larger the variation is, the more focused. In particular, in the method using heterodyne detection, since the phase information represents the direction of the inclination of the profile, an angle value of about ± 90 degrees is obtained in the vicinity of the focal point. For example, the contrast histogram of the signal intensity is sensitive to the in-focus state, and although this is used, the phase information can also be used as supplementary confirmation means.

  Since the moving direction of the in-focus position of the object can be known from the above calculation results, the focus moving direction can be determined by comparing the current frame information and the previous frame information. The in-focus state can be maintained by moving the objective lens in the moving direction or moving the moving stage up and down.

  On the other hand, in the above DPC method and the fusion method of the DPC method and the heterodyne method, light having a wavelength in the infrared region different from the wavelength of the signal detection laser is coaxially connected via a beam splitter between the scanning optical system and the objective lens. It is conceivable that the light is incident to That is, the light having the wavelength in the infrared region is synthesized by another optical system and irradiated on the target, and the target is fixed by performing laser trapping with the light in the infrared region.

  Here, in the DPC method, a color filter is used to separate the light in the infrared region from the light having the laser wavelength used. However, in the fusion method of the DPC method and the heterodyne method, the light in the infrared region is not modulated and does not affect the detection signal at all, so there is no need to use a color filter or the like. Therefore, by using light having a wavelength in the infrared region in this way, time required for image processing is not required, and thus higher-speed scanning is possible.

  As described above, according to the laser scanning microscope apparatus of the present invention, since cells and nanoparticles can be tracked, observed and measured in this way, information such as minute state changes can be acquired in real time. It has the big characteristic which the electron microscope which inactivates etc. inactivate and measures. In addition, a technique for applying a fine structure and movement of a living organism to a micromachine has been proposed and is being implemented. However, it becomes an extremely effective observation means for application of such a fine structure and movement of a living organism.

  As described above, if the DPC method or a method that combines the DPC method and the heterodyne method is used, even though a weak signal can be obtained with high accuracy, the three-dimensional profile information and the refractive index distribution information can be obtained in one scan. Since movement cannot be suppressed with cells, minute objects, etc., it has been difficult for these objects to fully demonstrate their observation and measurement capabilities.

  However, according to the laser scanning microscope apparatus of the present invention, the position information and the in-focus information are obtained by imaging the object using the two-dimensional scanning system and the data obtained by converting the signal data acquired by the light receiving element, and performing image processing. Can be obtained.

  Along with this, by controlling the position of the moving stage and objective lens on which the object is placed based on this information, high-speed measurement and observation are possible even for moving objects such as moving cells and microorganisms. In addition, the detailed movement of the moving object can be enlarged and observed, so that the observation and measurement ability of the laser scanning microscope apparatus can be fully exhibited. In addition, by combining the above optical system with a laser trap optical system using infrared laser light, the time required for image processing can be saved, so that higher-speed scanning is possible.

It is a block diagram of the transmission optical system in the DPC method which concerns on Example 1 of the laser scanning microscope apparatus of this invention. It is a block diagram of the reflective optical system in the DPC method which concerns on Example 1 of the laser scanning microscope apparatus of this invention. It is a block diagram of the transmission optical system which combined DPC method and heterodyne method which concern on Example 1 of the laser scanning microscope apparatus of this invention. It is a figure which expands and shows the objective lens of FIG. 3, and a measurement object peripheral part. It is explanatory drawing showing the irradiation area | region on the sample by the transmission optical system which combined DPC method and heterodyne method based on Example 1. FIG. It is a block diagram of the reflective optical system which combined DPC method and heterodyne method which concern on Example 1 of the laser scanning microscope apparatus of this invention. It is a block diagram of the transmission optical system in the DPC method which concerns on Example 2 of the laser scanning microscope apparatus of this invention. It is a block diagram of the reflective optical system in the DPC method which concerns on Example 2 of the laser scanning microscope apparatus of this invention. It is a block diagram of the transmission optical system which combined DPC method and heterodyne method which concern on Example 2 of the laser scanning microscope apparatus of this invention. It is a block diagram of the reflective optical system which combined DPC method and heterodyne method which concern on Example 2 of the laser scanning microscope apparatus of this invention. It is the schematic of the preparation applied to this invention.

