JP4478921B2 - 3D confocal laser microscope system - Google Patents

Description

  The present invention relates to a three-dimensional confocal laser microscope system, and more particularly to an improvement for observing a three-dimensional shape of a sample as an ultra-deep image.

The confocal microscope can obtain a slice image without making a thin section of the sample, and can construct an accurate three-dimensional stereoscopic image of the sample from the slice image. Therefore, the physiological reaction of living cells in the fields of organisms and biotechnology It is used for observation, morphology observation, or LSI surface observation in the semiconductor market (see, for example, Patent Document 1).
Such observation of a sample may require an ultra-deep image (or also called an all-focus image) that is entirely focused on the sample. For this reason, after obtaining a plurality of slice images at each focal position of the sample, they are synthesized by image processing to obtain an ultra-deep image. (For example, see Non-Patent Document 1)

JP 2002-72102 A "Ultra-depth color 3D shape measurement microscope VK-9500", catalog, Keyence Corporation, [searched on August 14, 2003], Internet <URL: http://www.keyence.co.jp/microscope/product/ VK9500 / index.html>

FIG. 3 is a configuration diagram of the confocal microscope described in Patent Document 1. The video rate camera 1, the confocal scanner 2, the microscope 3, the actuator 4 and the objective lens 5 are arranged on the same optical axis. The confocal scanner 2 has a nipou disk having a large number of pinholes and a corresponding microlens array, and is a compact add-on type employing a nipou disk system composed of a simple optical system.

  The confocal scanner 2 is attached to the camera port of the microscope 3. The confocal microscope uses a laser beam to input an image of the sample to the confocal scanner 2 via the objective lens 5, the actuator 4 and the microscope 3. The confocal scanner 2 obtains a confocal image of the sample and inputs it to the video rate camera 1.

Figure 4 is a time chart of various signals handled 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.

  When a video tape deck is used for the image processing apparatus 6, the video tape deck records the video signal 101 input from the image input terminal and the start signal 103 input from the audio input terminal simultaneously on the video tape for a long time. To do. 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 recorded simultaneously.

  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.

  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 a pulse width time of H level 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 the start signal 103 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. Based on the video signal 101 from the signal input terminal, the synchronization interface box 9 controls rotation synchronization of the Niipou disc by the confocal scanner 2, the start timing of acquisition of the video signal by the image processing device 6, and the objective lens of the optical control system. All the timings for starting the scanning of the focal position are synchronized.

  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.

  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.

  According to such a configuration, the rotation synchronization control of the Niipou disc, 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 synchronized with the video signal, The position 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.

  However, when an ultra-deep image is obtained in a conventional three-dimensional confocal laser microscope system, the objective lens is moved in the Z-axis direction by a lens moving mechanism such as the actuator described above as described in Non-Patent Document 1. Then, all the slice images obtained at the respective focal positions are acquired and then superposed to produce an ultra-deep image. For this reason, there was a problem of lack of real-time property.

  The present invention is intended to solve the problem of such a conventional confocal microscope system, and by scanning the objective lens at high speed, an ultra-deep image of the sample can be obtained in real time. The object is to realize a three-dimensional confocal laser microscope system.

The present invention is a three-dimensional confocal laser microscope system configured as follows.
(1) A confocal scanner that acquires a slice image of a sample as a confocal image, a video rate camera that converts the confocal image into a video signal, an image processing device that converts the video signal into image data, and a microscope An actuator for moving the focal position of the objective lens in the optical axis direction, and a control means for generating a scanning waveform signal for scanning the objective lens in the optical axis direction through the actuator, and slicing the sample in the depth direction A three-dimensional confocal laser microscope system configured to acquire an image,
The control means receives the video signal output from the video rate camera, outputs the video signal as it is, extracts the vertical synchronization signal included in the video signal , and based on this, the scanning waveform generation trigger signal and the image A signal controller for generating an acquisition trigger signal;
From the scanning period of the objective lens when observing the sample and the scanning distance in the optical axis direction, a waveform in which the tops of the isosceles triangles are formed in a semicircular shape is obtained by calculation, and this is used as an arbitrary waveform generator as a scanning waveform. An output waveform calculation device;
Arbitrary waveform generation that receives the scanning waveform generation trigger signal from the signal controller, generates a waveform in which the top of the isosceles triangle that has been sent from the waveform calculation device and stored in advance is formed in a semicircular shape, and is sent to the actuator driver And
An actuator driver that generates a drive signal for driving the actuator based on a scanning waveform signal output from the arbitrary waveform generator;
The pulse width of the image capture trigger signal is at least twice the period of the vertical synchronization signal included in the video signal , and the scanning waveform signal is generated at least once within the vertical synchronization signal period of the video signal. 3D confocal laser microscope system.

(2) The three-dimensional confocal laser microscope system according to (1), wherein the scanning waveform is a waveform generated by S-shaped control at a semicircular portion at the top of an isosceles triangle.

