JP3634343B2 - Digitally controlled scanning method and apparatus - Google Patents

Description

Technical field
The present invention relates to a sample observation device such as a scanning optical microscope, a device that performs light stimulation or light processing on an arbitrary point or minute region in a three-dimensional space in a sample observation region, or three-dimensional coordinate data of a surface shape or surface shape The present invention relates to a digitally controlled scanning method and apparatus that can be applied in a wide range such as an apparatus for measuring the length between two points on the surface of a sample and is excellent in versatility.
Background art
Scanning microscopes that employ confocal optical systems are widely used. Various types of scanning confocal microscopes have been put into practical use, such as a mode in which light is deflected using a photoacoustic element, a mode in which light is deflected by a galvanometer mirror, and a mode in which light is scanned by rotating a pinhole plate called a nippo desk Has been.
A scanning microscope using a micro light deflecting element (DMD) as a scanning means is published in “Optics Letters”, Vol. 22, 1997, 751 to 753. This type of scanning microscope is described, for example, in US Pat. No. 5,587,832.
Japanese Patent Application Laid-Open No. 11-194275 proposes a scanning microscope using a micro light deflecting element as a scanning means and an aperture correction technique.
However, the scanning microscope described in the above-mentioned US Pat. No. 5,587,832 that uses the micro light deflector as the scanning means has a wide illumination area of the sample because the illumination system is a projection illumination with a parallel beam of laser. It is illuminated and only images a single point within its wide illumination area. That is, since the light source and the detector are not conjugated with respect to the objective lens, and the illumination light is not condensed only at the observation point, the performance as a confocal microscope is not sufficiently exhibited.
Similarly, the scanning microscope described in Japanese Patent Application Laid-Open No. 11-194275 does not have a conjugate relationship between the light source and the detection point with respect to the objective lens, and a high spatial resolution as a confocal microscope cannot be obtained.
When a minute light deflection element is used as a reflection type scanning device, illumination light is tilted and introduced into the minute light deflection element, resulting in uneven illumination and uneven illumination in the observed image.
Furthermore, when laser light, which is highly coherent light as a light source, is used as the light source for the purpose of increasing the resolution, the light intensity becomes Gaussian with the center of the beam being maximized, so that the illumination unevenness increases and becomes complicated. There are problems such as.
The present invention has been made in view of the above-described conventional problems, and an object thereof is to provide a digitally controlled scanning method and apparatus that can correct illumination unevenness and realize an observation apparatus such as a laser confocal microscope with high spatial resolution. To do.
Another object of the present invention is to provide an apparatus for performing light stimulation / light processing on a three-dimensional arbitrary point in a sample observation region using the digital control scanning technique.
Another object of the present invention is to provide a length measuring device for an object on the surface based on the surface shape and the three-dimensional coordinate data of the surface shape using the digital control scanning technique.
Disclosure of the invention
In the digitally controlled scanning method according to the present invention, a real image of a light source is formed on a plurality of micromirrors of a micro light deflector (DMD), the tilt of each micromirror is scanned, and the tilted micromirror is used as a second light source. The objective lens illuminates an image in the sample, and the fluorescent or scattered light emitted from the imaging region of the sample is imaged by the objective lens on a scanned micromirror, and this is imaged. To do.
The digitally controlled scanning device according to the present invention comprises imaging optical means, illumination optical means, sample imaging means, and a computer.
In FIG. 1, the imaging optical means forms an image of fluorescence or scattered light emitted from a sample on the micromirror of the DMD 4 with an objective lens, and picks up the image through the imaging imaging lens 6 of the imaging optical system. It is configured. The imaging device 5 is configured by, for example, a charge coupled device (CCD).
The illumination optical means is made coaxial so that the optical axis overlaps with the imaging optical system, and a real image of the point light source is formed on the micromirror of the DMD 4 using the laser light introduced by using the dichroic mirror 7. The microlens two-dimensional array 12, the pinhole two-dimensional array plate 9, and the light source imaging lens 8 are configured such that one point light source is imaged on one micromirror. In the case of a planar light source, a minute portion of the planar light source image selectively extracted by tilting the micromirror is reduced and imaged by the objective lens. Only the imaging portion is illuminated on the sample space, and only the fluorescence emitted from the imaging portion is imaged.
