JP4962769B2 - 3D microscope system - Google Patents

Description

  The present invention relates to a three-dimensional microscope system capable of obtaining an accurate three-dimensional image, and more particularly to a three-dimensional microscope system that is stable with respect to ambient temperature.

  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, and LSI surface observation in the semiconductor market.

FIG. 5 is an explanatory diagram showing a state around the objective lens when a three-dimensional image is taken with a conventional confocal microscope. An observation sample (hereinafter referred to as a sample) 1 made of cells or the like is on a culture vessel bottom plate 2. The light 3 emitted from a laser diode (not shown) is irradiated onto the sample 1 via the objective lens 4 via the optical system. The light reflected by the sample 1 enters the microscope (not shown) through the objective lens 4 to obtain a confocal image of the sample 1. 2A and 2B are the front surface and the back surface (bottom surface) of the culture vessel bottom plate 2, respectively. By driving the objective lens 4 with the actuator 5, the focal position moves along the optical axis direction.

At the time of photographing a three-dimensional image, the actuator 5 is driven so that the distance Z from the back surface 2B of the culture vessel to the focal position of the objective lens 4 changes in proportion to time as shown in FIG. At this time, it is possible to acquire a continuous image in which the distance Z is shifted by a certain interval by photographing at a certain time interval.

  Prior art documents related to the above three-dimensional confocal microscope include the following.

JP 2005-148454 A

  However, the microscope inherently has thermal expansion and contraction, and a temperature change of about 5 ° C. causes the focal point, that is, the distance Z to be shifted by about 10 μm. Under these conditions, if a three-dimensional image is acquired at a fixed interval, the distance Z varies as shown in FIG. 7, so that an accurate three-dimensional image cannot be taken in the optical axis direction. There is.

The present invention is intended to solve such a problem. When acquiring a continuous three-dimensional image, it is possible to eliminate a gap between image acquisition intervals and to capture an accurate three-dimensional image in the optical axis direction. An object of the present invention is to provide a three-dimensional microscope system that can be used.

In order to achieve such a problem, the invention according to claim 1 of the present invention is:
In the three-dimensional microscope system in which the focal position of the objective lens is moved in the optical axis direction by the actuator, and the actuator is scanned in the optical axis direction based on the scanning waveform signal generated by the waveform generator,
A focus error detection optical system that outputs a focus error signal proportional to the amount of deviation in the optical axis direction from the in-focus position of the objective lens within a focusing operation pull-in range;
Control means for feedback- controlling the actuator so that a focus error signal output from the focus error detection optical system follows the scanning waveform signal;
Wherein the focus position, a constant target value signal of the control means in the observation at the time of normal controls therefrom relative to the back surface of the container bottom plate of matches and sample at an arbitrary position within the pull-in range, when 3-dimensional image acquired as the scanning waveform signal target value signal of the control means, characterized that you scanned within the pull-in range.

The invention according to claim 2
The three-dimensional microscope system according to claim 1,
The control means includes
A control circuit for outputting a signal for maintaining a focus error signal input from the focus error detection optical system at a target value;
Switching means for selecting one of a signal output from the control circuit and a scanning waveform signal output from the waveform generator;
A sample hold circuit for holding a position signal output from the actuator;
And an addition circuit for driving the actuator with a signal obtained by adding the output signal of the sample and hold circuit and the scanning waveform signal via the switching means.

The invention described in claim 3
The three-dimensional microscope system according to claim 1 or 2,
A confocal scanner that acquires a slice image of the sample as a confocal image;
And an image processing device that synthesizes the slice images based on a position signal output from the actuator to create a three-dimensional image.

The invention according to claim 4
The three-dimensional microscope system according to any one of claims 1 to 3,
The scanning waveform is a triangular wave.

The invention according to claim 5
The three-dimensional microscope system according to any one of claims 1 to 3,
The scanning waveform is a step wave.

  As is apparent from the above description, according to the present invention, the actuator is controlled so that the focus error signal follows the scanning waveform signal, so that the focus position of the objective lens can be accurately determined based on the focus position. Since the distance can be set, a three-dimensional image can be taken at an accurate interval in the optical axis direction.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a configuration block diagram showing an example of a three-dimensional microscope system according to an embodiment of the present invention. The same parts as those in FIG. 5 are given the same symbols.

In the three-dimensional microscope system, the laser light from the laser light source 21 is irradiated to the sample 1 (see FIG. 5) via the confocal scanner 22, the fluorescence microscope 10, the focus error detection optical system 6, and the objective lens 4, and the reflected light is The light enters the confocal scanner 22 through the objective lens 4, the focus error detection optical system 6, and the fluorescence microscope 10. The confocal scanner 22 obtains a confocal image of the sample 1 and enters the high-speed camera 23. The high speed camera 23 converts the confocal image into an image signal and inputs the image signal to the image processing unit 26. The confocal scanner 22 has a nipou disk having a large number of pinholes and a microlens array corresponding to the nipou disk, and is a compact add-on type employing a nipou disk system composed of a simple optical system.

