JP2003029151A - Confocal laser scanning microscope and control program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a confocal laser scanning microscope, which shortens the time for observation by rapidly acquiring the expected image of an observation sample and can prevent the wasteful color fading of the observation image by a laser beam, and to provide a control program for controlling the same. SOLUTION: The selective use of a system of thinning out the scanning in Y-axis direction and a system of not thinning out the same in the scanning by a laser beam in the X-axis and Y-axis directions on the observation sample 108 is made possible, and the diameter of a pinhole 110, arranged in a confocal position, is automatically adjusted according to the respective systems. Furthermore, control is performed by a control section 2, in such a manner that the light intensity of the laser beam can be adjusted according to the change in the diameter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a confocal laser scanning microscope apparatus and a control program.

[0002]

2. Description of the Related Art Conventionally, a scanning microscope apparatus as disclosed in JP-A-61-2199919 is known. This device narrows the laser light emitted from the light source to a minute spot in the objective lens and
Scan in the Y-axis direction. Light from the observation sample due to the irradiated spot, such as transmitted light, reflected light, or fluorescence from the observation sample due to excitation, is captured by a photodetector such as Photomal and converted into an electrical signal, and the electrical signal is monitored. To be displayed as an observation image.

In particular, in the optical system between the light from the observation sample due to the illuminated spot and the detector that captures this light, this light, for example, reflected light or fluorescence from the observation sample due to excitation, is returned to the incident optical path and dichroic. A method is known in which what is reflected by a mirror is condensed by a lens on a confocal surface and a pinfall is placed at this position to form a confocal optical system. It is called a laser scanning microscope device. By forming a confocal optical system in this way, it is possible to expect a confocal effect that blocks the extra light that is sent from around the in-focus position, resulting in a clearer image for thick observation samples. Can be obtained.

[0004]

However, the following two problems occur in determining the observation target area in the confocal laser scanning microscope and the confocal laser scanning microscope apparatus as described above.

The first problem is that since the scanning speed of the spot in the X-axis and Y-axis directions is about several hundred kilohertz to several thousand kilohertz, only a few to several tens of frames of image information can be acquired per second. This means that it is difficult to obtain the expected image for the observation sample in a short time.

The second problem is that the pinfall of this type of microscope is due to the method of staining the observation sample, the fluorescence wavelength from the observation sample, the magnification of the objective lens installed in the optical system,
And a diameter that is uniquely determined by the numerical aperture and is installed at a position (confocal plane) that is optically conjugate with the in-focus position, so that light from other than the in-focus position in the observation sample is excluded. Since it is designed to acquire observation images in a region with a narrow depth of focus,
It is difficult to capture a rough observation area with this confocal optical system.

Therefore, in order to avoid these two problems, first, the observation target area is roughly grasped by visual microscope observation without using laser light, and then the laser light is scanned with a spot on the observation target area. Generally, a method of adjusting the sensitivity of a photodetector or adjusting the position of a stage on which an observation sample is set while observing an observation image displayed on a monitor to acquire an expected image has been generally performed.

However, in contrast to the first scanning method for acquiring an observation image by scanning the laser beam as described above with the spot in the X-axis and Y-axis directions of the observation sample,
While keeping the speed of the spot to be scanned in the axial direction at the highest speed,
There is a second scanning method in which scanning lines in the Y-axis direction are decimated by 2, 4, or more scanning numbers, and a pseudo observation image is acquired only from the information of the scanned lines. By doing so, it is possible to shorten the acquisition time of the observation image by thinning out the scanning lines, which solves the first problem.

There is also known a scanning microscope apparatus having two optical systems, a confocal optical system in which a pinfall is arranged and an optical system in which it is not arranged. This apparatus is used for two optical systems, respectively. The above-mentioned photodetector is required, so that there is a drawback that the structure becomes large-scale.