The sample imaging means determines the position by moving up and down the objective lens 2 of the infinity correction optical system, the imaging lens 3 of the infinity correction optical system, and the objective lens 2 that form a real image of the sample 1 on the micromirror of the DMD 4. The objective lens Z position specifying mechanism 14 is configured. The light absorber 10 uses, for example, a black body to absorb light that is not used for illumination from a micromirror that does not tilt the DMD 4. The computer 11 incorporates a control program for realizing a scanning function, a spatial modulation function, and an intensity adjustment function while synchronizing the DMD 4 with the imaging device 5 and a user interface program necessary for the setting, and a pointing device 15. A display device 16 is attached.
A real image of the DMD 4 micromirror is picked up by the image pickup optical means, and this image data is transmitted to the computer 11. The computer 11 processes the image data and displays it. A real image of the point light source formed on the surface of the pinhole two-dimensional array plate 9 by the illumination optical system is formed on the micromirror of the DMD 4, and this real image forms the second light source. The micromirror described below has a predetermined angle. For example, when the sample is tilted by +10 degrees, the specimen is imaged and illuminated.
The sample imaging system illuminates the sample 1 by overlapping the optical path of the sample illumination and forming a real image of the point light source formed on the micromirror of the DMD 4 on the focal plane of the sample 1. At the same time, a real image of the focal plane of the sample 1 is formed on the micromirror of the DMD 4.
The DMD 4 has a digital optical switch function, and this switch function is controlled by a program built in the computer 11. This will be described in detail below. When the digital signal (1) is input to each micromirror, the micromirror tilts by a predetermined angle, and the optical path of the coaxial illumination optical system and imaging optical system is connected to the optical path of the sample imaging system. . When a digital signal (0) is input to each micromirror, the micromirror tilts a predetermined angle in the opposite direction to the tilt, and the optical path of the coaxial illumination optical system and imaging optical system forms a sample image. The optical path of the illumination optical system is switched to the optical path guided to the light absorber 10 by being blocked from the optical path of the system. That is, the light is absorbed and the sample 1 is not illuminated. For example, ± 10 ° or ± 12 ° is used as the tilt angle of the micromirror, which is determined in advance by the DMD specification.
In the optical system having the above optical path switching function, when a digital signal (1) is input to only one micromirror and a digital signal (0) is input to all the other micromirrors, one sample space is obtained. Only one point corresponding to the micromirror is illuminated, imaged, and imaged. When the position of this one micromirror is sequentially moved on one surface of the DMD (hereinafter referred to as scanning of the micromirror on the DMD), the focal plane of the sample space is scanned, and a scanned image is obtained. In the digital scanning control for moving the position of each micromirror, the condition setting of the micromirror scanned on the DMD can be performed on the software without changing the hardware.
The number of micromirrors to be scanned is not limited to one. If a shape and size are specified, a program that selects a group of tilting micromirrors corresponding to the shape and size can be provided, thereby enabling observation with coarse scanning that does not require high resolution. For example, two adjacent four micromirrors that are adjacent to each other in the vertical and horizontal directions can be designated as a micromirror group to be scanned and can be scanned.
Similarly, for applications that require resolution only in one of X and Y directions, slit scanning can be performed by designating micromirrors that scan in a straight line. In addition, one screen or a group of independent micromirrors can be simultaneously translated to scan one screen at high speed. In particular, the translation scan can scan a whole screen in a short time because a large number of micromirrors can be tilted at the same time as long as the spatial light modulation function for removing defocused light is maintained. Can do. In this example, high-speed scanning was demonstrated by translational scanning of 2000 micromirrors.
The Z position designation mechanism 13 of the objective lens 2 is configured by a piezo element or a voice coil motor as a driving means, outputs a Z position signal from the computer 11, converts it to an analog signal by the D / A converter 14, and applies it to the piezo element. To do. The focal plane of the objective lens 2 can be changed and moved by a minute amount by expansion and contraction of the piezo element in the Z-axis direction. Two-dimensional scanning is performed on another focal plane moved in the Z direction of the sample 1 (the thickness direction of the sample 1), and this is repeated to obtain a large number of optical sectioning images. Image processing is performed on the multiple optical sectioning images to obtain a three-dimensional reconstruction image.