The actuator 5 is attached between a mirror unit (not shown) of the fluorescence microscope 10 and the objective lens 4, and the length of the focal direction (optical axis direction) of the image is proportional to the drive signal input level by piezo driving. The focus position of the objective lens 4 is controlled. The actuator 5 includes a position sensor (also referred to as a displacement sensor) that detects its own position.

The actuator driver 9 controls the position of the actuator 5 by applying a drive signal to the actuator 5 such that the position signal fed back from the actuator 5 is equal to the position setting signal input from the control circuit 8.

The focus error detection optical system 6 outputs a focus error signal proportional to the amount of deviation from the focus position of the objective lens 4 (the position where the objective lens 4 can be focused, such as the back surface of the culture vessel bottom plate 2). Output. As the focus error detection optical system 6, for example, the focus error detection optical system shown in Japanese Patent Application No. 2006-098091 and 20 in FIG. 1 can be used.

When the waveform generator 7 receives the scanning waveform generation trigger signal from the synchronization signal controller 24, the waveform generator 7 generates a scanning waveform previously sent from the waveform calculator 25 and stored as a target value scanning signal for acquiring a three-dimensional image. Send to control circuit 8. However, a constant target value signal is output to the control circuit 8 during normal times.

The control circuit 8 constitutes control means for generating a signal for causing the focus error signal to follow a servo target value (hereinafter referred to as a target value). A subtracting means 81 for subtracting a focus error signal input from the focus error detecting optical system 6 from a servo target value (hereinafter referred to as a target value) signal input from the waveform generator 7, and an amplifying means 82 for amplifying the output of the subtracting means 81. The output of the amplification means 82 is sent to the actuator driver 9 as a position setting signal.

The synchronization signal controller 24 sends a scanning waveform generation trigger signal to the waveform generator 7 and sends a synchronization signal to the confocal scanner 22 by the image capture start signal sent from the image processing unit 26, and is sent from the waveform generator 7. By receiving the vertical synchronization signal and sending the image capture trigger signal to the high-speed camera 23, the timing of acquiring the image signal by the image processing unit 26, the scanning start timing of the focal position of the objective lens 4, and the Nipou disc by the confocal scanner 22 All rotation synchronization controls (not shown) are synchronized.

Based on the position signal in the optical axis direction of the objective lens 4 sent from the actuator driver 9, the image processing unit 26 obtains an accurate position of the slice image in the optical axis direction and combines the slice images to obtain a highly accurate three-dimensional image. Is generated.

The high-speed camera 23 is controlled in operation by a control signal from the computer 27.

Here, a part constituted by the laser light source 21, the confocal scanner 22, the high-speed camera 23, the synchronization signal controller 24, the waveform calculation unit 25, and the image processing unit 26 is referred to as a confocal image acquisition unit 20. The waveform calculation unit 25 and the image processing unit 26 are configured in the computer 27.

The operation of the three-dimensional microscope system having such a configuration will be described below.
In normal times, automatic focusing to a fixed position is performed by analog feedback control of the control circuit 8 based on the focus error signal output from the focus error detection optical system 6, and the observation sample is continuously observed at the fixed position. At the time of acquiring a three-dimensional image, automatic focusing is performed at time-varying positions following the target value scanning waveform by analog feedback control, and a slice image of the observation sample is acquired at each position.

FIG. 2 is an operation explanatory diagram for explaining the operation of automatic focusing. The vertical axis represents the signal intensity E of the focus error signal, and the horizontal axis represents the focus position Z. The arrows P and Q correspond to the front surface 2A and the back surface 2B of the culture vessel bottom plate 2 (FIG. 5), respectively, and the interval (PQ) between the arrows P and Q corresponds to the thickness of the culture vessel bottom plate 2. a and b are the focus pull-in widths on the front surface 2A and the back surface 2B, respectively (the pull-in range of the focusing operation: the range of the linear part that can be focused).

The operation in normal times will be described below.
First, the target value signal output from the waveform generator 7 is set to 0, and feedback control is performed by the control circuit 8 so that the focus error signal becomes 0 at the position of the arrow Q in FIG. The actuator 5 is driven. As a result, the focal position of the objective lens 4 coincides with the back surface 2B of the culture vessel bottom plate 2 (FIG. 5). In this case, for example, the objective lens 4 may be moved closer to the culture vessel bottom plate 2 and focused on the back surface 2B after focusing on the front surface 2A of the culture vessel bottom plate 2. According to this method, after being controlled to the position of the arrow P in FIG. 2, it is controlled to the position of the arrow Q by the second focusing.

