JPWO2003025656A1 - Digitally controlled scanning method and apparatus - Google Patents

Abstract

A micro-optical deflecting element (DMD) (4) having a plurality of two-dimensionally arranged micromirrors and tilting each micromirror with a digital signal is used to scan the tilt of each micromirror on the DMD (4). By doing so, a real image of a light source (point light source, flat light source) is formed on a tilted micromirror, and this real image is formed by an objective lens (2) to illuminate a point or a minute area in the space of the sample (1). Then, a real image of the fluorescent light or the scattered light emitted from this point or a minute area is formed on the tilted micromirror by scanning with the objective lens (2), and this is imaged with the charge-coupled device (CCD) (5). .

Description

Technical field
The present invention relates to a sample observation device such as a scanning optical microscope, a device for applying light stimulation or optical processing to an arbitrary point or a minute area in a three-dimensional space in a sample observation region, or a surface shape or three-dimensional coordinate data of a surface shape. The present invention relates to a digitally controlled scanning method and apparatus which can be applied to a wide range such as an apparatus for measuring a length between two points on a sample surface based on the method and is excellent in versatility.
Background art
Scanning microscopes employing a confocal optical system are widely used. Various types of scanning confocal microscopes have been put into practical use, such as a method of deflecting light using a photoacoustic element, a method of deflecting light with a galvanometer mirror, and a method of scanning light by rotating a pinhole plate called Nipkow Desk. Have been.
A scanning microscope using a micro light deflecting element (DMD) as a scanning means is disclosed in “Optics Letters”, Vol. 22, 1997, 751 to 753. Such a scanning microscope is described, for example, in US Pat. No. 5,587,832.
Japanese Patent Application Laid-Open No. H11-194275 proposes a scanning microscope in which a minute light deflecting element is used as a scanning means and an aperture correction technique is added.
However, the scanning microscope described in the above-mentioned US Pat. No. 5,587,832 in which the micro-light deflecting element is used as a scanning means has an illumination system that performs projection illumination using parallel rays of a laser, and thus illuminates the sample. The area is widely scattered and illuminated, and only captures a point within the wide illuminated area. That is, the light source and the detector do not have a conjugate relationship with the objective lens, and the illumination light is not focused only on the observation point, so that the performance as a confocal microscope is not sufficiently exhibited.
Similarly, the scanning microscope described in JP-A-11-194275 does not have a high spatial resolution as a confocal microscope because the light source and the detection point are not conjugate to the objective lens.
When a minute light deflecting element is used as a reflection-type scanning device, illumination light is inclined and introduced into the minute light deflecting element, resulting in non-uniform illumination and uneven illumination in an observed image.
Furthermore, if laser light, which is a highly condensable coherent light, is used as a light source as a light source for the purpose of increasing the resolution, the light intensity becomes a Gaussian distribution with the center of the beam being maximized, so that the illumination unevenness increases and becomes complicated. There is such a problem.
The present invention has been made in view of the above-described conventional problems, and has an object to provide a digitally controlled scanning method and apparatus capable of correcting illumination unevenness and realizing an observation apparatus such as a laser confocal microscope having a high spatial resolution. I do.
Another object of the present invention is to provide an apparatus for performing photostimulation and optical processing on any three-dimensional point in a sample observation area using the digital control scanning technique. Still another object of the present invention is to provide a device for measuring the length of an object on a surface based on the surface shape or three-dimensional coordinate data of the surface shape using the digital control scanning technique.
Disclosure of the invention
In the digital control scanning method according to the present invention, a real image of a light source is formed on a plurality of micromirrors of a minute light deflection element (DMD), and the tilt of each micromirror is scanned, and the tilted micromirror is used as a second light source. An image is illuminated on the sample with an objective lens, and fluorescence or scattered light emitted from an imaging region of the sample is imaged on a micromirror scanned by tilting with an objective lens, and the image is taken. I do.