By equipping the light source with observation and stimulation or processing, it is possible to configure a device for aiming intense light for stimulation or processing at any point in the observed sample space.
When a pointing device 15 such as a mouse attached to the computer 11 is used to designate an arbitrary point indicated on the display device 16, information on the Z coordinate corresponding to the position is sent to the Z position designation mechanism 13 of the objective lens 2, As the objective lens 2 moves to the designated position, the digital signal (1) is output to the micromirror corresponding to the two-dimensional information of the XD and Y of the DMD 4 and the micromirror tilts to move the designated sample space. An arbitrary position is determined, and this one point is stimulated or processed.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a first embodiment of a digitally controlled scanning device according to the present invention.
FIG. 2 is a schematic view of a second embodiment of the digitally controlled scanning device according to the present invention.
FIG. 3 is a schematic view of a third embodiment of the digitally controlled scanning device according to the present invention.
FIG. 4 is a block diagram of the light source.
FIG. 5 is a flowchart showing the control and user interface of the digitally controlled scanning device.
FIG. 6 is a flowchart of scanning and display.
FIG. 7 is a flowchart of uneven illumination correction.
FIG. 8 is a flowchart of 3D measurement / display.
FIG. 9 is a flowchart of the observation mode and stimulation / processing mode.
FIG. 10 is a flowchart of the processing mode.
FIG. 11 is a diagram showing a surface light source composed of a fiber bundle.
FIG. 12 is a diagram showing a surface light source composed of urecsite.
FIG. 13 is a diagram showing a surface light source composed of a surface light emitter.
FIG. 14 is a diagram showing a surface light source composed of a light scattering plate.
BEST MODE FOR CARRYING OUT THE INVENTION
Embodiments of the present invention will be described in more detail with reference to the accompanying drawings. FIG. 1 shows a first embodiment to which a digitally controlled scanning device according to the present invention is applied, and is a schematic diagram of a scanning fluorescence microscope employing a confocal optical system.
In FIG. 1, in this embodiment, DMD 4 in which 640 × 480 micromirrors installed at a real image position by a sample imaging system comprising objective lens 2 of sample 1 are integrated, and a number of mirror surfaces of DMD 4 An illumination optical system that forms a real image of a point light source, an imaging optical system that forms a real image of the mirror surface of the DMD 4 from an oblique direction, a light absorber 10 that absorbs light not used for sample illumination, and the DMD 4 and the CCD 5 are controlled synchronously. The computer 11 is made up of.
The illumination optical system includes an argon ion laser (wavelength 488 nm) (not shown), DMD4, pinhole two-dimensional array plate 9 having the same arrangement pitch as DMD4, microlens two-dimensional array 12 having the same arrangement pitch as DMD4, and light source imaging lens. 8. It is composed of a dichroic mirror 7 and illuminates from a direction perpendicular to the deflection axis of the micromirror of DMD 4 and at an angle of 70 degrees from the mirror surface. In order to increase the resolution, it is desirable to use a laser having a short wavelength and high coherency.
The pinhole two-dimensional array plate 9, the light source imaging lens 8, and the DMD 4 satisfy the Scheinproof condition. From this condition, the pinhole two-dimensional array plate 9 is tilted with respect to the optical axis, so that the position of each microlens is adjusted so as to match the corresponding pinhole.
The dichroic mirror 7 has a property of reflecting illumination light from the illumination optical system and transmitting fluorescence. By disposing the dichroic mirror 7 between the imaging lens 6 and the DMD 4, the fluorescence transmitted through the dichroic mirror 7 is imaged on the CCD 5 by the imaging lens 6. The imaging surfaces of the DMD 4 and the CCD 5 and the imaging lens 6 satisfy the Shineproof condition. The CCD 5 is exposed and read in synchronization with the DMD 4.