Next, when the waveform generator 7 makes the value of the target value signal of the control circuit 8 equal to the focus error signal value V corresponding to the distance Z in FIG. 5 (see FIG. 2), the feedback control of the control circuit 8 described above causes The focal position of the objective lens 4 moves from the back surface 2B of the culture vessel bottom plate 2 to a position inside the vessel by a distance Z, so that the sample 2 can be observed.

The operation at the time of acquiring a three-dimensional image will be described below with reference to the time chart of each signal shown in FIG.

When the waveform generator 7 receives the scanning waveform generation trigger signal from the synchronization signal controller 24 (FIG. 3 (3)), the waveform generator 7 generates a scanning waveform previously sent from the waveform calculator 25 and stored therein, and is scanned. The target value signal, that is, the target value scanning signal (FIG. 3 (4)) is sent to the control circuit 8. This target value scanning signal is obtained by converting the driving waveform (in the case of a staircase wave) of the actuator 5 in the prior art of FIG. 6 into a corresponding focus error signal scale. In FIG. 3 (4), the stepped waveform that gradually increases with time and returns to the base voltage at a predetermined voltage value is used as the target value scanning signal. This scanning waveform is a triangular wave. May be. The control circuit 8 performs feedback control so that the focus error signal received from the focus error detection optical system 6 follows this target value scanning signal, and drives the actuator 5 via the actuator driver 9 as shown in FIG. Thus, the objective lens 4 is scanned in the optical axis direction. The high-speed camera 23 sends the image of the sample 1 to the image processing unit 26 at the timing synchronized with the above scanning waveform by the image capture trigger signal (FIG. 3 (2)) sent from the synchronization signal controller 24. The image processing unit 26 obtains the actual position of the slice image of the observation sample 1 based on the position signal of each step of the staircase wave output from the position sensor of the actuator 5 and sent via the actuator 9, and obtains the three-dimensional image. Synthesize. For example, if the value V of the target value signal is made to correspond to the center of the cell nucleus that is the observation sample and scanned up and down, a three-dimensional image of the entire inside of the cell can be obtained.

The various signals output from the synchronization signal controller 24 are synchronized with the vertical synchronization signal (FIG. 3 (1)) sent from the waveform generator 7.

According to the three-dimensional microscope system configured as described above, the focal position of the objective lens 4 is set at an arbitrary position within the range of the focus pull-in width from the back surface 2B of the culture vessel bottom plate 2 that is the in-focus position. Therefore, even when thermal expansion or contraction occurs in the microscope, the image acquisition interval does not shift in the optical axis direction. Therefore, a three-dimensional image of the observation sample can be taken at an accurate interval in the optical axis direction. This is a very useful function in cell imaging in order to specify the exact location of intracellular proteins and to observe changes in cell shape.

In the above embodiment, the position of the actuator is cascade-controlled. However, if the relationship between the position of the actuator and the drive signal can be accurately grasped, the feedback loop in the actuator driver 9 is omitted and the loop is configured. Can be simplified.
As the high-speed camera 23, for example, a video rate camera may be used.

FIG. 4 is a modification of a part of the above three-dimensional microscope system, and is a partial configuration block diagram showing a means for acquiring a three-dimensional image at an arbitrary time point while performing automatic focusing on an observation sample for a long time. It is. The same parts as those in FIG. 1 are denoted by the same symbols, and redundant description is omitted.

In the controller 80, the control circuit 83 outputs a signal for keeping the focus error signal input from the focus error detection optical system 6 at the target value V constant. The changeover switch 83 constitutes a changeover means for selecting either the target value scanning signal given from the waveform generator 7 or the output signal of the control circuit 83. The sample hold circuit 85 samples and holds the position signal sent from the actuator 5. The adder circuit 86 outputs a signal obtained by adding the signal selected by the changeover switch 84 and the output signal of the sample hold circuit 85 as a drive signal for the actuator 5. Here, it is assumed that the actuator 5 can perform voltage control via the drive waveform input terminal and can monitor the expansion and contraction as a voltage signal by the position signal output from the monitor output terminal.

The operation of the three-dimensional microscope system configured as shown in FIG. 4 will be described below.

First, the operation when switching from the state of automatic focusing to acquisition of a three-dimensional image is shown below. In the automatic focusing state, the contact of the changeover switch 84 is at the position 2, and the output of the control circuit 83 is applied to the actuator 5 via the addition circuit 86, and analog feedback control by the control circuit 83 is performed. As a result, the output focus error signal output from the focus error detection optical system 6 becomes equal to the target value V, and the output of the control circuit becomes zero.

