JP3612538B2 - Confocal microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for controlling scanning in an optical axis direction in a confocal microscope apparatus.
[0002]
[Prior art]
As a conventional confocal microscope apparatus, for example, a Niipou type confocal microscope apparatus schematically shown in FIG. 5A is known.
As shown in the figure, this confocal microscope apparatus has a microlens array 101, a pinhole array 102 (nippo board) and an objective lens 103 for condensing laser light on a sample 20, and an objective lens 103 in an optical axis direction. The direction of reflected light from the sample passing through the actuator 104 for moving in the direction (Z direction in the figure), the camera 106 provided with the condenser lens 105, and the objective lens 103 and the pinhole array 102 is changed in the direction of the camera 106. And a beam splitter 107 to be operated.
[0003]
In such a configuration, the Z coordinate of the condensing point of the laser light is controlled by the position of the objective lens 103 in the Z direction, and the condensing point of the laser light is rotated by rotating the microlens array 101 and the pinhole array 102. The XY coordinates of are controlled. That is, the scanning point in the sample 20 photographed by the camera 106 can be three-dimensionally controlled by the position of the objective lens 103 in the Z direction and the rotation angles of the microlens array 101 and the pinhole array 102.
[0004]
Now, as a conventional scanning technique in such a confocal microscope apparatus, as shown in FIG. 5b, while rotating the microlens array 101 and the pinhole array 102 in synchronization with the vertical synchronization signal of the camera 106, There is known a technique for starting the operation of moving the objective lens 103 uniformly in a Z coordinate increasing direction for a period longer than a plurality of frame periods in synchronization with a camera vertical synchronization signal generated immediately after the trigger signal is input (for example, special No. 2002-72102).
[0005]
Prior art document information relating to the invention of this application includes the following.
[0006]
[Patent Document 1]
Japanese Patent Laid-Open No. 2002-72102
[Problems to be solved by the invention]
In the conventional technique, the timing for starting the movement of the objective lens 103 is synchronized with the vertical synchronization signal, but the movement after the movement is started is asynchronous with the vertical synchronization signal. For this reason, it is relatively difficult to accurately control the position of the scanning point in the Z direction for each video frame shot by the camera 106. More specifically, in the case of repeatedly shooting while moving the position in the Z direction, the shift in the Z direction position becomes cumulatively large, and such a shift cannot be confirmed or corrected.
[0008]
The conventional technique performs scanning in the XY directions while constantly changing the Z coordinate to photograph each scanning point. In addition, according to the method of shooting while constantly changing the Z coordinate in this way, it is possible to suppress the generation of Z coordinate points that are not scanned for all XY coordinates, and even if the structure is very small in the Z direction. It is possible to increase the probability that at least the part appears in the video.
[0009]
However, in this conventional technique, the coordinates of the objective lens 103 change uniformly even during a synchronization signal period such as a vertical synchronization signal period during which no image is taken by the camera 106. Then, since the Z coordinate range in the sample 20 corresponding to the range in which the objective lens 103 is moved during the synchronization signal period is not photographed at all, according to this conventional technique, a minute structure is missing in the Z direction. The probability that it will not be able to be suppressed sufficiently.
[0010]
On the other hand, depending on the application to which the confocal microscope apparatus is applied, there are cases where it is desired to generate an image frame obtained by photographing the XY plane of the sample with the Z coordinate fixed for each of different Z coordinates. For example, the set of video frames generated in this way becomes a voxel having an XYZ coordinate system as it is, and is therefore suitable for processing such as three-dimensional analysis of the sample 20.
[0011]
However, according to the conventional technique, since the Z coordinate is constantly changed even during the image capturing period of the camera 106, it is impossible to generate an image frame in which the XY plane of the sample is captured with the Z coordinate fixed.
Therefore, an object of the present invention is to further improve the accuracy of position control in scanning in the optical axis direction of a sample in a confocal microscope apparatus. In addition, another object of the present invention is to increase the probability that a minute structure can be captured in a captured video in a confocal microscope apparatus. Still another object of the present invention is to generate, for each of the different optical axis direction coordinates, an image frame in which the optical axis direction coordinates are fixed and a plane perpendicular to the optical axis of the sample is photographed in the confocal microscope apparatus. To do.