The operation of the DMD 4 micromirror will be described. When the digital signal (1) is input, the micromirror is tilted by 10 degrees from the position where there is no signal, and the optical path of the illumination optical system and the optical path of the sample imaging system are connected to each other in the sample. One point is illuminated. At the same time, the optical path of the sample imaging system and the optical path of the imaging optical system are connected, and the fluorescence emitted from one point in the illuminated sample is imaged on the CCD 5. A micromirror having a size of 16 μ × 16 μ exhibits a spatial light modulation function similar to that of a pinhole, thereby reducing stray light and out-of-focus light and increasing resolution.
When the digital signal (0) is input, the micromirror is tilted 10 degrees to the other side opposite to the above, the optical axis of the illumination system is switched to the optical path to the light absorber 10, and the illumination light is absorbed by the light absorber. 10 is absorbed.
According to the present embodiment, when the micromirrors that output the digital (1) signal are sequentially switched on the DMD 4, the tilting micromirrors are scanned, and the point for illumination imaging in the sample is scanned. When the scanning of one surface is completed on the DMD 4, the entire optical sectioning image is obtained on the CCD 5.
A large number of optical sectioning images can be obtained by moving the objective lens 2 slightly by the Z position designation mechanism 13 of the objective lens and shifting the focal plane.
Next, correction of illumination intensity unevenness will be described. The intensity distribution of the laser light has a Gaussian distribution in which the intensity of the central part of the beam is high and the intensity of the peripheral part is low. If this beam is used as illumination light as it is, a uniform image cannot be obtained. There are the following methods for correcting illumination unevenness unique to the apparatus due to the illumination light and optical elements and their arrangement. In the first method, the tilting time (hereinafter referred to as sampling time) of the tilting micromirror is controlled to be inversely proportional to the illumination intensity at the position of the micromirror. That is, in the sampling time control, observation is performed by extending the sampling time at a position where the illumination light is weak, and the illumination unevenness is corrected. The second method is to keep the sampling time constant for all micromirrors, apply PWM (pulse width modulation) to the micromirrors, repeat switching finely within the sampling time, and keep the integrated intensity within the sampling time constant. To correct the uneven illumination.
The first method will be described in detail. A uniform fluorescent screen is observed as an adjustment sample while keeping the time for tilting the micromirror constant. Then, the fluorescence intensity corresponding to each micromirror is obtained from the corresponding pixel of the obtained fluorescence image, and corrected so that the fluorescence intensity is all constant using the proportional relationship between the sampling time and the fluorescence intensity. Create a numerical table of the sampling time. During scanning, the sampling time for tilting individual micromirrors is controlled with reference to the created numerical table to correct illumination unevenness.
Next, the second method will be described in detail. The computer 11 is equipped with a program for controlling individual micromirrors using 10-bit (1024 gradation) PWM in the DMD 4. First, a uniform fluorescent screen is observed as an adjustment sample. Next, the fluorescence intensity corresponding to each micromirror is obtained from the corresponding pixel of the obtained fluorescent image, and a numerical table of PWM control data is created so that the fluorescence intensity is all constant. Thereafter, the created numerical table is referred to at the time of scanning, and the individual micromirrors are subdivided and tilted within a predetermined sampling time. Control of DMD by PWM is executed by regarding DMD as one display device. That is, when a value is written in the VRAM (video RAM) of an additional video card connected to these display devices as in the case of a CRT or liquid crystal display, the corresponding micromirror of the DMD is tilted and the written value is used. The intensity of 1024 gradations can be controlled.
The apparatus of the present invention can set scanning conditions without changing hardware. When scanning the micromirror group, the shape, size, number, or number of micromirror groups, when scanning in parallel, the number and arrangement of the micromirror groups, or when changing the scanning speed depending on the scanning position In addition, it is possible to flexibly implement the setting or setting change such as area designation when limiting the sample area to be scanned from the operation screen of the computer.
FIG. 2 shows a second embodiment of the digitally controlled scanning device according to the present invention.
The optical system of the present embodiment is an implementation of the present invention in consideration of facilitating adaptation based on an existing microscope. An example using a point light source will be described below, but a planar light source can also be used. In this embodiment, the pinhole two-dimensional array plate 9 and the microlens array 12 are arranged between the DMD 4 and the conjugating lens 3, scanned with the DMD 4 in a parallel light state, and then converted into a point light source. The optical system illuminates the DMD 4 with parallel light, and a fine parallel beam generated as a result of scanning and adjusting the intensity of the micro mirror of the DMD 4 is collected in each pin hole of the pin hole array 9 installed at the real image position of the microscope. It is configured to be incident on the microlens array 12 installed so as to emit light.
