Confocal microscope apparatus
A confocal microscope apparatus has a confocal scanner for scanning a sample with shifting a focal position of a light beam in a direction perpendicular to an optical axis, a moving mechanism for moving the focal position of the light beam in an optical axis direction, a camera for picking up an image of the sample with the light beam, and a movement control unit for controlling the moving mechanism to move the focal position of the light beam by a predetermined distance in the optical axis direction for every vertical synchronizing signal of the camera in synchronization with the vertical synchronizing signal. A high-speed three-dimensional image can be displayed in such that while measuring the sample, two or more slice images in such an arrangement on a common screen that their positions relative to the sample enables to be grasped.
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1. Field of the Invention
The present invention relates to a confocal microscope, which is enabled to measure a stereoscopic shape of a sample by combining an optical microscope and a confocal optical-scanner.
2. Description of the Related Art
For example, a Nipkow's disc type confocal microscope apparatus, as shown in
In the configuration, the Z-coordinate of the focused point of the laser light is controlled depending on the position of the objective lens 103 in the Z-direction, and the XY-coordinates of the focused point of the laser light is controlled by turning the microlens array 101 and the pinhole array 102. In other words, the scanning point in the sample 20 to be picked up by the camera 106 can be three-dimensionally controlled depending on the Z-direction position of the objective lens 103 and the turning angles of the microlens array 101 and the pinhole array 102.
In the such a scanning technique of the confocal microscope apparatus the operations to move the objective lens 103 uniformly in a Z-coordinate increasing direction for a longer period than a plurality of frame periods are started in synchronization with a vertical synchronizing signal of the camera, as produced just after the input of a trigger signal, while turning the microlens array. 101 and the pinhole array 102 in synchronization with the vertical synchronizing signal of the camera 106. This scanning technique is described, for example, in JP-A-2002-72102.
In the scanning technique, however, the timing for starting the movement of the objective lens 103 is synchronized with the vertical synchronizing signal, but the movement after the start is performed asynchronously of the vertical synchronizing signal. As a result, it is difficult to control the Z-direction position of the scanning point highly precisely for the individual video frames to be picked up by the camera 106. In the case of the repeated capturing with the movement of the Z-direction position, more specifically, the discrepancy of the Z-direction position is so cumulatively enlarged that the discrepancy can be neither confirmed nor corrected.
In the related art described above, moreover, the individual scanning points are captured by scanning in the XY-directions while changing the Z-coordinate at all times. According to the capturing method by thus changing the Z-coordinate at all times, moreover, the Z-coordinate point can be prevented from being unscanned for all the XY-coordinates so that even a micro structure in the Z-direction can enhance the probability of its appearance at least in the captured images.
In the related art, the coordinates of the objective lens 103 change uniformly, too, even for the time period of the synchronizing signal such as the vertical synchronizing signal, when the capturing is not done in the camera 106. However, that Z-coordinate range in the sample 20, which corresponds to the range for the objective lens 103 to have moved for the synchronizing signal period, is not captured in the least. According to the related art, therefore, a micro structure in the Z-direction may drop out.
Depending on the application of the confocal microscope apparatus, on the other hand, the video frames having picked up the XY-plane of the sample with the Z-coordinate being fixed may be desirably produced individually for the different Z-coordinates. For example, a set of video frames thus produced become as they are the voxels having the XYZ-coordinate system so that they are suited for the processing such as the three-dimensional analysis of the sample 20.
According to the related art thus far described, however, the Z-coordinate always changes, too, for the video pickup period of the camera 106 so that the video frames having picked up the XY-plane of the sample with the Z-coordinate being fixed cannot be produced.
SUMMARY OF THE INVENTIONAn object of the invention is to provide a confocal microscope apparatus that improves the precision of the scanning position control of a sample in the optical axis direction.
Another object of the invention is to provide a confocal microscope apparatus that enhances the probability of grasping a micro structure in an image picked up.
A further object of the invention is to provide a confocal microscope apparatus that creates video frames captured by picking up a plane normal to the optical axis of a sample with the coordinate in the optical axis direction being fixed, individually for the coordinates in the different optical axis directions.