(3) The three-dimensional confocal laser microscope system according to (1) or (2), wherein the image processing means integrates or averages the acquired slice images .

The present invention has the following effects.

According to the first aspect of the present invention, a confocal scanner that acquires a slice image of a sample as a confocal image, a video rate camera that converts the confocal image into a video signal, and an image that converts the video signal into image data. A processing apparatus, an actuator for moving the focal position of the objective lens of the microscope in the optical axis direction, and a control means for generating a scanning waveform signal for scanning the objective lens in the optical axis direction via the actuator, A three-dimensional confocal laser microscope system configured to acquire a slice image in the depth direction of
The control means receives the video signal output from the video rate camera, outputs the video signal as it is, extracts the vertical synchronization signal included in the video signal , and based on this, the scanning waveform generation trigger signal and the image A signal controller for generating an acquisition trigger signal;
From the scanning period of the objective lens when observing the sample and the scanning distance in the optical axis direction, a waveform in which the tops of the isosceles triangles are formed in a semicircular shape is obtained by calculation, and this is used as an arbitrary waveform generator as a scanning waveform. An output waveform calculation device;
Arbitrary waveform generation that receives the scanning waveform generation trigger signal from the signal controller, generates a waveform in which the top of the isosceles triangle that has been sent from the waveform calculation device and stored in advance is formed in a semicircular shape, and is sent to the actuator driver And
An actuator driver that generates a drive signal for driving the actuator based on a scanning waveform signal output from the arbitrary waveform generator;
The pulse width of the image capture trigger signal is set to be twice or more the period of the vertical synchronization signal included in the video signal , and the scanning waveform signal is generated at least once within the vertical synchronization signal period of the video signal. The objective lens can be scanned at a higher speed than the imaging speed to obtain an ultra deep image of the sample in real time, and the objective lens can be scanned at a higher speed than the imaging speed of the camera to obtain an ultra deep image of the sample in real time. By driving the actuator with a waveform with a semicircular top, the displacement is linear in time, so the exposure time is uniform depending on the cross section of the sample, and the brightness information of the resulting ultra-deep image However, an accurate three-dimensional confocal laser microscope system can be realized.

According to the second aspect of the present invention, the scan waveform is a waveform in which the semicircle at the top of the isosceles triangle is generated by S-shaped control, whereby the acceleration value is limited to a constant value, and the actuator or the microscope housing. The vibration of the body can be suppressed.

According to the invention described in claim 3 , since the image processing means integrates or averages the acquired slice images, the super-deep image is obtained and the brightness information of the cross section of the sample is uniform. It is possible to observe the image that is being converted into real time. This is effective mainly when observing a dark sample (low reflectance).

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing an embodiment of a three-dimensional confocal laser microscope system according to the present invention. In FIG. 1, the same parts as those in FIG. 4 are denoted by the same reference numerals, and the description thereof is omitted.

In FIG. 1, 10 is a signal controller, 11 is a waveform calculator, 12 is an actuator driver, and 20 is a sample.
The signal controller 10 receives a video signal output from a video rate camera (hereinafter simply referred to as a camera) 1 and outputs the video signal as it is, and also extracts a vertical synchronization signal from the video signal, and based on this, various signals are extracted. Generate a trigger signal.
When the arbitrary waveform generator 7a receives the scanning waveform generation trigger signal from the signal controller 10, the arbitrary waveform generator 7a generates a waveform in which the tops of the isosceles triangles sent from the waveform calculation device 11 and stored in advance are formed in a semicircular shape. To the actuator driver 12.

The waveform calculation device 11 calculates the waveform in which the top of the isosceles triangle is formed in a semicircular shape from the scanning period of the objective lens 5 when observing the sample 20 and the scanning distance in the optical axis direction, and scans it. The waveform is output to the arbitrary waveform generator 7a.
The actuator driver 12 generates a drive signal for driving the actuator 4 based on the scanning waveform signal output from the arbitrary waveform generator 7a.
Here, a part constituted by the waveform calculation device 11, the arbitrary waveform generator 7a, the actuator driver 12, and the signal controller 10 is referred to as control means.

  Next, the operation of such a configuration will be described with reference to the time chart of each signal shown in FIG. When the video signal (including the vertical synchronization signal) shown in FIG. 2A is sent from the camera 1 to the control signal device 10, the signal controller 10 sends the video signal to the image processing device 6 as it is. The vertical sync signal is extracted from the video signal, and the sync signal is sent to the confocal scanner 2, and various trigger signals, that is, the scan waveform generation trigger signal [FIG. 2 (c)], the image capture trigger signal [FIG. (E)] is generated.