FIG. 3 shows a third embodiment of the digitally controlled scanning device according to the present invention.
The optical system of the present embodiment is an implementation of the present invention in consideration of facilitating adaptation based on an existing microscope. An example using a point light source will be described below, but a planar light source can also be used. The epi-illumination device of the existing microscope is modified and the light source and the imaging system are assembled. Further, the DMD 4 is installed at a predetermined angle, for example, 10 degrees so that the micromirror deflected to the real image position of the sample 1 is orthogonal to the optical axis. A point light source generated by the microlens array 12 and the pinhole array 9 is imaged on the DMD 4 via the half mirror 17 and picked up by the CCD 5. In this optical system, the following three relationships satisfy the Shineproof condition. The first is the relationship between the DMD 4, the objective lens 2, and the sample focal plane placed on the stage 18. The second is the relationship among the DMD 4, the light source imaging lens 8, and the pinhole array 9. The third is the relationship between the DMD 4, the imaging lens 6, and the CCD 5.
FIG. 4 shows a configuration example of an observation light source and a stimulus / processing light source. The light source is a laser light source alone or a combination of a laser light source and an ultraviolet light source. In the case of only the laser light source, a mechanism for changing the laser output in two stages, for observation and for stimulation and processing, is used by switching between these laser outputs. In the case of a combination of a laser light source and an ultraviolet light source, the laser is used by removing the optical path switching element 19 from the optical path during observation, while the optical path switching element 19 is inserted into the optical path during stimulation and processing, and ultraviolet rays are introduced into the optical path.
FIG. 5 is a flowchart showing the control and user interface of the apparatus. When the apparatus is activated, first, a D / A converter that controls the piezo elements constituting the Z position designation mechanism of the DMD 4, the CCD 5, and the objective lens 2 is initialized, and a menu screen is displayed. The following items are displayed on this menu screen, and each function is executed by selection.
[Scan / Display]
FIG. 6 shows a flow chart of scanning and display. The exposure of the CCD 5 is started, and the micromirror is scanned on the DMD 4. After the scanning is completed, the exposure of the CCD 5 is terminated, and the image is read and displayed. As a method for correcting illumination unevenness, a sampling time adjustment method or an intensity adjustment method using PWM can be selected.
[Lighting unevenness correction]
FIG. 7 shows a flowchart of illumination unevenness correction. A standard fluorescent sample, which is a uniform fluorescent plate, is mounted as an adjustment jig and measured. From the result, the fluorescent light intensity I (x, y) at the position (x, y) of the micromirror on the DMD 4 is obtained. The reciprocal of this fluorescence intensity I (x, y) is calculated for all the micromirrors, and a correction table is created. The correction table is referred to when the illumination unevenness is corrected in the scanning / display subroutine.
[Setting the number of scanning units and translation units]
This is a method of scanning one micromirror independently, or a method of grouping a plurality of micromirrors, setting the group unit as a scanning unit, and sequentially moving the unit to scan. The unit sets, for example, four 2 × 2 micromirrors. In addition, when a plurality of units are arranged in a lattice pattern and the plurality of units are moved simultaneously and scanned (translated), the number of units to be translated is set.
[Scanning speed setting]
Set the micromirror sampling time.
[3D measurement / display]
FIG. 8 shows a flowchart of 3D measurement / display. A plurality of optical sectioning images are acquired with the input number and interval values. At this time, the focal plane is moved by the distance designated by the interval value by the Z position designation mechanism of the objective lens for each sheet. By this operation, a specified number of image data is acquired, and a three-dimensional reconstructed image is displayed based on this image data.
[Specify Z position of objective lens]
A driving means of the Z position designation mechanism of the objective lens 2, for example, a piezo element is operated by giving a Z position control signal to move the focal plane of the objective lens 2 to the Z position.