A further object of the invention is to provide a confocal microscope apparatus that creates video frames captured by picking up a plane normal to the optical axis of the sample with the coordinate in the optical axis direction being fixed, individually for the coordinates in the different optical axis directions, and to display a three-dimensional image at a high speed, thereby to grasp the whole image while measuring a sample.
A further object of the invention is to provide a confocal microscope apparatus that grasps slice images in each section and their stereoscopic relations precisely.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described in detail with reference to the accompanying drawings.
As shown in
The present confocal microscope apparatus is further provided with an illuminating light source 12 so that it also functions as an optical microscope apparatus with the illuminating light source 12 and the optical system housed in the body portion 1.
The scan control in the present confocal microscope apparatus will be described below.
On the other hand, the vertical synchronizing signal VSYNC of the camera 5 is outputted to the confocal scanner unit 4 and the Z-axis scan control device 9. In synchronization with the vertical synchronizing signal VSYNC inputted, the (not-shown) rotation control unit of the confocal scanner unit 4 drives the microlens array and the pinhole array so rotationally as to scan the whole XY-area once for every image pickup periods of the individual video frames. Here, the image pickup period portion such a period in one video frame period as excludes at least the vertical synchronizing signal period. Moreover, the image pickup period may exclude a horizontal synchronizing signal period and the period, for which the pixels before and after the horizontal synchronizing signal period and the vertical synchronizing signal period.
In synchronization with the vertical synchronizing signal VSYNC inputted, as shown in
On the other hand, the actuator 8 integrates the pulses of the movement control signal CNT inputted from the Z-axis scan control device 9, to produce the drive signals which keep constant values for the periods of the vertical synchronizing signals VSYNC but uniformly increase for the image pickup periods of the video frames, as shown in
In response to the reset signal RST from the Z-axis scan control device 9, moreover, the actuator 8 returns the drive signals to the initial value.
By the operations thus far described, the scanning points in the sample 20 and the individual video frames are given such relations as are shown in
Reverting to
One configuration example of the Z-axis scan control device 9 will be described in the following.
In
A first counter 92 is reset with the TRIGER signal to count the vertical synchronizing signals VSYNC from 0. A first decoder 93 decodes the counted value of the first counter 92. When this counted value reaches the predetermined value N, the first decoder 93 outputs a reset enable signal to a reset output circuit 94 and a mask circuit 95, and outputs a counter reset signal to the first counter 92. The reset output circuit 94 produces, when fed with the reset enable signal, the reset signal RST of a predetermined pulse length, and outputs the reset signal RST to the actuator 8. When the first counter 92 is fed with a counter reset signal, on the other hand, it is reset to 0 in synchronization with the input of the next vertical synchronizing signal VSYNC.
A second counter 96 counts clock signals of the predetermined period T outputted by an oscillator 97, from 0. A second decoder 98 decodes the counted value. When this counted value becomes M, the second decoder 98 outputs a pulse mask signal to the mask circuit 95 and outputs a stop signal to the second counter 96. Only for the time period while the reset enable signal is not outputted from the first decoder 93 and while the pulse mask signal is not outputted from the second decoder 98, the mask circuit 95 outputs the clock signal of the predetermined period T outputted from the oscillator 97, as the pulse of the movement control signal CNT to the actuator 8. Here, the second counter 96 stops the counting operation, when fed with the stop signal, until the vertical synchronizing signal VSYNC is inputted. When the vertical synchronizing signal VSYNC is inputted, the second counter 96 resets the count value to 0, and starts the counting operation.
The foregoing configuration of the Z-axis scan control device 9 is just one example, and can adopt another. In a configuration, for example, a PLL can be used to produce a pulse signal of a 1/M period having an image pickup period synchronized with the vertical synchronizing signal, and this pulse signal can be outputted as the movement control signal CNT only for the image pickup period. Alternatively, the Z-axis scan control device 9 may also be constructed as a CPU circuit so that the foregoing operations of the Z-axis scan control device 9 may be executed in the software manner.
Next, the drive signal is produced in the actuator 8 by integrating the pulses of the movement control signal CNT inputted from the Z-axis scan control device 9, as has been described hereinbefore. This integration may be made by the well-known analog integration circuit. Another analog integration circuit can be constructed, as shown in
Now, the confocal microscope apparatus of the embodiment thus far described may further execute the following scanning sequence.