  When the signal controller 10 receives the image capture start signal shown in FIG. 2B, the signal controller 10 outputs the first vertical synchronization signal after the image capture start signal becomes LOW as the scanning waveform generation trigger signal [FIG. ]] Is sent to the arbitrary waveform generator 7a, and the vertical synchronization signal is sent to the image processing device 6 as an image capture trigger signal shown in FIG. The image capture start signal is a signal that the operator inputs to the signal controller at an arbitrary timing from a host controller such as a personal computer, and the pulse width is at least twice the period of the vertical synchronization signal of the video signal. is there.

In the arbitrary waveform generator 7a, when the scanning waveform generation trigger signal is received from the signal controller 10 , the top of the isosceles triangle shown in FIG. The formed waveform is generated and sent to the actuator driver 12.

The actuator 4 is driven by a drive signal from the actuator driver 12 and scans the objective lens 5 in the optical axis direction according to the waveform of FIG. The image processing apparatus 6 acquires an ultra-deep image of the sample 20 in synchronization with this.
Since the imaging speed of one frame of the camera 1 is sufficiently slow with respect to the scanning of the objective lens, the slice images of the sample scanned at each focal position are captured in an overlapped manner, and the image processing device 6 converts the video signal into image data. To obtain an ultra-deep image.

In the series of operations described above, the top of an isosceles triangle that is a scanning waveform for driving the actuator 4 is formed in a semicircular shape within one period of the video signal [FIG. 2A] (frame period of the camera 1). Waveform [Fig. 2 (d)]
There are three. Thereby, the entire cross section of the sample can be exposed in one frame of the video signal, and an ultra-deep image is obtained as one image.
By driving the actuator 4 with a waveform in which the apex of the isosceles triangle is formed in a semicircular shape , the displacement becomes linear in time, so that the exposure time becomes uniform depending on the cross section of the sample, and the obtained ultra-deep image Brightness information is accurate.

It should be noted that there is at least one waveform in which the top of the isosceles triangle formed in one period of the video signal is formed in a semicircle, and the vertical synchronization signal period of the video signal and the top of the isosceles triangle are in a semicircle. The relationship between the periods of the formed waveforms is as follows.
Vertical synchronization signal period of video signal = n × period of a waveform in which the top of an isosceles triangle is formed in a semicircle (n is an integer)

Further, at the turning point of the waveform in which the top of the isosceles triangle is formed in a semicircular shape, the above-described waveform calculation device 11 performs the calculation by the S-shaped control in order to limit the acceleration value to a constant value, whereby the actuator 4 In addition, it is possible to generate a waveform in which the top of an isosceles triangle that can suppress the vibration of the microscope casing is formed in a semicircular shape .

  The present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.

It is a block diagram which shows one Example of the three-dimensional confocal laser microscope system which concerns on this invention. It is a time chart of each signal concerning the present invention. It is a block diagram which shows an example of the conventional confocal microscope. It is a time chart of each signal which the confocal microscope of FIG. 3 handles .

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Video rate camera 2 Confocal scanner 3 Microscope 4 Actuator 5 Objective lens 6 Image processing apparatus 7a Arbitrary waveform generator 10 Signal controller 11 Waveform calculation apparatus 12 Actuator driver 20 Sample

Claims (3)

A confocal scanner that acquires a slice image of a sample as a confocal image, a video rate camera that converts the confocal image into a video signal, an image processing device that converts the video signal into image data, and an objective lens of a microscope An actuator that moves the focal position in the optical axis direction and a control means that generates a scanning waveform signal for scanning the objective lens in the optical axis direction via this actuator, and obtains a slice image in the depth direction of the sample A three-dimensional confocal laser microscope system configured to be able to
The control means receives the video signal output from the video rate camera, outputs the video signal as it is, extracts the vertical synchronization signal included in the video signal , and based on this, the scanning waveform generation trigger signal and the image A signal controller for generating an acquisition trigger signal;
From the scanning period of the objective lens when observing the sample and the scanning distance in the optical axis direction, a waveform in which the tops of the isosceles triangles are formed in a semicircular shape is obtained by calculation, and this is used as an arbitrary waveform generator as a scanning waveform. An output waveform calculation device;
Arbitrary waveform generation that receives the scanning waveform generation trigger signal from the signal controller, generates a waveform in which the top of the isosceles triangle that has been sent from the waveform calculation device and stored in advance is formed in a semicircular shape, and is sent to the actuator driver And
An actuator driver that generates a drive signal for driving the actuator based on a scanning waveform signal output from the arbitrary waveform generator;
The pulse width of the image capture trigger signal is at least twice the period of the vertical synchronization signal included in the video signal , and the scanning waveform signal is generated at least once within the vertical synchronization signal period of the video signal. 3D confocal laser microscope system. The three-dimensional confocal laser microscope system according to claim 1, wherein the scanning waveform is a waveform generated by S-shaped control at a semicircular portion at the top of an isosceles triangle.   The three-dimensional confocal laser microscope system according to claim 1, wherein the image processing unit integrates or averages the acquired slice images.

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