  Embodiments 1 and 2 of the laser scanning microscope apparatus according to the present invention will be described below in detail with reference to the drawings.

  Hereinafter, an optical system of the DPC method which is effective by specifically applying the above optical system and an optical system combining the DPC method and the heterodyne method will be described. Here, in the laser scanning microscope apparatus according to the present embodiment, an object is imaged based on the obtained signal such as the difference output and the scanning signal, and the image signal is used in the sample using an image processing technique. This is for tracking an object that can move, and will be specifically described below.

  FIG. 1 shows a block diagram of a transmission optical system in the DPC method, which will be described in detail below based on this block diagram. As shown in FIG. 1, the light beam from the laser light source 1 is collimated by the collimator lens 2 and is incident on the two-dimensional scanning device 6. The two-dimensional scanning device 6 is a device that two-dimensionally scans light on a surface, and includes, for example, a MEMS, a galvanometer mirror, a resonant mirror, and the like.

  The parallel luminous flux from the collimator lens 2 is incident on the objective lens 11 through the pupil transmission lens system 10 that transmits the pupil position of the two-dimensional scanning device 6 to the pupil position of the objective lens 11, and then converges on the sample S. The The light converged on the sample S becomes transmitted light and enters the light receiving element 9. The light receiving element 9 has a structure including a photoelectric conversion unit (not shown) that generates each beat signal subjected to photoelectric conversion. The light receiving element 9 is connected to the data processing control unit 14, and data processing is performed by the data processing control unit 14 based on information relating to light reception sent from the light receiving element 9. At this time, the light receiving element 9 is arranged at a position substantially becoming a far field from the sample S, and is divided light receiving elements 9A and 9B which are divided into two symmetrically with respect to the optical axis L. However, it may be divided into three or more.

  As a result, the parallel light beam on the optical axis L is separated into 0th order diffracted light and ± 1st order diffracted light by the refractive index distribution and unevenness of the sample S, and these separated lights are received by the light receiving element 9 while interfering with each other. The Along with this, the refractive index distribution and unevenness information of the sample S are converted into interference information between the 0th-order diffracted light and the ± 1st-order diffracted light. At this time, the above information of the sample S is reflected on the difference output between the two divided light receiving elements 9A and 9B that are symmetrical with respect to the optical axis L, and sent to the data processing control unit 14.

  On the other hand, the sample S is mounted on a moving stage 12 that is orthogonal to the optical axis L and that can be moved by a motor or the like along the X-axis direction and the Y-axis direction orthogonal to each other. The objective lens 11 can also be moved up and down along the optical axis L by a motor or the like. The moving stage 12 and the objective lens 11 are respectively connected to the data processing control unit 14, and the data processing of the data processing control unit 14 and the scanning information sent to the two-dimensional scanning device 6 from the data processing control unit 14. Based on this, the data processing control unit 14 can make the information into the form of an image or data. Accordingly, the data processing control unit 14 can match the object in the sample S by following the viewpoint of the laser scanning microscope apparatus.

  On the other hand, FIG. 2 is a block diagram of a reflection optical system in the DPC method, and differs from the transmission optical system shown in FIG. 1 in that a beam disposed between the collimator lens 2 and the two-dimensional scanning device 6. A part of the light beam is taken out by the splitter 7, and the light receiving element 9 including the divided light receiving elements 9A and 9B divided into at least two parts respectively receives the light to detect the difference output therebetween. At this time, the reflected parallel light from the sample S is substantially far-field information.

Next, FIG. 3 and FIG. 6 show block diagrams of an optical system combining the DPC method and the heterodyne method proposed by the inventors, which will be described below. Here, FIG. 3 shows a block diagram of a transmission optical system that combines these methods, and FIG. 6 shows a block diagram of a reflection optical system that combines these methods.
These optical systems differ from the optical systems shown in FIGS. 1 and 2 by creating two light beams that are very close to the acoustooptic device 3 and making the magnification ratio appropriate so that the beam shown by the solid line in FIG. The sample S can be irradiated with two beams that are very close to each other and have the same diameter as a beam LB indicated by LA and a dotted line.