A digitally controlled scanning device according to the present invention includes an imaging optical unit, an illumination optical unit, a sample imaging unit, and a computer.
In FIG. 1, an imaging optical unit is an imaging device 5 that forms an image of fluorescence or scattered light emitted from a sample on a micromirror of a DMD 4 by an objective lens, and images this through an imaging imaging lens 6 of an 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 coaxial so that the optical axis overlaps with the imaging optical system, and forms a real image of a point light source on a micro mirror of the DMD 4 using laser light from a laser light source introduced using a dichroic mirror 7. A microlens two-dimensional array 12, a pinhole two-dimensional array plate 9, and a light source imaging lens 8 are configured so that one point light source forms an image on one micromirror. In the case of a plane light source, a minute portion of the plane light source image selectively extracted by tilting the micromirror is reduced and formed by the objective lens. Only the imaged portion is illuminated on the sample space, and only the fluorescent light emitted from the imaged portion is imaged.
The sample imaging means determines the position of 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 which form an actual image of the sample 1 on the micromirror of the DMD 4. It comprises a Z-position designating mechanism 14 for the objective lens. The light absorber 10 absorbs light that is not used for illumination from the non-tilt micromirror of the DMD 4 using, for example, a black body. The computer 11 incorporates a control program for realizing a scanning function, a spatial modulation function, and an intensity adjustment function and a user interface program necessary for setting the same while synchronizing the DMD 4 with the imaging device 5. A display device 16 is attached.
The real image of the micromirror of the DMD 4 is captured by the imaging optical unit, and the image data is transmitted to the computer 11. The computer 11 performs image processing on the image data and displays it. The 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 micro mirror of the DMD 4, and this real image forms the second light source. For example, when the sample is tilted by +10 degrees, the sample is illuminated with an image.
The sample imaging system illuminates the sample 1 by forming an actual image of a point light source formed on the micromirror of the DMD 4 on the focal plane of the sample 1 so as to overlap the optical path of the sample illumination. At the same time, a real image of the focal plane of the sample 1 is formed on the micro mirror of the DMD 4.
The DMD 4 has a digital optical switch function, and the switch function is controlled by a program stored in the computer 11. Details will be described below. When a digital signal (1) is input to each micromirror, the micromirror is tilted by a predetermined angle, and the optical paths of the illumination optical system and the imaging optical system that are coaxial are connected to the optical path of the sample imaging system. . When a digital signal (0) is input to each micromirror, the micromirror is tilted at a predetermined angle in a direction opposite to the tilt, and the optical paths of the illumination optical system and the imaging optical system that are coaxialized form a sample image. The optical path of the illumination optical system is cut off from the optical path of the system, and is switched to the optical path guided to the light absorber 10. That is, light is absorbed and the sample 1 is not illuminated. The tilt angle of the micromirror is, for example, ± 10 ° or ± 12 °, which is predetermined according to the DMD specification.
In the optical system having the above-described optical path switching function, when a digital signal (1) is input to only one micromirror and a digital signal (0) is input to all other micromirrors, one micromirror is used. 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 software without changing hardware.
The number of micromirrors to be scanned is not limited to one. If a shape and size are designated, a program for selecting a tilting micromirror group corresponding to the shape and size is provided, so that observation with coarse scanning that does not require high resolution is possible. For example, two adjacent vertical and horizontal micromirrors can be designated as a micromirror group to be scanned, and scanning observation can be performed.
Similarly, for applications requiring resolution in only one of the X and Y directions, a slit scan can be performed by specifying a single row of micromirrors that scan linearly. In addition, a single screen or an independent group of micromirrors can be simultaneously translated and a single screen can be scanned at high speed. In particular, in the parallel scanning, if a sufficient distance is maintained to maintain a spatial light modulation function for removing out-of-focus light, a large number of micromirrors can be tilted at the same time. Can be. In the present embodiment, high-speed scanning was demonstrated by translational scanning of 2000 micromirrors.