[0012]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a confocal scanner that scans the focal position of a light beam in a direction perpendicular to the optical axis with respect to the sample, and the focal position of the light beam in the optical axis direction with respect to the sample. A moving mechanism that scans, a camera that captures an image of the sample by the light beam, and the vertical synchronization signal of the camera, the focal position of the light beam is moved to the moving mechanism in the direction of the optical axis for each vertical synchronization signal. A confocal microscope apparatus provided with a movement control unit for moving a predetermined distance is provided.
[0013]
According to such a confocal microscope apparatus, since the focal position of the light beam is moved in synchronization with each vertical synchronizing signal, the scanning point is always set to a certain coordinate range for each video frame in the optical axis direction. Can fit inside. Therefore, the deviation of the focal position in the optical axis direction does not accumulate, and the scanning of the sample in the optical axis direction can be controlled more accurately.
[0014]
According to the present invention, in the confocal microscope apparatus, the movement control unit causes the moving mechanism to keep the focal position of the light beam constant at least during the vertical synchronization signal period of the camera. In the period excluding at least the vertical synchronization signal period, the moving mechanism moves the focal position of the light beam by a predetermined distance in the optical axis direction.
[0015]
In this way, by avoiding the movement of the scanning point in the optical axis direction during the vertical synchronization signal period in which imaging is not performed, the generation of a coordinate range in the optical axis direction that is missing from the imaging target is generated. It can be kept small. Therefore, it is possible to improve the probability that a minute structure can be captured in the captured video.
Further, according to the present invention, in such a confocal microscope apparatus, the movement control unit causes the movement mechanism to keep the focal position of the light beam constant at least during the pixel imaging period of the camera. A confocal microscope apparatus characterized by causing the moving mechanism to move the focal position of the light beam by a predetermined distance in the optical axis direction during a period excluding at least a pixel imaging period.
[0016]
In this way, by moving the scanning point in the optical axis direction only during a period other than at least during the pixel imaging period, the video frames obtained by photographing the plane perpendicular to the optical axis of the sample with the optical axis direction coordinates fixed are different. It can be generated for each of the optical axis direction coordinates.
[0017]
In this case, the movement control unit may cause the moving mechanism to move the focal position of the light beam by a predetermined distance in the optical axis direction during the vertical synchronization signal period of the camera.
Further, in the above confocal microscope apparatus, in parallel with the imaging of the camera, an image obtained by the three-dimensional representation of the sample is reconstructed and displayed from the image captured by the camera, and is newly displayed by the camera. It is preferable to provide display means for updating the image based on the three-dimensional representation with the captured image. By doing in this way, the image by the three-dimensional representation of a sample can be observed in what is called real time.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described.
FIG. 1 shows a configuration of a confocal microscope apparatus according to the present embodiment.
As shown in the figure, the confocal microscope apparatus includes a main body 1, a stage 2 on which a sample 20 is placed, a laser light source 3, a confocal scanner unit 4, a 66 frame / second double-speed camera adopting IEEE 1394 as a communication standard, and the like. High-speed camera 5, image intensifier 6 for adding image enhancement and high-speed shutter function to camera 5, objective lens 7, actuator 8 for moving objective lens 7 in the optical axis direction, Z-axis scanning control device 9, video capture It has an image processing apparatus 10 and a display apparatus 11 such as a computer having an interface. The confocal scanner unit 4 accommodates the above-described microlens array, pinhole array, beam splitter, and rotation control unit that rotationally drives the microlens array and the pinhole array.
[0019]
In addition, the confocal microscope apparatus includes an illumination light source 12 and functions as an optical microscope apparatus by the illumination light source 12 and the optical system housed in the main body 1.
Hereinafter, scanning control in the confocal microscope apparatus will be described.
FIG. 2 shows a scanning sequence of the confocal microscope apparatus.