Specifically, as shown in FIG. 3, a laser light source 1 that emits laser light and an acousto-optic device (AOD) 3 that is an optical modulator to which the operation is controlled by connecting an AOD driver 4. A collimator lens 2 is arranged between them. Further, the acoustooptic device 3 includes a pupil transmission magnifying lens system 5 composed of two groups of lenses, a beam splitter 7 that separates and emits input laser light, and two-dimensionally scans the input laser light 2. The dimension scanning devices 6 are arranged in order.
Further, a pupil transmission lens system 10 composed of two groups of lenses is positioned adjacent to the beam splitter 7, and an objective lens 11 is disposed next to the objective lens 11 so as to face it. That is, these members are arranged along the optical axis L which is the optical path of the laser beam.

  On the other hand, a light receiving element 8 that is an optical sensor is disposed at a position that is orthogonal to the direction in which the optical axis L passes and is adjacent to the right side of the beam splitter 7. A light receiving element 9 that is also an optical sensor is disposed on the lower side, which is the back side of the sample S. The light receiving elements 8 and 9 are constituted by divided light receiving elements 8A, 8B, 9A, and 9B that are divided into two symmetrically with respect to the optical axis L, respectively. The light receiving elements 8 and 9 have a structure including a photoelectric conversion unit (not shown) that creates each photoelectrically converted beat signal.

  Further, these light receiving elements 8 and 9 are connected to a signal comparator 13 for comparing signals from these light receiving elements 8 and 9, respectively, and the modulation signal itself applied to the acoustooptic element 3 and the output generated by the light receiving element 9. The signal comparator 13 detects the phase difference between the two lights obtained based on the beat signal and the amplitude of the beat signal itself from the beat signal which is a signal. The signal comparator 13 is connected to a data processing control unit 14 that finally processes data to obtain a profile of the sample S and the like.

  Accordingly, two DSB-modulated signals having the carrier frequency fc and the modulation frequency fm are input from the outside to the acoustooptic device 3 via the AOD driver 4 to create these two light beams that are very close to each other. it can. The light beams emitted in two very close directions are transmitted through the pupil transfer lens system 5 that transmits the substantial pupil position of the acoustooptic device 3 to the pupil position of the two-dimensional scanning device 6, and the two-dimensional light is scanned on the surface. The light enters the objective lens 11 through the pupil transmission lens system 10 for transmitting the scanning device 6 and the pupil position of the two-dimensional scanning device 6 to the pupil of the objective lens 11.

  Similar to the above, this sample S is mounted on a moving stage 12 that is orthogonal to the optical axis L and that can be moved by a motor or the like along the X-axis direction or the Y-axis direction orthogonal to each other. Yes. The objective lens 11 can also be moved up and down along the optical axis L by a motor or the like. The moving stage 12 and the objective lens 11 are respectively connected to the data processing control unit 14, and based on the data processing of the data processing control unit 14, the viewpoint of the laser scanning microscope apparatus with respect to the object S1 in the sample S. It is possible to match by following.

  As a result of the above, the light beam converged by the objective lens 11 scans the surface of the sample S as two very close spots with the optical axis L shown in FIG. The frequency of these two beams LA and LB is “light frequency + carrier frequency fc ± modulation frequency fm”, and these two spots are two signals of frequency fc + fm and frequency fc−fm. By heterodyne detection of these signals, a signal reflecting the unevenness information and refractive index distribution of the sample S can be obtained.

  Accordingly, the approximate center of the irradiation areas A and B where the two beams LA and LB centered at two points separated by the center distance Δx are defined as a boundary line C in FIG. Since the sandwiched two beams LA and LB are irradiated onto the sample S and the position of the object S1 is detected, the object S1 that can move within the sample S can be followed.