The Z position designation mechanism 13 of the objective lens 2 is constituted by a piezo element or a voice coil motor as a driving means, outputs a Z position signal from the computer 11, converts the Z position signal into an analog signal by a D / A converter 14, and applies the analog signal to the piezo element. I do. The focal plane of the objective lens 2 can be changed and changed little by little 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 large number of optical sectioning images to obtain a three-dimensional reconstructed image.
By equipping a light source for observation and stimulation or processing, it is possible to configure a device for aiming at an intense light for stimulation or processing at an arbitrary point in the observed sample space.
When an arbitrary point indicated on the display device 16 is pointed by a pointing device 15 such as a mouse attached to the computer 11, 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, a digital signal (1) is output to the micromirror corresponding to the two-dimensional information of X and Y of the DMD 4, and the micromirror is tilted so that the micromirror is tilted. An arbitrary position is determined, and this one point is stimulated or processed.
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 fluorescent microscope employing a confocal optical system.
In FIG. 1, in this embodiment, a DMD 4 in which 640 × 480 micromirrors are installed at a real image position of a sample 1 formed by an objective lens 2 of a sample 1 and a large number of mirrors are provided on the mirror surface of the 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 synchronous control of the DMD 4 and the CCD 5 Computer 11.
The illumination optical system includes an argon ion laser (wavelength 488 nm) (not shown), a DMD 4, a pinhole two-dimensional array plate 9 having the same arrangement pitch as the DMD 4, a microlens two-dimensional array 12 having the same arrangement pitch as the DMD 4, a light source imaging lens 8. The dichroic mirror 7 illuminates the micromirror of the DMD 4 from a direction perpendicular to the deflection axis of the micromirror and forming an angle of 70 degrees from the mirror surface. In order to further increase the resolution, it is desirable to use a laser having a short wavelength and a high coherence.
The pinhole two-dimensional array plate 9, the light source imaging lens 8, and the DMD 4 satisfy the Scheimpflug condition. Under these conditions, the pinhole two-dimensional array plate 9 is inclined with respect to the optical axis, so that the position of each microlens is adjusted so as to coincide with the corresponding pinhole.
The dichroic mirror 7 has a property of reflecting illumination light from the illumination optical system and transmitting fluorescence. By arranging 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 Scheimpflug condition. The CCD 5 is exposed and read out in synchronization with the DMD 4.
The operation of the micro mirror of the DMD 4 will be described. When the digital signal (1) is input, the micromirror is tilted 10 degrees to one side from the position where there is no signal, the optical path of the illumination optical system and the optical path of the sample imaging system are connected, and 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 fluorescent light emitted from one point in the illuminated sample is imaged on the CCD 5. A micromirror having a size of 16 μ × 16 μ exerts a spatial light modulation function similar to that of a pinhole, and reduces stray light and defocused light to increase resolution.
When the digital signal (0) is input, the micromirror is tilted 10 degrees to the other side in the opposite direction 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 in 10.
According to the present embodiment, when the micromirrors that output digital (1) signals are sequentially switched on the DMD 4, the tilting micromirrors are scanned, and the points in the sample to be illuminated and imaged are scanned. When scanning of one surface is completed on the DMD 4, an entire optical sectioning image is obtained on the CCD 5.
A number of optical sectioning images can be obtained by slightly moving the objective lens 2 by the objective lens Z-position designating mechanism 13 and shifting the focal plane for measurement.
Next, correction of illumination intensity unevenness will be described. The intensity distribution of the laser light has a Gaussian distribution in which the intensity at the center of the beam is high and the intensity at the periphery is low. If this beam is used as it is as illumination light, a uniform image cannot be obtained. The following method is used to correct the illumination unevenness inherent to the device due to the illumination light and the optical elements and their arrangement. In the first method, the tilting time of the tilting micromirror (hereinafter referred to as sampling time) is controlled so as 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 uneven illumination is corrected. In the second method, PWM (pulse width modulation) is applied to the micromirrors while the sampling time is kept constant for all the micromirrors, and switching is repeated finely within the sampling time to keep the integrated intensity within the sampling time constant. Value and correct uneven lighting.