[Stimulation / processing mode]
FIG. 9 shows a flowchart of the observation mode and the stimulation or processing mode. After executing the observation mode of FIG. 5, the stimulus / processing mode is executed. Since the stimulation mode and the machining mode can be realized by the same process, only the machining mode will be described here. The stimulation mode can be realized by changing the “processing point” in FIG. 10 to the “stimulation point”.
In FIG. 10, 3D measurement / display in the observation mode is executed, and a point (x, y, z) to be stimulated or processed is designated using the pointing device 15 on the displayed 3D reconstructed image. The focal plane of the objective lens 2 is moved to the Z position of this point, and the micromirror corresponding to the X and Y positions is tilted by applying the digital signal (1) to aim the target stimulus and / or processing point. To do. Next, the light source for stimulation / processing is irradiated with intense light having increased illumination light intensity, ultraviolet light separately provided, and the like, and condensed on the stimulation / processing point.
Next, embodiments of the light source will be described. A surface light source can be used as the light source. Since it is not the illumination of coherent light that has been narrowed down to the diffraction limit, the spatial resolution is inferior to that of the embodiment, but it can be manufactured at low cost. A minute portion (16 μ × 16 μ) of the planar light source image cut out by the micromirror is reduced and imaged by the objective lens 2 to excite the sample, and only the fluorescence emitted from this imaging area is imaged.
FIGS. 11 to 14 show various surface light sources, and the light source surface of the installed surface light source, the light source imaging lens 8 and the DMD 4 satisfy the Scheimpflug condition.
The surface light source shown in FIG. 11 is composed of an optical fiber bundle 20, light is incident from one end surface, and the other end surface is used as a planar light source. The other end face is cut obliquely so as to satisfy the Shine Playfuff condition.
In the surface light source shown in FIG. 12, the urecsite 21 is installed so that the fiber direction coincides with the optical axis, and the exit end is cut obliquely with respect to the optical axis so as to satisfy the Scheinproof condition. Urecsite 21 is a natural mineral and is a collection of transparent inorganic fibers having a diameter of several microns, and is used as a fiber plate.
The surface light source shown in FIG. 13 is composed of a surface light emitter 22 such as EL (electroluminescence).
The surface light source shown in FIG. 14 is composed of a light scattering plate 23.
In addition, as the surface light source, an integrated parallel optical device in which a large number of semiconductor lasers and LEDs are arranged on a plane can be used.
Since the objective lens 2 of the infinity correction optical system is used, a scanning fluorescence microscope capable of zooming can be obtained by installing an intermediate zooming optical device.
Application examples for other uses will be described. In the above embodiment, when a half mirror is used instead of the dichroic mirror for separating fluorescence and excitation light, the reflected light and scattered light of the sample can be observed. Therefore, the scanning metal frequently used for observation of the semiconductor integrated circuit substrate It can be applied to a microscope. The metal microscope is capable of not only image observation but also two-dimensional measurement such as the line width of the wiring on the semiconductor integrated circuit substrate and the diameter of the hole. If three-dimensional measurement is required, a large number of optical sectioning images can be taken and displayed on the three-dimensional reconstruction image.
When the digital control scanning technology of the present invention is applied to measurement of an opaque sample such as a semiconductor, the scattered light from the focal position of the illumination light is detected, and the spatial light modulation function of the DMD is used to detect the light from the position deviated from the focal point. Since the scattered light is excluded, the intensity of the scattered light detected when the focal point of the illumination light coincides with the sample and the sample surface is maximized. In order to detect the peak of the scattered light intensity, the computer 14 constituting the control means moves the objective lens 2 up and down to search for a position where the intensity of the scattered light is maximized (software) or the objective. The lens 2 is vibrated, and peak tracking means (software) is added to control the objective lens 2 so as to always follow the maximum position of the scattered light intensity from the change in the scattered light intensity associated therewith. The X and Y coordinates of the point on the sample surface are determined by the scanned micromirror, and the Z coordinate can be determined from the above-described peak detection or peak tracking control. Since the shape of the sample surface can be measured by scanning the entire surface of the micromirror on the DMD 4, a three-dimensional surface shape measuring apparatus can be realized. In measuring the shape of the sample surface, the whole or a part of the observation field can be designated. When measuring the whole, the micromirror is scanned over the entire area of the DMD 4. When a part of the field of view is measured, the micromirror scan is limited to the DMD 4 partial area by designating the measurement area on the screen of the user interface.