  In the present embodiment, in order to specifically perform heterodyne detection, a part of the irradiated modulation signal is extracted by the beam splitter 7, a reference signal is obtained by the light receiving element 8, and the reference signal and the light receiving element divided into two are obtained. A differential output is obtained from the signal detected at 9, phase difference information and intensity information are acquired by the signal comparator 13, and sent to the data processing control unit 14. The data processing control unit 14 displays the phase difference information and intensity information acquired together with the scanning information sent to the two-dimensional scanning device 6 as an image or data form on a display (not shown) or accumulates it as data in a memory. To do.

  However, the light receiving element 8 is not necessarily required, and may be compared with a signal output to the acoustooptic element 3, that is, a modulation signal itself applied to the acoustooptic element 3. In this case, a delay occurs due to a circuit system, an acoustooptic device, or the like. However, if it is corrected in advance, it does not have a great influence on phase difference detection or the like.

  In addition, two very close spot lights that scan the surface of the sample S on the surface are lights having different frequencies from each other. However, a magnification optical system such as the pupil transfer lens system 5 or 10 is substantially used. This makes it possible to make the spot very close even at a high frequency. Thus, high-speed information acquisition can be performed by high-speed scanning.

  On the other hand, FIG. 6 is a block diagram of a reflection optical system combining the DPC method and the heterodyne method. The difference from the transmission optical system shown in FIG. The difference output is detected by taking out the light beam by the splitter 7 and receiving the light beam by the light receiving element 9 including the divided light receiving elements 9A and 9B divided into two. At this time, the reflected parallel light from the sample S is substantially far-field information.

  On the other hand, the two lights obtained in this way can have a very low degree of separation by the above-described method, and are substantially the same as the information scanned with one beam. On the other hand, the DPC method described above is a method of scanning with one beam and obtaining a differential output of at least two divided light receiving elements arranged in the far field.

  That is, in comparison with the DPC method, in the method further using the present heterodyne method, the phase change and the intensity change can be detected with high accuracy by detecting the heterodyne, and the light received by the light receiving element 9 is reduced. Even if it is very weak, it can be detected with high accuracy by increasing the gain of the detection circuit system, and since the detected signal is only the modulation signal, it is not affected by disturbance light. High-precision detection can be performed.

  In addition, by using an optical system that once separates the 0th-order diffracted light and ± 1st-order diffracted light in the light receiving element portion of the optical system as described above, information with a higher spatial frequency, that is, a significant improvement in lateral resolution Can be planned.

  The signal obtained using the above optical system is a signal having no bias of the differential interference optical system, and reflects the inclination of the object S1 with respect to the plane formed by the XY axes in FIG. Positive and negative are determined. Further, the laser light is two-dimensionally scanned by the two-dimensional scanning device 6 and sent to the sample S side. The light from the sample S is received by the light receiving element 9 and the signal comparator 13 receives the phase difference information and The intensity information is acquired and sent to the data processing control unit 14. Then, by combining the data based on the differential signal from the differential output and the scanning information at the time of two-dimensional scanning, the data processing control unit 14 can obtain the image information of the object S1.

  When acquiring this image information, in the DPC method, a differential output can be raised and displayed in advance by applying a bias. Further, in the fusion method of the DPC method and the heterodyne method, since the intensity information and the phase information can be divided, the inclination information can be expressed as phase information, and the intensity information can be displayed as the absolute value of the differential output signal. .

  By such data processing, the image information can be converted into several types of information, but optically, the spatial frequency of the object S1 in the sample S is maximized in the just focus state. Therefore, the contrast change of intensity increases as the just focus is achieved. In other words, the intensity contrast histogram increases in variation as the focus is adjusted. Therefore, the degree of variation can be quantified and used as an evaluation amount of the focus degree.

Here, according to the DPC method and the fusion method of the DPC method and the heterodyne method, three-dimensional profile information can be acquired by one scan, and the following processing can be considered based on the information.
First, according to the optical system described above, three-dimensional information can be acquired in real time, so that a moving object and a stationary background can be easily separated between frames of a video signal constituting image information. For example, the data processing control unit 14 can perform a difference between frames, leave a pixel having a change, and identify an area of a certain size or more as a moving object area. In this way, in the first process, first, a target area where the target object S1 is present is selected from among several areas in the sample S.

