JP4158077B2 - Confocal microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a confocal microscope, and more particularly to a confocal microscope that records a highly accurate confocal image and facilitates image analysis.
[0002]
[Prior art]
The confocal microscope adopts the Niipou disc method that uses pinholes to increase the spatial resolution, squeezes the light to a minute pinpoint, irradiates the sample, eliminates excess scattered light and blur from the sample, One point in a three-dimensional space can be measured accurately. The confocal microscope can obtain a tomographic (slice) image without making an ultrathin section of a sample, and can construct an accurate three-dimensional stereoscopic image from the slice image data. Therefore, the physiology of living cells in the field of biological and biotechnology can be obtained. Used for reaction observation, morphology observation and LSI surface observation in the semiconductor market.
[0003]
FIG. 4 is a block diagram of a conventional confocal microscope. The confocal scanner 2 is attached to the camera port of the microscope 3, and the sample image is input to the video rate camera 1 as a confocal image via the microscope 3, the actuator 4, and the objective lens 5, and the image is observed. To do. The video rate camera 1 inputs the converted video signal 101 to the confocal scanner 2 and the image processing device 6. The image processing device 6 starts capture for converting and recording the video signal 101 into image data. The confocal scanner 2 performs rotation synchronization control in synchronization with the video signal 101. The arbitrary waveform generator 7 inputs a scanning signal 104 to the controller 8, and the controller 8 generates a control signal 105 based on the scanning signal 104 and controls the actuator 4. The actuator 4 adjusts the position on the optical axis of the objective lens 5 with respect to the microscope 3.
[0004]
[Problems to be solved by the invention]
The start of capture of the image processing device 6 and generation of the scanning signal 104 of the arbitrary waveform generator 7 are performed independently. The image generation timing at which the video signal 101 is generated, the rotation synchronization control by the confocal scanner 2, the capture start timing by the image processing device 6, and the scan start timing of the image in the focal direction by the arbitrary waveform generator 7 are mutually Do not synchronize. For this reason, the start timing of image acquisition by the image processing device 6 and the start timing of scanning in the focal direction of the image by the arbitrary waveform generator 7 vary in a maximum data acquisition time of 32 msec, and individual confocal images Therefore, the reliability of slice images obtained from a plurality of confocal images is reduced.
[0005]
The present invention has been made to solve the problems of the conventional techniques as described above, and an object thereof is to provide a confocal microscope capable of obtaining a highly reliable slice image.
[0006]
[Means for Solving the Problems]
In order to achieve the above object, a confocal microscope of the present invention includes a confocal scanner having a nipou disk and acquiring a sample image as a confocal image, a video rate camera for converting the confocal image into a video signal, An image processing device that converts the video signal into image data, and an optical control system that controls a focal position of an objective lens and designates a depth position in a focal direction of an image acquired by the confocal scanner, and slices a sample In the confocal microscope for acquiring an image, the rotation synchronization control of the Niipou disc, the start timing of acquisition of a video signal by the image processing device, and the start timing of scanning of the focal position of the objective lens by the optical control system, The slice image and the scan start timing are recorded simultaneously in synchronization with the signal.
[0007]
In the confocal microscope of the present invention, the rotation synchronization control of the Nipo disk, the start timing of video signal acquisition by the image processing device, and the scan start timing of the focal position of the objective lens by the optical control system are all synchronized with the video signal. As a result, the positional accuracy of the confocal image is improved, and variations in individual acquisition times are eliminated when a plurality of slice images are acquired, so that a highly reliable slice image can be obtained.
[0008]
The confocal microscope of the present invention includes a confocal scanner having a nipou disk and acquiring a sample image as a confocal image, a video rate camera for converting the confocal image into a video signal, and the video signal as image data. And an optical control system that controls the focal position of the objective lens and designates the depth position in the focal direction of the image obtained by the confocal scanner, and obtains a slice image of the sample. In a microscope, in response to an external signal, generation of a video signal by the video rate camera, rotation synchronization control of the Niipou disc, acquisition of a video signal by the image processing device, and a focal position of an objective lens by the optical control system The scanning is started and the slice image and the scanning start timing are simultaneously recorded .