According to the present invention, while adopting the second scanning method, the second problem is solved by adjusting the confocal effect, and at the same time, the change of the laser intensity accompanying the adjustment of the confocal effect is controlled. An object of the present invention is to provide a confocal laser scanning microscope apparatus capable of preventing unnecessary discoloration of a tissue observation sample by laser light and a program for controlling the same.

[0011]

The present invention is premised on being applied to a confocal laser scanning microscope. Then, the confocal laser scanning microscope apparatus which is one of the aspects of the present invention, the observation sample on the stage is made to scan the spot where the laser light is narrowed down, and the light from the observation sample by the spot is condensed. A confocal laser scanning microscope apparatus for arranging a pinfall at the condensing position and acquiring an observation image of the observation sample based on light passing through the pinfall, wherein the observation sample is scanned in a fixed direction. The first scanning method in which the X-axis scanning line is repeatedly scanned in the Y-axis direction which is parallel to the observation sample and orthogonal to the scanning line, and the X-axis scanning line is set to the Y-axis with the maximum scanning speed in the scanning method. A means for acquiring two observation images of the second scanning method which is thinned out in the axial direction, and the acquisition means is caused to selectively execute one of the first and second scanning methods, and the selected scanning method is executed. And control means for adjusting the diameter of the pinhole in accordance with the method, configured to have.

According to the above arrangement, the diameter of the pinfall can be changed, so that the diameter can be made larger than the size at which the confocal effect can be obtained in the second scanning method, and the observation target area is determined. An observation image with an easy thickness can be obtained. In the control means,
When the observation sample is configured to have a function of adjusting the laser light intensity according to the diameter of the pinfall, the observation sample can be irradiated with the laser light at an appropriate light intensity.

According to another aspect of the present invention, there is provided a control program for a microscope, wherein an observation sample on a stage is caused to scan a spot in which a laser beam is narrowed down, and the light from the observation sample is condensed by the spot. And is used for an apparatus for controlling a confocal laser scanning microscope which obtains an observation image of the observation sample on the basis of light passing through the pinfall, the pinfall being arranged at a position where the light is condensed. A first X-axis scanning line of the confocal laser scanning microscope which is scanned in a certain direction of the observation sample is repeatedly scanned in a Y-axis direction parallel to the observation sample and orthogonal to the scanning line. Either the scanning method or the second scanning method in which the scanning speed in the scanning method is maximized and the X-axis scanning lines are thinned out in the Y-axis direction is selectively executed to select the scanning method.査方 in which to perform the control of the diameter of the pinhole to the control device in accordance with the equation.

A control program for adjusting the laser light intensity according to the diameter of the pin fall may be used. Further, the control program may be such that the information of the observation sample is acquired by executing the second scanning method and the focus is adjusted to the focus position determined by the observer based on the information.

According to the above-mentioned control program, the observer can collectively obtain the means for quickly obtaining the expected image in the environment in which this control program operates. Further, in this environment, the observer can select the in-focus position of the observation material, and can further adjust the intensity of the laser light with which the observation sample is irradiated.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (First Embodiment) FIG. 1 schematically shows the structure of a confocal laser scanning microscope apparatus to which the present invention is applied. The numbers given to the respective parts of the structure are used. The configuration of this device will be described below.

In FIG. 1, reference numeral 1 denotes a confocal laser scanning microscope main body, and this confocal laser scanning microscope main body 1
A control unit 2 such as a PC is connected to the. Further, the control unit 2 is connected with a keyboard, an operation panel 3 including a pointing device such as a trackball, a joystick or a mouse, and a memory 4, and an image monitor 5 is connected to the memory 4.

Further, the confocal laser scanning microscope body 1
Regarding the configuration (hereinafter referred to as the main body 1), the laser light source 101 is built in the back of the main body 1,
1, a mirror 102 is arranged above the main body 1, a dichroic mirror 103 is arranged in front of the mirror 102, and a lens 10 is arranged below the dichroic mirror 103.
9 is arranged, and further below is a confocal aperture 110 which is a pin fall.
Below that is the photodetector 111. A scanning unit 104 is located in front of the dichroic mirror 103 and in front of the main body 1. This scanning unit 104
Has an unillustrated galvanometer mirror for X-axis scanning and an unillustrated galvanometer mirror for Y-axis scanning. A revolver 105 is located below the scanning unit 104.
An objective lens 106 is attached to the. A stage position detector 112 is mounted in front of the lower part of the main body 1, a stage 107 is mounted thereon, and an observation sample 108 is placed on the stage 107.