Next, after specifying the calculation area of the object S1 as described above, the data processing control unit 14 obtains 2 pieces of intensity information obtained from observation of the object S1 based on the differential output of the object area in the second process. The centroid coordinates X and Y, which are centroid positions in the X-axis direction and the Y-axis direction orthogonal to the optical axis L and orthogonal to each other, are detected by the following simple calculation.
X = Σxnnx / Σnx
Y = Σynny / Σny
Here, xn, yn are the x coordinate and y coordinate corresponding to the binarized pixel, and nx, ny are the number of pixels occupying the x, y coordinate.
This calculation is performed for each frame, and based on the values of the center of gravity coordinates X and Y calculated by the calculation of each frame, the x and y values of the moving stage 12 that is the mounting stage on which the sample S is placed are changed to change the object. S1 can be moved with respect to the optical axis L. For example, if the object S1 is controlled so as to be always positioned on the optical axis L which is the center of the visual field, the object S1 can be almost stopped at the viewpoint which is the center of the visual field.

  On the other hand, since the object S1 such as a cell or plankton moves three-dimensionally, in addition to the two-dimensional tracking, the objective lens 11 or the moving stage 12 is not moved in the focus direction (Z-axis direction in FIG. 4). And a blurred image. For this reason, the variation amount of the histogram of the intensity information, which is the above-described evaluation parameter of the focus degree, is acquired for each frame, the data processing control unit 14 evaluates this variation degree in the third process, and this variation amount is large. Then, the objective lens 11 or the moving stage 12 is moved up and down, and control is performed so as to maintain the in-focus state.

  For example, when a contrast histogram of the signal intensity detected from the object S1 is created, the larger the variation, the better the focus. In particular, in the method using heterodyne detection, since the phase information represents the direction of the inclination of the profile, an angle value near ± 90 degrees is obtained near the focal point. Accordingly, the contrast histogram of the signal intensity is sensitive with respect to the in-focus state, and although this is used, the phase information can also be used as supplementary confirmation means.

  At this time, in order to specify the direction in which the variation is large, the objective lens 11 is always finely oscillated in the vertical direction in the fourth process so as to be in a so-called wobbling state. It is possible to move 11. Since there are various other methods for realizing the in-focus state based on such image information, they may be applied. For example, as one method, the more in-focus state, the clearer the image becomes, and the substantial spatial frequency becomes higher. For this reason, the output itself on the light receiving element away from the optical axis of the light receiving element becomes large. By acquiring this information, it is possible to determine the degree of the in-focus state. Further, since the tilt information becomes larger in the focused state, the intensity information obtained by the above heterodyne detection becomes larger, so that it can be used to determine the degree of the focused state.

  As described above, if the three-dimensional tracking is performed by the image processing method from the obtained signal output, the object S1 is relatively easily positioned at the center of the field of view and is brought into a focused state. be able to. In this way, the movements of microorganisms and cells can be directly observed three-dimensionally, and it is possible to acquire the relationship between the organs and movements in the cells and various information.

In this embodiment, unlike the first embodiment, the object S1 is physically trapped to facilitate observation and measurement. In this embodiment, in addition to the optical system shown in the first embodiment, an optical system that performs so-called laser trapping is added.
7 shows a block diagram of the transmission optical system in the DPC method, FIG. 8 shows a block diagram of the reflection optical system in the DPC method, and FIG. 9 shows a block of the transmission optical system in which the DPC method and the heterodyne method are combined. FIG. 10 shows a block diagram of a reflection optical system in which the DPC method and the heterodyne method are combined. These optical systems are substantially the same as those described in the first embodiment, and differences from the first embodiment will be described below.

  Each optical system of the present embodiment has a near-infrared laser light source 15 that emits infrared light, which is laser light, and a collimator lens 16 that collimates the infrared light from the near-infrared laser light source 15. doing. Further, a sub beam splitter 17 for making the infrared light that has passed through the collimator lens 16 and converted into parallel light incident on the objective lens 11 together with the signal light from the laser light source 1 includes the pupil transfer lens system 10 and the objective lens. 11. For this reason, this sub beam splitter 17 irradiates the object S1 in a spot shape with this infrared light that is coaxially incident on the optical axis L of each optical system. As a result, the object S1 can be trapped and fixed on the moving stage 12 by a known light pressure.