Also, a length measuring means (software) that captures the entire three-dimensional coordinate data of the sample surface into the computer and calculates the distance between two points, line width, step, hole diameter, depth, etc. specified on the displayed image ) Can be added to realize a length measuring device. Note that the above-described peak detection software and peak tracking software can also be realized by hardware using an electronic circuit.
Moreover, it can be applied to a microscopic fluorescence spectrum measuring apparatus by installing a spectroscope and a photoelectric detector. With this apparatus, it is possible to obtain a spectrum of fluorescence emitted from any one point or a plurality of designated points in the sample.
As another application, operations such as processing can be performed with the same apparatus after observation, and a highly convenient apparatus can be provided. Specific examples include a cell photostimulation device that photostimulates the targeted organ in the cell, a micro-cell surgical device that denatures the targeted portion of the cell by light irradiation, and an integrated circuit that burns the targeted portion with a high-intensity light source A trimming device, a microscopic stereolithography device, and the like are applicable.
Industrial applicability
The present invention has a scanning function, a spatial light modulation function, and a light intensity adjustment function in the DMD to realize a high-resolution scanning microscope, as well as a stimulating device, a processing device, a three-dimensional surface shape measuring device, a length measuring device, and the like. Made it possible to apply.
When a real image of a point light source is formed on a micromirror on a DMD, a confocal optical system using coherent light is formed, and high-speed scanning with high resolution and high contrast is possible. When a real image of a planar light source is formed on a micromirror on a DMD, the resolution is worse than when a point light source is used, but a minute portion (16 μ × 16 μ) of the planar light source image cut out by the micromirror is an objective lens. The sample is reduced and imaged in order to excite the sample, and only the fluorescence emitted from this imaging region is imaged. This action mechanism can provide an inexpensive scanning device that can be easily adjusted with less noise such as stray light.
Since the DMD is digitally controlled through a user interface, the scanning apparatus itself can flexibly realize condition setting and setting change. The scanning speed can be changed for each pixel, and the intensity of illumination light can be adjusted. In addition, since the DMD is digitally controlled by a computer, the user can change the scanning condition setting from the program using the keyboard and mouse without changing the hardware according to the usage condition.

Claims (29)

A real image of a light source is formed on a plurality of micromirrors of a micro light deflecting element (DMD), the tilt of each micromirror is scanned, and the illumination is imaged in a sample with an objective lens using the tilted micromirror as a second light source. A digitally controlled scanning method characterized in that the fluorescence or scattered light emitted from the imaging region of the sample is imaged by the objective lens on the scanned micromirror, and this is imaged. 2. The digitally controlled scanning method according to claim 1, wherein the light source is a point light source that emits coherent light. 2. The digitally controlled scanning method according to claim 1, wherein the light source is a flat light source. 4. The digitally controlled scanning according to claim 1, wherein when the micromirror is scanned on the DMD, light intensity modulation by pulse width modulation is performed for each micromirror to correct illumination unevenness. Method. 4. The digitally controlled scanning method according to claim 1, wherein when the micromirror is scanned on the DMD, the sampling time is expanded and contracted for each micromirror to correct the illumination unevenness. A micro light deflecting element (DMD) having a plurality of micro mirrors arranged two-dimensionally and tilting each micro mirror with a digital signal;
A real image of a point light source or a planar light source is formed on the micromirror, the tilt of each micromirror is scanned, and the micromirror tilted by the scanning is used as a second light source to connect points or microscopic areas in the sample with the objective lens. Illumination optical means for image illumination;
Sample imaging means for forming a real image of the sample by fluorescence or scattered light emitted from a point or a minute area in the sample illuminated by the illumination optical means on the micromirror tilted by the scanning by the objective lens;
Imaging optical means for imaging a real image of a point or a micro area in the sample imaged on the micromirror;
Control means for controlling scanning, spatial modulation, and intensity adjustment while synchronizing the DMD with imaging optical means;