The main scanning sequence is started by a TRIGER signal output from the Z-axis scanning control device 9 shown in FIG. The camera 5 receives the TRIGER signal as an external trigger signal, and synchronizes the vertical synchronization signal with the TRIGER signal as shown in FIG.
[0020]
The vertical synchronization signal V SYNC of the camera 5 is output to the confocal scanner unit 4 and the Z-axis scanning control device 9. Then, the rotation control unit of the confocal scanner unit 4 synchronizes with the input vertical synchronization signal V SYNC so as to scan the entire XY range once for each imaging period of each video frame and the pinhole array. Is driven to rotate. Here, the imaging period refers to a period excluding at least the vertical synchronization signal period in one video frame period. Note that the imaging period may not include a horizontal synchronization signal period or a period in which pixels that are before and after the horizontal synchronization signal period and the vertical synchronization signal period are not imaged.
[0021]
On the other hand, as shown in FIG. C, the Z-axis scanning control device 9 synchronizes with the input vertical synchronization signal V SYNC and applies a predetermined number of pulses to a predetermined number of actuators during the imaging period of each video frame. The stage which outputs as CNT is performed. Further, the input vertical synchronization signal V SYNC is counted, and when the predetermined count is reached, the pulse output is stopped, and a stage for outputting the reset signal RST to the actuator 8 is executed as shown in FIG. If the next vertical synchronization signal V SYNC is input, a predetermined number of video frame periods and a predetermined number of pulses having a predetermined cycle during the imaging period of each video frame are used as the movement control signal CNT in the same manner as described above. The sequence consisting of the stage that outputs to the actuator 8 and the stage that outputs the reset signal RST to the actuator 8 are repeated while stopping the pulse output for one video frame period.
[0022]
On the other hand, the actuator 8 integrates the pulse of the movement control signal CNT input from the Z-axis scanning control device 9, and as shown in FIGS. E and f, the period of the vertical synchronization signal V SYNC maintains a constant value, A driving signal that uniformly increases during the imaging period is generated, and the objective lens 7 is moved in the Z direction by this driving signal. Here, the amount of movement of the objective lens 7 is proportional to the magnitude of the drive signal. If the amount of movement of the objective lens 7 is not linearly proportional to the magnitude of the drive signal, the objective lens 7 does not move during the period of the vertical synchronization signal V SYNC and moves uniformly during the imaging period of the video frame. A drive signal having such a waveform is generated. As a mechanism for moving the objective lens 7, a mechanism using a piezo element or the like can be used.
[0023]
When the reset signal RST is input from the Z-axis scanning control device 9, the actuator 8 returns the drive signal to the initial value.
With the above operation, the relationship between the scanning point in the sample 20 and each video frame is as shown in FIG. That is, in each video frame period (T1 to T8 in the figure), a video in a different Z coordinate range is captured for each video frame period. Here, in the present embodiment, the objective lens 7 is moved in the Z direction only during the imaging period of the video frame as described above. Therefore, there is little occurrence of a Z coordinate range in which any XY coordinate is not photographed.
[0024]
Now, referring back to FIG. 1, the image processing apparatus 10 captures and stores each video frame VIDEO output from the camera 5, and repeats the process of combining and displaying it on the display device 11. Here, the image processing device 10 is supplied with a timing signal indicating the timing of the TRIGER signal from the Z-axis scanning control device 9, and the image processing device 10 applies to each video frame and its video frame according to this timing signal. Recognizing the correspondence with the Z-direction order of the scanning plane of the sample 20 that has been imaged, the video frames are synthesized or arranged according to the recognized order to reconstruct a three-dimensional image (voxel) of the sample 20 and reconstruct the three-dimensional image. The process of displaying the image by the three-dimensional expression projected on the virtual two-dimensional screen by an appropriate rendering algorithm (for example, volume rendering) on the display device 11 is repeated. That is, the image processing apparatus 10 performs real-time display of an image based on a three-dimensional representation of the sample 20.
[0025]
Hereinafter, a configuration example of the Z-axis scanning control device 9 will be described.
FIG. 3 shows a configuration example of the Z-axis scanning control device 9.