Further, in the control unit 2, a change amount setting unit 2a and a calculation / processing unit 2b are housed inside.
Next, the function of each part of this apparatus will be described. In the main body 1, the laser light emitted from the laser light source 101 is reflected by the mirror 102, advances toward the dichroic mirror 103, passes through the dichroic mirror 103, and enters the scanning unit 104. The incident laser light is scanned in the X-axis and Y-axis directions by a control signal sent from the control unit 2. Also, every time one line of scanning in the X-axis direction is completed, a control signal indicating the end of scanning is sent to the control unit 2. The laser light scanned in the X-axis and Y-axis directions is condensed by the objective lens 106 and is irradiated onto the observation sample 108 as spot light. Light from the observation sample 108 due to this spot light, for example, reflected light or fluorescence from the observation sample due to excitation is returned to the incident optical path, reflected by the dichroic mirror 103, and condensed via the lens 109.
A confocal aperture 110 is arranged at this condensing position, and the light passing through the confocal aperture 110 is captured by a photodetector 111 such as a photomul. An electric signal corresponding to the amount of the captured light is output to the control unit 2.

The stage position detector 112 detects the position information (X, Y, Z) of the stage 107 and outputs it to the controller 2. Further, a command to start scanning laser light, a command to input an image, a command to adjust the sensitivity to the photodetector 111, and the like are output from the operation panel 3 to the control unit 2 according to an instruction from an observer.

The memory 4 is transferred from the photodetector 111 via the control unit 2 and the position information storage unit for storing the position of the final line of the observation sample 108 and the line position after the movement of the stage 107. And an image storage unit for storing image information. The control unit 2 controls the entire apparatus, and outputs a signal for controlling scanning to the scanning unit 104 in response to an instruction from the operation panel 3 to instruct scanning, and sends the signal from the photodetector 111. The electric signal of the incoming observation image is transferred to the memory 4, and the image and the operation menu are displayed on the image monitor 5. Further, the applied voltage, the gain, the offset, etc. in the photodetector are set in accordance with the sensitivity adjustment command input from the operation panel 3.

Next, the operation of acquiring the expected image in the first embodiment will be described. First, the observer inputs a command to the operation panel 3 to scan the observation sample by the above-mentioned second scanning method. The control unit 2 receives this command and starts the process of the flowchart shown in FIG. The operation outputs a command to automatically open and maximize the diameter of the confocal aperture 110 (S20).
0), it is determined whether or not the diameter of the confocal aperture 110 which operates in response to the instruction has actually become maximum (S201). When it is determined that the diameter is adjusted to the maximum, a command to start scanning on the observation sample is output (S202), and laser light is emitted from the laser light source 101. In addition, corresponding to this maximum diameter,
It is determined whether the intensity of the emitted laser light is adjusted to the optimum value (S203).

Regarding the adjustment of the diameter, the value of the diameter at which the confocal effect is obtained is the confocal aperture 11.
It is a value smaller than the maximum value of the diameter of 0, and it is desirable to set it within the maximum and minimum range of this confocal aperture 110. Further, it is more preferable to set the value within the range of the maximum value of the diameter of the confocal aperture 110 from the value of the diameter at which the confocal effect is obtained.