As shown in
The vertical synchronizing signal VSYNC of the camera 5 is outputted to the confocal scanner unit 4 and the Z-axis scan control device 9. In synchronization with the vertical synchronizing signal VSYNC inputted, the rotation control unit of the confocal scanner unit 4 drives the microlens array and the pinhole array so rotationally as to scan the whole XY-area once for every image pickup periods of the individual video frames.
In synchronization with the vertical synchronizing signal VSYNC inputted, as shown in
The actuator 8 integrates the pulses of the movement control signal CNT inputted from the Z-axis scan control device 9, to produce the drive signals which increase for the vertical synchronizing signal period but keep constant values for the image pickup periods of the video frames, as shown in
In response to the reset signal RST from the Z-axis scan control device 9, moreover, the actuator 8 returns the drive signals to the initial value.
By the operations thus far described, the scanning points in the sample 20 and the individual video frames are given such relations as are shown in
According to the scanning sequence thus far described, the objective lens 7 is moved in the Z-direction only for the image pickup period of the video frames. Therefore, the video frames having picked up the XY-plane of the sample 20 with the fixed Z-coordinate can be created individually for the different Z-coordinates.
As described hereinbefore, the confocal microscope apparatus is enabled to improve the precision of the scanning position control of the sample better in the optical axis direction. Moreover, the confocal microscope apparatus is enabled to enhance the probability of grasping a micro structure in the image picked up. Still moreover, the confocal microscope apparatus is enabled to create the video frames, which are picked up by picking up a plane normal to the optical axis of the sample with the coordinate in the optical axis direction being fixed, individually for the coordinates in the different optical axis directions.
Another embodiment of the invention will be described in the following. In the case a stereoscopic image of the sample is to be attained with the confocal microscope apparatus using the confocal scanner, a number of slice images are obtained at different positions in the optical axis direction, as described above, and are made stereoscopic by the CG (Computer Graphics) technique. FIG. 6 is a display example of the three-dimensional image of a Californian purple sea urchin measured by that method. By this display, the whole image of the sample can be grasped.
However, this case has the following problems.
(1) The CG processing takes time at least several minutes to several hours. This image processing after the CG has to be performed after the measurement. It is difficult to grasp the whole image during the measurement, to decide the propriety of the sample and to select the best portion of measurement.
(2) The shapes in the individual sections cannot be precisely grasped with perspective views. The shapes of the individual sections can be precisely grasped neither too much nor too less by using the two-dimensional images (or the slice images), as shown in
In
The processing portion 400 is provided with a display screen 410 and is enabled to read the image data outputted from the camera 300 and subject them to a predetermined processing and to display the image on the display screen 410. A personal computer is usually used as that processing portion 400.
Numeral 500 designates a drive portion for moving an objective lens 110 of the microscope 100 in the optical axis direction. For example, a piezo-element (PZT) is used as the drive portion 500.
Numeral 600 designates a stage controller for controlling the drive portion 500 on the basis of an instruction coming from the processing portion 400.
Here, the components of
In this configuration, the operations to obtain the slice images of the sample 20 placed on the microscope 100 are identical to those of the confocal microscope apparatus of the related art, and their description is omitted.
While the objective lens 110 is moved in the optical axis direction by activating the drive portion 500, the confocal slice images are picked up at the individual optical axis heights by the camera 300. The processing portion 400 transforms the images (in the top plan view) obtained from the camera 300 into the perspective images (or the corresponding images) picked up obliquely downward, and display them on the screen 410.
These transformations into the perspective views may be made merely by drawing pixels of coordinates Xi and Yi at the plane coordinates Xj and Yj of a predetermined perspective view, so that the transformations can be processed at a high speed.
For images of an inclination of 30 degrees, the coordinates Xj and Yj are determined, for example, on the basis of the following Formulas:
Xi=Xj cos θ−Yj sin θ;
Yi=Xj sin θ−Yj cos θ,
wherein θ=30°.
The coordinates Xj and Yj can be determined merely by the product/sum operations, if the processing portion 400 has the cos 30° as the table of constants. The product/sum operations can be processed at high speeds.