The first method will be described in detail. While keeping the time for tilting the micro mirror constant, a uniform fluorescent plate is observed as a sample for adjustment. Next, the fluorescence intensity corresponding to each micromirror is obtained from the corresponding pixel of the obtained fluorescence image, and correction is made so that the fluorescence intensity becomes a constant value by utilizing the fact that the sampling time and the fluorescence intensity are proportional. Create a numerical table of the sampling time. At the time of scanning, the sampling time for tilting each micromirror 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 has a program for controlling each micromirror using 10-bit (1024 gradation) PWM in the DMD 4. First, a uniform fluorescent screen is observed as a sample for adjustment. Next, the fluorescent intensity corresponding to each micromirror is obtained from the corresponding pixel of the obtained fluorescent image, and a numerical table of the PWM control data is created so that all the fluorescent intensities become constant. Thereafter, at the time of scanning, the created numerical table is referred to, and each micromirror is subdivided and tilted within a fixed sampling time. The control of the DMD by PWM is executed by regarding the DMD as one display device. That is, like a CRT or a liquid crystal display, when a value is written to a VRAM (video RAM) of an additional video card to which these display devices are connected, the corresponding micro mirror of the DMD tilts, and the written value is used. Control of the intensity of 1024 gradations becomes possible.
The apparatus of the present invention can set scanning conditions without changing hardware. When scanning a group of micromirrors, when changing the shape, size and number of the groups, or when scanning a plurality of micromirrors in parallel, when changing the scanning speed according to the number and arrangement, or the scanning position The setting can be flexibly changed on the operation screen of the computer by changing the setting or specifying the area when limiting the sample area to be scanned.
FIG. 2 shows a second embodiment of the digitally controlled scanning device according to the present invention.
The optical system according to the present embodiment implements the present invention in consideration of facilitating remodeling based on an existing microscope. An example using a point light source will be described below, but a flat light source can also be used. In the present embodiment, the pinhole two-dimensional array plate 9 and the microlens array 12 are arranged between the DMD 4 and the imaging lens 3, and the DMD 4 scans in a parallel light state, and then turns into a point light source. The optical system illuminates the DMD 4 with parallel light, and focuses a fine parallel beam resulting from scanning and intensity adjustment by the micro mirror of the DMD 4 onto each pinhole of the pinhole 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 according to the present embodiment implements the present invention in consideration of facilitating remodeling based on an existing microscope. An example using a point light source will be described below, but a flat 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. Also, 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. The 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 is imaged by the CCD 5. In this optical system, the following three relationships satisfy the Scheimpflug 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 between the DMD 4, the light source imaging lens 8, and the pinhole array 9. Third is the relationship between the DMD 4, the imaging lens 6, and the CCD 5.
FIG. 4 shows a configuration example of a light source for observation and a light source for stimulation / processing. 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 using only the laser light source, a mechanism for changing the laser output in two stages for observation and stimulation / processing is provided, and these laser outputs are switched and used. In the case of the combination of the laser light source and the ultraviolet light source, the optical path switching element 19 is removed from the optical path at the time of observation and a laser is used. On the other hand, at the time of stimulation / processing, the optical path switching element 19 is inserted into the optical path and ultraviolet rays are introduced into the optical path.
FIG. 5 is a flowchart showing the control of the apparatus and the user interface. When the apparatus is started, first, the D / A converter for controlling the DMD 4, the CCD 5, and the piezo element constituting the Z position designation mechanism of 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 flowchart of scanning and displaying. The exposure of the CCD 5 is started, and the micro mirror is scanned on the DMD 4. After the scanning, the exposure of the CCD 5 is completed, 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 is configured to be selectable.
[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 a jig for adjustment 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 the fluorescence intensity I (x, y) is calculated for all micromirrors, and a correction table is created. The correction table is referred to at the time of correcting illumination unevenness in the above-described scanning / display subroutine.