A digitally controlled scanning device comprising: 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a fiber bundle that emits light incident from one end face from the other end face. 8. The digitally controlled scanning device according to claim 7, wherein the other end face of the fiber bundle that emits light is cut obliquely. 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a urecsite that emits light incident from one end face from the other end face. 10. The digitally controlled scanning device according to claim 9, wherein the other end face of the urexite that emits light is cut obliquely. 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a surface light emitter using electroluminescence. 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a light scattering plate irradiated with light. 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a surface emitting laser that is a two-dimensional array of parallel light sources. 7. The digitally controlled scanning device according to claim 6, wherein the planar light source is a surface emitting LED which is a two-dimensional array of parallel light sources. 7. The digital control scanning apparatus according to claim 6, wherein when the control means scans the micromirror on the DMD, the light intensity is adjusted by pulse width modulation for each micromirror to correct illumination unevenness. 7. The digitally controlled scanning device according to claim 6, wherein when the control means scans the micromirror on the DMD, the sampling time is extended and contracted for each micromirror to correct illumination unevenness. 7. The dichroic mirror in which the illumination optical means is installed so that the optical axis of the optical system of the imaging optical means overlaps, and the micro light utilizing laser light from the laser light source introduced by using the dichroic mirror. A digitally controlled scanning device comprising a two-dimensional array of microlenses for forming a real image of a point light source on a mirror, a two-dimensional array plate of pinholes, and a light source imaging lens. 7. The pinhole two-dimensional array plate is installed at a real image position of a microscope, a microlens array that focuses on each pinhole, and a DMD is installed so that scanning light is incident on the microlens array. A digitally controlled scanning device. In claim 6, the sample conjugate position of the objective lens, as micromirrors tilted the DMD is perpendicular to the optical axis, and placed inclined at a predetermined angle to DMD, the half mirror installed in the middle of the DMD and the objective lens A digitally controlled scanning device characterized in that a real image of a point light source or a flat light source is formed on a DMD through the image, and the real image on the DMD is imaged and illuminated in a sample by an objective lens. 7. The sample imaging means according to claim 6, wherein the objective lens of the infinity correction optical system that forms a real image of the sample on the micromirror, the imaging lens of the infinity correction optical system, and positioning of the objective lens in the Z-axis direction. A digitally controlled scanning device comprising a Z-position specifying mechanism for performing 7. The digitally controlled scanning device according to claim 6, wherein the control means detects the peak of the intensity of scattered light emitted from the sample and obtains the Z position of the surface based on the peak detection. A digitally controlled scanning device according to any one of claims 6 to 21, and a light source for photostimulation for stimulating a point in a sample space illuminated by illumination optical means of the digitally controlled scanning device. Features a light stimulator. 23. The photostimulation apparatus according to claim 22, wherein the illumination optical means comprises a sample illumination light source and a light stimulation light source as the same light source, and is capable of switching between them. 23. The light stimulation means according to claim 22, wherein the illumination optical means comprises a sample illumination light source and a light stimulation light source as separate light sources, and comprises means for introducing the light stimulation light source into the sample illumination optical system. apparatus. A digitally controlled scanning device according to any one of claims 6 to 21, and an optical processing light source that performs optical processing on a point in a sample space illuminated by illumination optical means of the digitally controlled scanning device. An optical processing device. 26. The optical processing apparatus according to claim 25, wherein the illumination optical means comprises a light source for sample illumination and a light source for optical processing as the same light source, and these can be switched. 26. The optical processing according to claim 25, wherein the illumination optical means comprises a light source for sample illumination and a light source for light processing as separate light sources, and includes means for introducing the light source for light processing into the sample illumination optical system. apparatus. The digitally controlled scanning device according to claim 20 and the control means of the digitally controlled scanning device detect the peak of the intensity of scattered light emitted from the sample surface, and based on the Z position of the sample surface obtained by the peak detection. A surface shape measuring apparatus characterized by obtaining a sample surface shape. 21. The digitally controlled scanning device according to claim 20 and the control means of the digitally controlled scanning device perform peak detection of scattered light intensity emitted from the sample, and the entire three-dimensional coordinates of the sample surface obtained by the peak detection A length-measuring device that takes in data and obtains a length between two designated points on a displayed image.

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