Hereinafter, in the scanning sequence shown in FIG. 2, the Z-axis scanning control device 9 uses a predetermined number N of pulses with a predetermined period T as the movement control signal CNT during a predetermined number N of video frame periods and the imaging period of each video frame. A description will be given on the assumption that the sequence of the stage that outputs to the actuator 8 and the stage that stops the pulse output for one video frame period and outputs the reset signal RST to the actuator 8 is repeated.
[0026]
In FIG. 3, the sequence control unit 91 generates the above-described TRIGER signal in response to a request from the image processing apparatus 10 or a user operation.
The first counter 92 is reset by the TRIGER signal and counts the vertical synchronization signal V SYNC from 0. The first decoder 93 decodes the count value of the first counter 92, and when the count value reaches a predetermined value N, outputs a reset enable signal to the reset output circuit 94 and the mask circuit 95, and outputs the counter reset signal to the first counter 92. Output. When the reset enable signal is input, the reset output circuit 94 generates a reset signal RST having a predetermined pulse length and outputs it to the actuator 8. Further, when the counter reset signal is input, the first counter 92 is reset to 0 in synchronization with the input of the next vertical synchronization signal VSYNC.
[0027]
The second counter 96 counts from 0 the clock signal with a predetermined period T output from the oscillator 97. The second decoder 98 decodes the count value, and when the count value reaches M, outputs a pulse mask signal to the mask circuit 95 and outputs a stop signal to the second counter 96. The mask circuit 95 outputs a clock signal having a predetermined cycle T output from the oscillator 97 only during a period when the reset enable signal is not output from the first decoder 93 and the pulse mask signal is not output from the second decoder 98. It outputs to actuator 8 as a pulse of movement control signal CNT. Here, when the stop signal is input, the second counter 96 stops counting until the vertical synchronization signal V SYNC is input. When the vertical synchronization signal V SYNC is input, the second counter 96 resets the count value to 0 and counts. Start operation.
The above-described configuration of the Z-axis scanning control device 9 is merely an example, and other configurations can be adopted as the configuration of the Z-axis scanning control device 9. For example, a configuration may be adopted in which a pulse signal having a 1 / M cycle of the imaging period synchronized with the vertical synchronization signal is generated using a PLL, and this pulse is output as the movement control signal CNT only in the imaging period. Further, the Z-axis scanning control device 9 may be configured as a CPU circuit, and the above-described operation of the Z-axis scanning control device 9 may be implemented by software.
[0028]
Next, the generation of the drive signal in the actuator 8 is performed by integrating the pulse of the movement control signal CNT input from the Z-axis scanning control device 9 as described above. This integration may be performed by a known analog integration circuit. For example, a counter 81 that counts the pulses of the movement control signal CNT as shown in the figure, and a D / A converter that performs D / A conversion on the counter value of the counter 81. The A / A converter 82 and the driver circuit 83 that amplifies the output of the D / A converter 82 can be used. The counter 81 is reset by a reset signal RST input from the Z-axis scanning control device 9.
[0029]
The embodiment of the present invention has been described above.
In the confocal microscope apparatus in the above embodiment, the following scanning sequence may be further executed.
As shown in the figure, this scanning sequence is also started by a TRIGER signal output from the Z-axis scanning control device 9 shown in FIG. The camera 5 receives the TRIGER signal as an external trigger signal, and synchronizes the vertical synchronization signal with the TRIGER signal as shown in FIG.
[0030]
The vertical synchronization signal V SYNC of the camera 5 is output to the confocal scanner unit 4 and the Z-axis scanning control device 9. Then, the rotation control unit of the confocal scanner unit 4 synchronizes with the input vertical synchronization signal V SYNC so as to scan the entire XY region once for each imaging period of each video frame and the pinhole array. Is driven to rotate.