[Setting the number of scanning units and translation units]
This is a method in which one micromirror is independently scanned, or a method in which a plurality of micromirrors are grouped, a group unit is set as a scanning unit, and the unit is sequentially moved to perform scanning. The unit sets, for example, four 2 × 2 micromirrors. Further, when a plurality of units are arranged in a grid and scanning (translation) is performed by simultaneously moving the plurality of units, the number of units to be translated is set.
[Scan speed setting]
Set the sampling time of the micromirror.
[3D measurement / display]
FIG. 8 shows a flowchart of 3D measurement / display. A plurality of optical sectioning images are acquired based on the input number and the interval value. At this time, the focal plane is moved by a distance specified by the interval value by the Z position specifying mechanism of the objective lens for each lens. By this operation, a designated number of image data is obtained, and a three-dimensional reconstructed image is displayed based on the image data.
[Designation of Z position of objective lens]
A Z-position control signal is applied to a driving means of a Z-position designating mechanism of the objective lens 2, for example, a piezo element, to operate the objective lens 2, thereby moving 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 stimulation / machining mode is executed. Since the stimulation mode and the processing mode can be realized by the same processing, only the processing mode will be described here. The stimulation mode can be realized by changing the “processing point” in FIG. 10 to a “stimulation point”.
In FIG. 10, 3D measurement / display in the observation mode is executed, and a point (x, y, z) at which stimulation or processing is performed is designated on the displayed 3D reconstructed image using the pointing device 15. The focal plane of the objective lens 2 is moved to the Z position at this point, and the digital mirror (1) is applied to the micromirror corresponding to the X and Y positions to tilt the micromirror, thereby aiming at a target stimulus and / or processing point. I do. Next, the stimulating / processing point is irradiated with intense light having an increased luminous intensity or separately provided ultraviolet light from the stimulating / processing light source to condense it at the stimulating / processing point.
Next, an embodiment of the light source will be described. A surface light source can be used as the light source. Since the illumination is not coherent light that has been stopped 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 plane 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 imaged 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 face, and the other end face is used as a flat light source. The other end face is cut diagonally so as to satisfy the conditions of Shine Leaf.
In the surface light source shown in FIG. 12, the urexite 21 is installed so that the direction of the fiber coincides with the optical axis, and the exit end is cut obliquely with respect to the optical axis so as to satisfy the Scheimpflug condition. Urexite 21 is a natural mineral, in which transparent inorganic fibers having a diameter of several microns are accumulated, and is used as a fiber plate.
The surface light source shown in FIG. 13 includes a surface light emitter 22 such as an EL (electroluminescence).
The surface light source shown in FIG.
In addition, as a surface light source, an integrated parallel optical device in which many semiconductor lasers or LEDs are arranged on a plane can be used.
Since the objective lens 2 of the infinity correcting optical system is used, a scanning fluorescent microscope capable of changing the magnification can be obtained by installing an intermediate magnification changing optical device.
Examples of application to other uses will be described. In the above embodiment, when a half mirror is used instead of the dichroic mirror for separating the fluorescence and the excitation light, the reflected light and the scattered light of the sample can be observed, so that the scanning metal used for observation of the semiconductor integrated circuit substrate is often used. Applicable to microscope. The metallographic microscope can perform not only image observation but also two-dimensional length measurement such as a line width of a wiring on a semiconductor integrated circuit substrate and a diameter of a hole. When three-dimensional length measurement is required, a large number of optical sectioning images can be taken and can be obtained on a three-dimensional reconstructed image.