In the case a plurality of slice images are to be displayed, they are drawn as they are at a spacing in the optical axis direction while being held at their relative positions in the optical axis direction, as shown in
Thus, the confocal microscope apparatus of this embodiment can grasp the precise slice images of the sample at the individual optical axis heights and the stereoscopic relations of the samples as a whole.
The invention may be exemplified by the changes/modifications, as will be enumerated in the following.
(1) In the case a plurality of slice images are to be obtained, the XY-plane of the sample may be captured by the aforementioned scanning sequence with the Z-coordinate being fixed.
(2) The number of display sheets should not be limited to four but can be any from two to several tens.
(3) The display angle can be 0 to 360° individually in the longitudinal and latitudinal directions.
(4) For the image display, all the images need not be displayed, but some may be thinned out. For example, the confocal optical scanner can raise the speed up to 1,000 sheets/second, but the display cannot be recognized by the human eyes even if it is made at a speed exceeding a human-recognizable video rate (about 30 sheets/second) In this case, the display of one sheet per 1,000/30=33 (sheets) is sufficient.
(5) Alternatively, the image display need not display all the slice images being measured but may display only a representative image, as shown in
As shown in
(6) The display image should not be limited to a monochromatic display but may be a multicolor display.
(7) The measurement of sizes and the grasp of shapes are facilitated if known markers such as graduations or circles or known scales are displayed together with the slice images.
(8) Even the map display format shown in
(9) The drive of the objective lens 110 should not be limited to that of the piezo-element but may be exemplified by a stage drive or that of a magnetic actuator.
(10) The sample 20 should not be limited to a living organism with a fluorescent light but may be a semiconductor surface or a mechanical part with a reflecting mirror.
(11) A more proper display can be obtained if the angle or number of displays can be changed during the measurement/display.
(12) The image display may be updated for each slice image at any time when the slice image is measured, or the slice images displayed in the display screen may be updated all at once when their measurement was ended.
According to the confocal microscope apparatus of the embodiment shown in the configuration diagram of
(1) The three-dimensional display at a high speed can be easily realized to grasp the whole image easily while the sample is being measured.
(2) It is possible to grasp the slice images of the individual sections and their stereoscopic relations precisely.
Claims
1-5. (canceled)
6. A confocal microscope display device for displaying a plurality of slice images of a sample picked up at different positions in an optical axis direction by using an optical microscope and a confocal scanner, comprising:
- a moving portion for moving an objective lens of the optical microscope in the optical axis direction; and
- a processing portion for reading the slice images of the sample obtained from the confocal scanner, to display the slice images on a screen,
- wherein while measuring the sample, the processing portion displays two or more slice images in such an arrangement on a common screen that their positions relative to the sample enables to be grasped.
7. (canceled)
8. The confocal microscope display device according to claim 6,
- wherein the processing portion displays the slice images one-dimensionally or two-dimensionally in such an arrangement that their positions relative to the sample enables to be grasped.
9. The confocal microscope display device according to claim 6,
- wherein the display number of the slice images enables to be changed while measuring the sample.
10. The confocal microscope display device according to claim 6,
- wherein the slice images are presented in a perspective view so that the processing portion displays the sample and each of the slice images at coincident relative positions in the optical axis direction.
11. The confocal microscope display device according to claim 6,
- wherein the display angle of the perspective view of the slice images enables to be changed while measuring the sample.
12. The confocal microscope display device according to claim 6,
- wherein the processing portion displays sizes, axes, frames or marker images over the slice images.
13. The confocal microscope display device according to claim 6,
- wherein the moving portion includes a piezo-element, stage drive portion or a magnet actuator.
14. The confocal microscope display device according to claim 6,
- wherein the processing portion includes a personal computer.
Type: Application
Filed: Apr 14, 2006
Publication Date: Aug 31, 2006
Applicants: TOKAI UNIVERSITY EDUCATIONAL SYSTEM (Tokyo), JAPAN SCIENCE AND TECHNOLOGY AGENCY (Kawaguchi), YOKOGAWA ELECTRIC CORPORATION (Musashino-shi)
Inventors: Hideyuki Ishida (Hadano-shi), Takeo Tanaami (Musashino-shi)
Application Number: 11/403,832
International Classification: G02B 27/40 (20060101); H01J 3/14 (20060101); G02B 27/64 (20060101);