When switching to 3D image acquisition, the position signal output from the actuator 5 is sampled and held in the sample hold circuit 85 by the sampling pulse S in the above state. Next, after the contact of the changeover switch 83 is switched to 1, a sample hold value is output from the sample hold circuit 85, and a position signal corresponding to the target value V becomes a drive input of the actuator 5. In this state, when a scanning waveform for obtaining a three-dimensional image is output from the waveform generator 7, the focal position of the objective lens 4 is scanned based on the initial position Z corresponding to the target value V by open loop control. Scan following the above. By capturing an image in the same manner as in the embodiment of FIG. 1, it is possible to capture a three-dimensional image based on the in-focus position (Z = 0).

Switching from the state in which the three-dimensional image is being acquired to automatic focusing is performed by switching the contact position of the changeover switch 84 to 2 after returning to the initial position after performing the scanning. Here, when performing automatic focusing, control is not performed unless the initial position of the objective lens immediately before the start of feedback control is within the linear portion of the ZE graph shown in FIG. However, since the above three-dimensional image acquisition time is sufficiently short with respect to the time when the microscope undergoes thermal expansion due to a change in ambient temperature, the output value of the sample hold circuit 85 at the time of switching is an automatic value before the three-dimensional image acquisition. It is almost the same as when focusing.

According to the three-dimensional microscope system configured as described above, as in the case of FIG. 1, a three-dimensional image having accurate Z-axis information is acquired while maintaining a reference point for scanning when acquiring a three-dimensional image. be able to.

Moreover, the software of the waveform generator 7 can be simplified by realizing switching between automatic focusing and three-dimensional image acquisition by hardware.

In each of the above embodiments, automatic focusing is performed by analog feedback control, but may be performed by computer software.

Further, it can be applied to a three-dimensional microscope system other than the confocal microscope.

1 is a configuration block diagram showing an embodiment of a three-dimensional microscope system according to the present invention. It is operation | movement explanatory drawing for demonstrating the operation | movement of the automatic focusing of the apparatus of FIG. It is a time chart for demonstrating the operation | movement at the time of the three-dimensional image acquisition of the apparatus of FIG. It is a block diagram which shows the modification of the three-dimensional microscope system which concerns on the Example of FIG. It is explanatory drawing which shows the mode of the objective lens periphery in the case of taking a three-dimensional image with the conventional confocal microscope. It is explanatory drawing for demonstrating the operation | movement at the time of the three-dimensional image photography of the apparatus of FIG. It is a time chart for showing a focal position in the case of taking a three-dimensional image with a conventional confocal microscope.

Explanation of symbols

4 Objective lens 5 Actuator 6 Focus error detection optical system 7 Waveform generator 8 Control means 22 Confocal scanner 83 Control circuit 84 Switching means 85 Sample hold circuit 86 Addition circuit

Claims (5)

In the three-dimensional microscope system in which the focal position of the objective lens is moved in the optical axis direction by the actuator, and the actuator is scanned in the optical axis direction based on the scanning waveform signal generated by the waveform generator,
A focus error detection optical system that outputs a focus error signal proportional to the amount of deviation in the optical axis direction from the in-focus position of the objective lens within a focusing operation pull-in range;
Control means for feedback- controlling the actuator so that a focus error signal output from the focus error detection optical system follows the scanning waveform signal;
Wherein the focus position, a constant target value signal of the control means in the observation at the time of normal controls therefrom relative to the back surface of the container bottom plate of matches and sample at an arbitrary position within the pull-in range, as time three-dimensional image acquisition the scanning waveform signal target value signal of the control means, 3D microscope system characterized that you scanned within the pull-in range. The control means includes
A control circuit for outputting a signal for maintaining a focus error signal input from the focus error detection optical system at a target value;
Switching means for selecting one of a signal output from the control circuit and a scanning waveform signal output from the waveform generator;
A sample hold circuit for holding a position signal output from the actuator;
2. The three-dimensional microscope system according to claim 1, further comprising an adding circuit that drives the actuator with a signal obtained by adding the output signal of the sample hold circuit and the scanning waveform signal via the switching means. A confocal scanner that acquires a slice image of the sample as a confocal image;
The three-dimensional microscope system according to claim 1, further comprising: an image processing device that synthesizes the slice images based on a position signal output from the actuator to create a three-dimensional image.   4. The three-dimensional microscope system according to claim 1, wherein the scanning waveform is a triangular wave.   4. The three-dimensional microscope system according to claim 1, wherein the scanning waveform is a staircase wave.

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