When the digital control scanning technique 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 scattered light from the position defocused. Since the scattered light is excluded, the scattered light intensity detected when the focus 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 maximum, or a peak detection means (software). The lens 2 is vibrated, and peak follow-up means (software) for controlling the objective lens 2 to always follow the maximum position of the scattered light intensity from the change in the scattered light intensity accompanying the vibration is added. The X and Y coordinates of a point on the sample surface are determined by the micromirror to be scanned, and the Z coordinate can be determined from the peak detection or peak following control described above. Since the shape of the sample surface can be measured by scanning the entire surface of the DMD 4 with the micromirror, a three-dimensional surface shape measuring device can be realized. The shape measurement of the sample surface can also designate the whole or a part of the observation visual field. When measuring the whole, the micromirror is scanned over the entire area of the DMD 4. When measuring a part of the field of view, the scanning of the micromirror is performed only on the partial area of the DMD 4 by specifying the measurement area on the screen of the user interface.
In addition, the computer takes in the three-dimensional coordinate data of the entire sample surface and calculates the distance, line width, step, hole diameter, depth, etc. between two points specified on the displayed image (software By adding the parentheses, a length measuring device can be realized. Note that the above-described peak detection software and peak tracking software can also be realized by hardware using an electronic circuit.
Further, by additionally installing a spectroscope and a photoelectric detector, it can be applied to a microscopic fluorescence spectrum measuring device. In this apparatus, a spectrum of fluorescence emitted from any one point or a plurality of designated points in a sample can be obtained.
As another application, an operation such as processing can be performed by the same device after observation, and a highly convenient device can be provided. Specific examples include a cell light stimulator that stimulates a target organ in a cell with light, a microcellular surgery device that denatures a target part in a cell with light irradiation, and an integrated circuit that burns out a target part with a high-intensity light source And a micro-stereolithography device.
Industrial applicability
The present invention provides a DMD with a scanning function, a spatial light modulation function, and a light intensity adjustment function to realize a high-resolution scanning microscope, and to a stimulating device, a processing device, a three-dimensional surface shape measuring device, a length measuring device, and the like. Application enabled.
When forming a real image of a point light source 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 plane light source is formed on a micro mirror on a DMD, the resolution is lower than when a point light source is used. However, a minute portion (16 μ × 16 μ) of the plane light source image cut out by the micro mirror is an objective lens. Is excited to excite the sample, and only the fluorescent light emitted from the imaging region is imaged. With this operation mechanism, it is possible to provide an inexpensive scanning device which is easy to adjust and has little noise such as stray light.
Since the DMD is digitally controlled via a user interface, the scanning device itself can flexibly set and change conditions. The scanning speed can be changed for each pixel, and the intensity of illumination light can be adjusted. Further, since the DMD is digitally controlled by a computer, the user can change the setting of the scanning conditions from a program using a keyboard or a mouse without changing the hardware in accordance with the usage conditions.
[Brief description of the drawings]
FIG. 1 is a schematic view of a digitally controlled scanning device according to a first embodiment of the present invention.
FIG. 2 is a schematic view of a digitally controlled scanning device according to a second embodiment of the present invention.
FIG. 3 is a schematic view of a digitally controlled scanning device according to a third embodiment of the present invention.
FIG. 4 is a configuration diagram of a light source.
FIG. 5 is a flowchart showing the control of the digitally controlled scanning device and the user interface.
FIG. 6 is a flow chart of scanning / display.
FIG. 7 is a flowchart of illumination unevenness correction.
FIG. 8 is a flowchart of 3D measurement / display.
FIG. 9 is a flowchart of the observation mode and the stimulation / processing mode.
FIG. 10 is a flowchart of the processing mode.
FIG. 11 is a diagram showing a surface light source constituted by a fiber bundle.
FIG. 12 is a diagram showing a surface light source composed of urexite.
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 constituted by a light scattering plate.