[0031]
On the other hand, as shown in FIG. C, the Z-axis scanning control device 9 executes a stage that outputs a pulse to the actuator 8 as the movement control signal CNT during the vertical synchronization signal period in synchronization with the input vertical synchronization signal VSYNC. To do. Further, the input vertical synchronization signal is counted, and when the predetermined count is reached, the pulse output of the movement control signal CNT is stopped, and a stage for outputting the reset signal RST to the actuator 8 is executed as shown in FIG. When the next vertical synchronization signal V SYNC is input, a stage for outputting a pulse to the actuator 8 as a movement control signal CNT during a predetermined number of video frame periods and vertical synchronization signal periods, as described above, and one video frame While stopping the pulse output of the period movement control signal CNT, the sequence including the stage that outputs the reset signal RST to the actuator 8 is repeated.
[0032]
The actuator 8 integrates the pulse of the movement control signal CNT input from the Z-axis scanning control device 9 and increases the vertical synchronization signal period as shown in FIGS. E and f, and the image frame imaging period is maintained constant. A signal is generated, and the objective lens 7 is moved in the Z direction by this drive signal. Here, the amount of movement of the objective lens 7 is proportional to the magnitude of the drive signal.
[0033]
When the reset signal RST is input from the Z-axis scanning control device 9, the actuator 8 returns the drive signal to the initial value.
With the above operation, the relationship between the scanning point in the sample 20 and each video frame is as shown in FIG. That is, in each video frame period (T1 to T8 in the figure), an image on the XY plane having a specific Z coordinate that is separated for each video frame in each video frame period is captured.
According to the scanning sequence as described above, the objective lens 7 is moved in the Z direction only during the straight period of the video frame. Therefore, a video frame obtained by photographing the XY plane of the sample 20 with the Z coordinate fixed can be generated for each of different Z coordinates.
[0034]
【The invention's effect】
As described above, according to the present invention, in the confocal microscope apparatus, it is possible to further improve the accuracy of position control for scanning the sample in the optical axis direction. In the confocal microscope apparatus, it is possible to increase the probability that a minute structure can be captured in the captured image. Further, in the confocal microscope apparatus, a video frame in which a plane perpendicular to the optical axis of the sample is captured with the optical axis direction coordinates fixed can be generated for each of the different optical axis direction coordinates.
[Brief description of the drawings]
FIG. 1 is a diagram schematically showing a configuration of a confocal microscope apparatus according to an embodiment of the present invention.
FIG. 2 is a diagram showing a scanning sequence according to the embodiment of the present invention.
FIG. 3 is a block diagram illustrating a configuration example of a Z-axis scanning control device and an actuator according to the embodiment of the present invention.
FIG. 4 is a diagram showing another scanning sequence according to the embodiment of the present invention.
FIG. 5 is a diagram showing a configuration and a scanning sequence of a conventional confocal microscope apparatus.
[Explanation of symbols]
1: main body, 2: stage, 3: laser light source, 4: confocal scanner unit, 5: camera, 6: image intensifier, 7: objective lens, 8: actuator, 9: Z-axis scanning control device, 10 : Image processing device, 11: Display device, 12: Illumination light source, 20: Sample, 81: Counter, 82: D / A converter, 83: Driver circuit, 91: Sequence control unit, 92: First counter, 93: First 1 decoder, 94: reset output circuit, 95: mask circuit, 96: second counter, 97: oscillator, 98: second decoder.

Claims (2)

A confocal scanner that scans the focal position of the light beam in a direction perpendicular to the optical axis with respect to the sample;
A moving mechanism for scanning the focal position of the light beam with respect to the sample in the optical axis direction;
A camera that captures an image of the sample by the light beam;
A movement control unit that moves the focal position of the light beam by a predetermined distance in the direction of the optical axis to the moving mechanism for each vertical synchronizing signal period in synchronization with the vertical synchronizing signal of the camera;
The movement control unit moves the focal position of the light beam in the optical axis direction to the moving mechanism only during an image capturing period of the camera with the light beam, and captures an image with the light beam of the camera. The confocal microscope apparatus is characterized in that the moving mechanism does not move the focal position of the light beam in the optical axis direction during a period excluding . The confocal microscope apparatus according to claim 1 ,
In parallel with the imaging of the camera, the image of the sample is reconstructed from the image captured by the camera and displayed, and the image newly captured by the camera is displayed by the 3D representation. A confocal microscope apparatus having display means for updating

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