[0009]
In the confocal microscope of the present invention, an apparatus for generating an external signal can arbitrarily change the rotation speed of the Niipou disk by controlling the confocal scanner.
[0010]
In the confocal microscope of the present invention, it is preferable that the optical control system includes a piezo actuator that controls a focal position of the objective lens. In this case, scanning of the focal position of the objective lens by the optical control system becomes fast and highly accurate.
[0011]
In the confocal microscope of the present invention, the image processing apparatus records a slice image in an image area of a recording medium, and records a scan start timing of a focal position of the objective lens in an audio area of the recording medium. preferable. In this case, since the first frame of the slice image can be detected by searching the scanning start timing of the focal position of the lens recorded in the audio area of the recording medium, analysis and editing of the recorded slice image is facilitated.
[0012]
It is also a preferred aspect of the present invention that the recording medium is a video tape. In this case, by adopting a video deck as the image processing apparatus, a system capable of recording slice images for a long time can be easily configured.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, a confocal microscope of the present invention will be described with reference to the drawings based on an embodiment of the present invention. FIG. 1 is a block diagram of a confocal microscope according to a first embodiment of the present invention. The confocal microscope according to this embodiment includes a video rate camera 1, a confocal scanner 2, a microscope 3, an actuator 4, an objective lens 5, an image processing device 6, an arbitrary waveform generator 7, a controller 8, and a synchronization interface box 9. Consists of.
[0014]
The video rate camera 1, the confocal scanner 2, the microscope 3, the actuator 4, and the objective lens 5 are disposed on the same optical axis. The confocal scanner 2 is a compact add-on type that employs a nipou disk system having a simple optical system and having a nipou disk having 20,000 pinholes and a corresponding microlens array. By attaching the confocal scanner 2 to the camera port of the microscope 3, the handheld microscope 3 is upgraded to a confocal microscope. The confocal microscope inputs an image of the sample into the confocal scanner 2 via the objective lens 5, the actuator 4, and the microscope 3. The confocal scanner 2 creates a confocal image from the sample image and inputs it to the video rate camera 1 for image observation.
[0015]
FIG. 2 is a time chart of various signals handled by the confocal microscope of FIG. The video rate camera 1 converts the confocal image into a video signal 101, and inputs the video signal 101 to the confocal scanner 2, the signal input terminal of the synchronization interface box 9, and the image input terminal of the image processing device 6. The confocal scanner 2 performs rotation synchronization control of the Niipou disc in synchronization with the video signal 101.
[0016]
When a video tape deck is used for the image processing apparatus 6, the video tape deck simultaneously applies the video signal 101 input from the image input terminal and the start signal 103 input from the audio input terminal to the video tape for a long time. Record. In addition, the scanning signal 104 can be recorded instead of the starting signal 103. If a digital video tape deck is employed, the starting signal 103 can be recorded in another area other than the image input area such as a superimpose. . On the video tape, the confocal image that changes in real time and the scanning start timing of the focal position of the objective lens 5 are simultaneously recorded. In this case, since the first frame of the slice image can be detected by searching the scanning start timing of the focal position of the objective lens recorded in the audio area of the recording medium, analysis and editing of the recorded slice image is facilitated. If a video deck is adopted as the image processing apparatus, a system capable of recording slice images for a long time can be easily configured.
[0017]
The synchronous interface box 9 extracts either the even-numbered pulse train or the odd-numbered pulse train of the video signal 101 and creates an internal A signal. The arbitrary waveform generator 7 generates a trigger signal 102 which is an H level pulse signal, inputs the trigger signal 102 to the trigger input terminal of the synchronous interface box 9, and uses it for the start timing of scanning of the focal plane.