Claims (29)

A real image of a light source is formed on a plurality of micromirrors of a micro light deflecting element (DMD), and the tilt of each micromirror is scanned, and the tilted micromirror is used as a second light source to form an image on a sample with an objective lens. A digitally controlled scanning method characterized in that the tilt or the tilt is imaged on a scanned micromirror by an objective lens with fluorescence or scattered light emitted from an imaging region of the sample, and the image is taken. 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. The digital control scanning according to any one of claims 1 to 3, wherein when scanning the micromirrors on the DMD, light intensity modulation by pulse width modulation is performed for each micromirror to correct illumination unevenness. Method. 4. The digital control scanning method according to claim 1, wherein when scanning the micromirrors on the DMD, illumination time unevenness is corrected by expanding and contracting a sampling time for each micromirror. A micro-optical deflecting element (DMD) having a plurality of two-dimensionally arranged micro mirrors, each of which is tilted by a digital signal;
A real image of a point light source or a flat light source is formed on the micromirror, and the tilt of each micromirror is scanned. The micromirror tilted by the scanning is used as a second light source to form a point or a small area in the sample with an objective lens. Illumination optical means for illuminating the image;
Sample imaging means for imaging 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 a micromirror tilted by the scanning with the objective lens,
Imaging optical means for capturing a real image of a point or a minute area in the sample formed 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 flat 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 surface of the fiber bundle for emitting light is obliquely cut. 7. The digitally controlled scanning device according to claim 6, wherein the flat light source is an urexite that emits light incident from one end surface from the other end surface. 10. The digitally controlled scanning device according to claim 9, wherein the other end surface of the urexite for emitting light is obliquely cut. 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 flat 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 digitally controlled scanning device according to claim 6, wherein when the control means scans the micromirrors 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 unit scans the micromirrors on the DMD, the sampling time is expanded and contracted for each micromirror to correct illumination unevenness. 7. The dichroic mirror according to claim 6, wherein the illumination optical means is installed so that the optical axis thereof overlaps with the optical system of the imaging optical means, and the laser light from the laser light source introduced by using the dichroic mirror is used for micro illumination. 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. In claim 6, a two-dimensional array plate of pinholes is installed at a real image position of a microscope, and a microlens array that focuses on each pinhole and a DMD is installed so that scanning light is incident on the microlens array. Digitally controlled scanning device. 7. The half mirror according to claim 6, wherein the DMD is installed at a predetermined angle so that the tilted micro mirror of the DMD is orthogonal to the optical axis of the micro mirror at the sample conjugate position of the objective lens, and is installed between 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 via a light source, and the real image on the DMD is formed 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 the positioning of the objective lens in the Z-axis direction. A digitally controlled scanning device comprising: a Z position designation mechanism for performing 7. The digital control scanning apparatus according to claim 6, wherein the control means detects the peak of the intensity of the 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 applying photostimulation to a point in a sample space illuminated by illumination optical means of the digitally controlled scanning device. Characteristic photostimulator. 23. The light stimulating device according to claim 22, wherein the illumination optical means comprises the same light source as the light source for illuminating the sample and the light source for stimulating light, and these can be switched. 23. The optical stimulus according to claim 22, wherein the illumination optical means comprises a light source for illuminating the sample and a light source for stimulating light, and a means for introducing the light stimulating light source into the optical system for illuminating the sample. apparatus. A digitally controlled scanning device according to any one of claims 6 to 21, and an optical processing light source for performing optical processing on a point in a sample space illuminated by illumination optical means of the digitally controlled scanning device. Characteristic optical processing equipment. 26. An optical processing apparatus according to claim 25, wherein the illumination optical means comprises a sample illumination light source and an optical processing light source, which are formed of the same light source, and which can be switched. 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 separately from each other, and has means for introducing the light source for optical processing into the sample illumination optical system. apparatus. 21. The digitally controlled scanning device according to claim 20, wherein the control means of the digitally controlled scanning device detects a peak of the intensity of the scattered light emitted from the sample surface, and uses the Z position of the sample surface obtained by the peak detection as a basis. A surface shape measuring apparatus characterized in that a sample surface shape is determined in advance. 21. The digitally controlled scanning device according to claim 20, wherein the control means of the digitally controlled scanning device detects a peak of the scattered light intensity emitted from the sample, and three-dimensional coordinates of the entire sample surface obtained by the peak detection. A length measuring device for acquiring data and obtaining a length between two designated points on a displayed image.

Images