  Here, in order to separate the signal light and the infrared light for obtaining the signal, in the DPC method shown in FIGS. 7 and 8, the color filter 18 that is an infrared cut filter includes the light receiving element 9 and the infrared light. It is arranged at a position in front of the light receiving element 9 which is between the light introduction optical path and only the signal light passes through the color filter 18.

  On the other hand, in the fusion method of the DPC method and the heterodyne method shown in FIGS. 9 and 10, the signal light is electrically modulated, whereas the infrared light is not modulated. Therefore, automatic detection by electrical heterodyne detection is performed. Therefore, only signal light is detected. For this reason, in the fusion method of the DPC method and the heterodyne method, there is no need to use a special optical element such as the color filter 18.

  As described above, according to the present embodiment, the object S1 that can be trapped with infrared light is a small cell or a nanoparticle or polymer in a liquid. Normally, it is difficult to observe by moving spontaneously or by Brownian motion, but the object S1 can be easily captured by using the method described above.

  In addition, according to the present embodiment, time required for image processing is not required, so that higher-speed scanning is possible. Further, when infrared light is introduced from an objective lens having a low NA from the opposite direction to the signal light and signal light is introduced by an objective lens having a high NA, observation and measurement may be performed while capturing the object S1 with high resolution. it can.

Next, an example of the preparation 21 for fixing the object S1 on the moving stage 12 in each of the above embodiments will be described with reference to FIG.
Specifically, using a material with low birefringence such as glass or silicon, a preparation 21 having a spherical tip 21A and a stretchable material as shown in FIG. 11 is employed. A structure in which the preparation 21 is softly adsorbed to a cover glass (not shown) like a suction cup, and several uneven portions 22 having a diameter and depth different from the size of the object S1 are provided between the tips 21A. And

  That is, the preparation 21 serves to roughly confine the solution containing the object S1 between the cover glass and the cover glass. In this way, since the range of the object S1 can be limited to some extent from the beginning, the object S1 can be easily identified, the calculation speed can be improved, and the efficiency of the optical trap pressure can be improved.

Instead of moving the objective lens in the Z-axis direction as in the above embodiment, the objective lens may be moved in the XYZ three-axis directions by the moving stage 12. Moreover, the table used as the support surface of the moving stage 12 is naturally formed of a transparent glass material or the like if it is a transmission system optical.
The embodiments according to the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention.

  The present invention is suitable for various types of microscopes as well as laser scanning microscope apparatuses that perform observation and measurement of the surface shape of an opaque object by scanning with laser light, and observation and measurement of the surface or internal structure of a transparent object at high speed. Is.

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Collimator lens 3 Acousto-optic device 4 AOD driver 5 Pupil transmission lens system 6 Two-dimensional scanning device 7 Beam splitter 8, 9 Light receiving element 10 Pupil transmission lens system 11 Objective lens 12 Moving stage 13 Signal comparator 14 Data processing Control unit 15 Near-infrared laser light source 16 Collimator lens 17 Sub beam splitter 18 Color filter 21 Preparation 22 Concavity and convexity S Sample S1 Object

Claims (13)