[0018]
The synchronization interface box 9 creates an internal B signal in synchronization with the falling edge of the trigger signal 102. The internal B signal is a pulse signal having an H level pulse width time of about 35 msec and slightly longer than the video rate time of the video rate camera 1. The synchronous interface box 9 generates a start signal 103 by performing an AND operation on the inverted signal of the internal A signal and the internal B signal, and inputs it to the synchronous input terminals of the image processing device 6 and the arbitrary waveform generator 7. The image processing device 6 starts capture for converting and recording the video signal 101 into image data in synchronization with the rising edge of the start signal 103 from the synchronization input terminal. The synchronization interface box 9 is based on the video signal 101 from the signal input terminal, and the rotation synchronization control of the Niipou disc by the confocal scanner 2, the start timing of the video signal acquisition by the image processing device 6, and the objective lens by the optical control system All the scanning start timings of the focal positions are synchronized.
[0019]
When the video rate camera 1 (for example, a digital camera) in which the timing of image generation for generating the video signal 101 is synchronized with an external trigger signal from the outside is employed instead of the above configuration, the external device replaces the start signal 103. The external trigger signal is input to the video rate camera 1, the image processing device 6, and the arbitrary waveform generator 7, and the image generation timing, the Nipo disk rotation synchronization control, the video signal acquisition start timing, and the objective The scanning start timing of the focal position of the lens is synchronized. The confocal scanner 2 is arbitrarily variable in the number of rotations based on control from an external device. In this case, the synchronization interface box 9 can be omitted, and the synchronization rotational speed of the confocal scanner changes under the control of the external device.
[0020]
The arbitrary waveform generator 7 starts scanning the focal position of the objective lens 5 by the optical control system in synchronization with the rise of the start signal 103. The arbitrary waveform generator 7 generates a scanning signal 104 and inputs it to the controller 8. The scanning signal 104 is a sawtooth signal that increases linearly from L level to H level in a certain time. The controller 8 inputs the scanning signal 104 to the actuator 4. The actuator signal 105 is an actual actuator position signal, and after having fully extended, there is an overshoot after returning to the origin at once, and during this time, there is a dead zone.
[0021]
The actuator 4 is attached between the objective lens revolver of the microscope 3 and the objective lens 5, and the length in the focal direction of the image changes in proportion to the level of the scanning signal 104 by piezo driving, and the focal position of the objective lens 5 To control. The confocal microscope acquires a slice image of the sample by scanning the focal plane based on the scanning signal 104.
[0022]
According to the above embodiment, the rotation synchronization control of the Niipou disc, the start timing of video signal acquisition by the image processing device, and the start timing of scanning of the focal position of the lens by the optical control system are all synchronized with the video signal. As a result, the positional accuracy of the confocal image is improved, and variations in individual acquisition times are eliminated when acquiring a plurality of slice images, so that a highly reliable slice image can be obtained.
[0023]
FIG. 3 is a time chart of various signals handled by the confocal microscope according to the second embodiment of the present invention. In this embodiment, a personal computer is adopted as the image processing apparatus 6 of the previous embodiment. The personal computer includes a video capture board that records images, control software that controls the video capture board, a RAM as a temporary recording medium, and a hard disk (HDD) as a peripheral storage device. Further, the peripheral storage device may be a DVD, a CD-RW, or an MO.
[0024]
The personal computer starts the first image recording in synchronization with the rise of the start signal 103 at time t0. The input video signal 101 is converted into image data for the p number of sheets, the image data is temporarily recorded in the RAM, and the image data on the RAM is transferred to the HDD at time t1 when the recording to the RAM is completed. At the time t2 when the recording to the HDD is completed, the first image recording is finished. The second image recording starts at time t2 and ends at time t4 in the same manner as described above.
[0025]
The time Ta is a processing time for converting the video signal 101 into image data for p sheets and recording it in the RAM, and is determined by the number p of designated image data. The time Tb is a transfer time for transferring the image data on the RAM to the HDD, and is determined by the size of the image data on the RAM and the HDD head scan time. The times Ta and Tb are measured in advance, and the period Tc of the trigger signal 102 is designed as Tc = Ta + Tb.