A laser light source that emits laser light;
A scanning optical element for two-dimensionally scanning laser light;
An objective lens that emits laser light to a sample having a pupil position and a symmetrical object that can move three-dimensionally;
A light receiving element for receiving reflected light or transmitted light from the sample;
A data processing control unit that obtains information on an output difference from the light receiving element and performs data processing, and matches a viewpoint to an object in the sample based on the data processing,
A laser scanning microscope apparatus comprising: A laser light source that emits laser light;
An optical modulator that emits light in different directions while modulating the laser light into two lights having different frequencies;
A scanning optical element for two-dimensionally scanning two lights;
An objective lens that emits two lights to a sample having a pupil position and a symmetrical object that can move three-dimensionally;
A pupil transfer lens system that is positioned between the light modulator and the objective lens and causes the light emitted in the mutually different directions by the light modulator to approach each other on the sample;
A light receiving element that receives reflected light or transmitted light from the sample and photoelectrically converts it to generate a beat signal; and
A signal comparator for detecting a phase difference between two lights obtained based on a beat signal of the light receiving element and an amplitude of the beat signal itself;
A data processing control unit that obtains phase information based on the phase difference detected by the signal comparator and intensity information based on the amplitude to perform data processing, and matches a viewpoint to an object in the sample based on the data processing,
A laser scanning microscope apparatus comprising: A beam splitter that partially separates light from the light modulator and reflects reflected light from the sample;
The light receiving element is
A first light receiving element for receiving the light separated by the beam splitter;
A second light receiving element for receiving reflected light from the sample reflected by the beam splitter;
Including
3. The laser scanning microscope apparatus according to claim 2, wherein a beat signal between the modulation signal received by the first light receiving element and the output signal received by the second light receiving element is created by a signal comparator. A beam splitter for partially separating light from the light modulator;
The light receiving element is
A first light receiving element for receiving the light separated by the beam splitter;
A second light receiving element for receiving transmitted light from the sample;
Including
3. The laser scanning microscope apparatus according to claim 2, wherein a beat signal between the modulation signal received by the first light receiving element and the output signal received by the second light receiving element is created by a signal comparator. The signal comparator is
By creating a beat signal between the modulation signal applied to the optical modulator and the output signal of the light receiving element, the phase difference between the two lights obtained based on the beat signal and the amplitude of the beat signal itself are obtained. The laser scanning microscope apparatus according to claim 2, wherein the laser scanning microscope apparatus is detected.   The optical modulator includes an acousto-optic device that makes the laser beam emitted from the laser light source incident, and a signal generator that applies a carrier AC signal (fc) and a sine wave signal (fm) to the acousto-optic device. The laser scanning microscope apparatus according to claim 2, wherein:   The scanning optical element is a one-dimensional scanning element of a galvanometer mirror or a resonant mirror, a two-dimensional scanning optical system comprising two one-dimensional scanning devices and a pupil transfer lens system, or a one-dimensional or two-dimensional micromirror device. The laser scanning microscope apparatus according to claim 1, wherein the laser scanning microscope apparatus is provided.   The light-receiving element or the second light-receiving element is a divided light-receiving element that is divided into at least two parts in a direction perpendicular to a separation direction of light emitted in different directions by the light modulator. The laser scanning microscope apparatus according to any one of 3 to 7.   The output of the light receiving element is the sum signal of all the divided light receiving elements of the divided light receiving elements divided into two or more of the light receiving elements, or the difference between the divided light receiving elements at the corresponding positions of the divided light receiving elements divided into two or more. The laser scanning microscope apparatus according to claim 1, wherein the laser scanning microscope apparatus is obtained from a signal. A laser scanning microscope apparatus in which the sample is mounted on a mounting table,
A first step of identifying a target area where the target object exists;
A second step of binarizing the intensity information of the target region to detect the center of gravity;
A third step of evaluating the degree of variation in the histogram of the intensity information;
A fourth process for identifying the direction in which the variation is large;
Each process including
By moving the mounting table, the object in the sample is moved to the center of gravity calculated from the second process,
10. The laser scanning microscope apparatus according to claim 1, wherein the objective lens is moved in a direction in which the variation is large by a fourth process.   The laser scanning microscope apparatus according to claim 10, wherein in the fourth step, the objective lens is moved in a direction in which variation of the histogram is large by minutely vibrating the objective lens. A near-infrared laser light source that emits infrared light; and
A collimator lens that collimates infrared light from the near-infrared laser light source;
A sub-beam splitter disposed on the optical axis between the pupil transmission lens system and the objective lens or between the pupil transmission lens system and the two-dimensional scanning device and reflecting infrared light to the sample side When,
The laser scanning microscope apparatus according to claim 1, comprising:   13. The laser scanning microscope apparatus according to claim 12, wherein an infrared cut filter is disposed between the light receiving element and an infrared light introduction optical path.

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