[0026]
The size of the storage area that can be used with the current RAM is at most about 1 GB. For example, when an image has a screen size of 640 × 480 pixels and a gradation has an 8-bit gray scale level, when the image is captured at a video rate, the recordable time is about 90 seconds, and particularly in observation of dynamics of living organisms. You may not be satisfied. Further, if the recordable time is lengthened by image compression, image quality is deteriorated and image data is lost. Therefore, the temporarily recorded image data on the RAM is transferred to the peripheral storage device.
[0027]
If commercially available image analysis software is installed in a personal computer, an accurate three-dimensional structure can be reconstructed from the acquired slice image, and the operability of image analysis is improved.
[0028]
According to the above-described embodiment, the image data can be analyzed from the confocal image collected in real time by transferring the temporarily recorded image data on the RAM to the peripheral storage device, so that a long time lapse measurement can be performed. .
[0029]
As described above, the present invention has been described based on the preferred embodiment. However, the confocal microscope of the present invention is not limited to the configuration of the above-described embodiment example. Confocal microscopes with modifications and changes are also within the scope of the present invention.
[0030]
【The invention's effect】
As described above, in the confocal microscope of the present invention, the rotation synchronization control of the Niipou disk, the acquisition timing of the video signal by the image processing device, and the scanning start timing of the focal position of the lens by the optical control system are all By recording the slice image and the scan start timing at the same time as synchronizing with the video signal, the position accuracy of the confocal image is improved, and there is no variation in individual acquisition time when acquiring a plurality of slice images. Since a highly reliable slice image is obtained and the first frame of the slice image can be detected, analysis and editing of the recorded slice image is facilitated.
[0031]
In addition, the device that generates the external signal can control the confocal scanner to arbitrarily change the rotation speed of the Nipo disk.
[Brief description of the drawings]
FIG. 1 is a block diagram of a confocal microscope according to a first embodiment of the present invention.
FIG. 2 is a time chart of various signals handled by the confocal microscope of FIG.
FIG. 3 is a time chart of various signals handled by a confocal microscope according to a second embodiment of the present invention.
FIG. 4 is a block diagram of a conventional confocal microscope.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Video rate camera 2 Confocal scanner 3 Microscope 4 Actuator 5 Objective lens 6 Image processing apparatus 7 Arbitrary waveform generator 8 Controller 9 Synchronous interface box 101 Video signal 102 Trigger signal 103 Start signal 104 Scan signal 105 Actuator signal

Claims (5)

A confocal scanner having a nipou disc and acquiring a sample image as a confocal image, a video rate camera for converting the confocal image into a video signal, an image processing device for converting the video signal into image data, and an objective lens An optical control system that controls the focal position of the image and specifies the depth position in the focal direction of the image acquired by the confocal scanner, and a confocal microscope that acquires a slice image of the sample,
The slice synchronization control is performed in synchronization with the video signal while the rotation synchronization control of the Nipkow disk, the video signal acquisition start timing by the image processing apparatus, and the scanning start timing of the focal position of the objective lens by the optical control system are performed. A confocal microscope characterized by simultaneously recording an image and scanning start timing . A confocal scanner having a nipou disc and acquiring a sample image as a confocal image, a video rate camera for converting the confocal image into a video signal, an image processing device for converting the video signal into image data, and an objective lens An optical control system that controls the focal position of the image and specifies the depth position in the focal direction of the image acquired by the confocal scanner, and a confocal microscope that acquires a slice image of the sample,
In response to an external signal, generation of a video signal by the video rate camera, rotation synchronization control of the Niipou disc, acquisition of a video signal by the image processing device, and scanning of the focal position of the objective lens by the optical control system are started. And simultaneously recording the slice image and the scanning start timing . The optical control system includes a piezo actuator that controls the focal position of the objective lens.
The confocal microscope according to claim 1 or 2 The image processing apparatus records the slice image in an image area of a recording medium, and records a scan start timing of the focal position of the objective lens in an audio area of the recording medium.
The confocal microscope according to claim 1. The recording medium is a video tape;
The confocal microscope according to claim 4.

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