The intensity of the laser light described here is basically proportional to the diameter of the confocal aperture.
When the observation target is a fluorescence image, it is necessary to take into consideration the efficiency of being excited by the fluorescent substance. Whether or not the intensity of the laser beam is adjusted to the optimum value is repeatedly monitored until the scanning of the observation sample is completed, and it is determined whether or not the scanning of the observation sample is completed (S20
4) When it is determined that the process is completed, whether or not the optical path for visually observing reflected light (not shown in FIG. 1), that is, the BI optical path is set without using the laser light next time is set. Is determined (S205). When set to this BI optical path, the magnification of the objective lens 106 installed in this optical path, the staining method of the observation sample 108, and the observation sample 1
The optimum value of the diameter of the confocal aperture 110 determined by the fluorescence wavelength from 08 and the numerical aperture is calculated by the following calculation formula I or a calculation formula equivalent thereto, and the diameter is automatically adjusted to the resulting value. A command is issued (S206).

[0025]

[Equation 1]

Subsequently, it is judged whether or not the diameter of the confocal aperture 110 has been adjusted to the value calculated in S206 (S207), and when the adjustment is completed, visual observation can be performed here. Then, the position (current stage position) information of the stage 107 at this point and the preset inspection range, inspection step amount, and number of inspection steps are acquired (S208). Then, an instruction to move the stage to the start position of this inspection range is output (S209), and it is determined whether the stage 107 has moved to this start position (S210). Upon moving to the start position, an inspection start command in the inspection range is output, and the brightness value at each step position according to the step amount acquired in S208 is acquired (S211). The end of the inspection is
It is judged whether or not the stage 107 has reached the inspection end position (S212), and when it reaches, a graph showing the relationship between the Z-axis inspection range and the luminance value at each step position shown in FIG. An IZ graph) is created from the brightness value at each step position acquired in S211 and displayed on the operation panel 3 (S213). The observer determines the expected focus position of the Z axis (hereinafter referred to as the expected focus position) based on this IZ graph, and inputs the expected focus position to the operation panel 3. The control unit 2 determines whether or not this input has been made (S214), and if it has been input, an instruction is issued to move the stage 107 to the expected focus position (S21).
5), it is determined whether or not the movement of the stage 107 is completed (S216). When the movement is completed, the preset threshold value range of the brightness value is acquired (S21).
7). Then, the upper limit of the brightness value at the expected focus position,
When the upper limit of the brightness value at the expected focus position is lower than the lower limit of the threshold range of the brightness value, the gain is adjusted to the optimum brightness by comparing with the lower limit of the threshold range of the brightness value set in advance (S218). The value is automatically adjusted (S219), and this processing ends. If it is determined in S218 that the upper limit of the brightness value at the expected focus position is not below the lower limit of the threshold range of the brightness value, the upper limit of the brightness value at the expected focus position and S217
It is compared with the upper limit of the threshold value range of the brightness value acquired in (S2
20). If the upper limit of the brightness value at the expected focus position exceeds the upper limit of the preset threshold range of the brightness value, the gain is automatically adjusted to the optimum brightness value (S221), and this processing ends. . In this state, if the operation panel 3 outputs a command to the control unit 2 to start scanning with the spot of the laser light on the observation sample 108, it becomes possible to perform the scanning at the in-focus position expected by the observer and obtain the expected image. To be (Second Embodiment) The schematic configuration of the confocal laser scanning microscope in the present embodiment uses the configuration of the first embodiment, and the function thereof is the same as that of the first embodiment. To do.

The operation up to acquiring the expected image in this embodiment under these conditions will be described. First, the observer inputs a command to the operation panel 3 to scan the observation sample by the above-described second scanning method. The control unit 2 receives this command and starts the process of the flowchart shown in FIG. In the flowchart shown in FIG. 4, the processing until it is determined in S212 that the detection of the brightness value in each step is ended based on the information obtained in S208 is the same as the content of FIG. 2 described above. S30 shown after S212
The operation from 0 has contents different from those in FIG. 2, and the contents are the IZ graph shown in FIG. 5 created based on the luminance information acquired in the processes of S208 to S212. A command to start the peak detection process used is output (S300). In FIG. 5, the operator detection position is the Z position detected by the observer, which corresponds to the stage position acquired in S208. Further, the peak here does not indicate only the maximum luminance value in the inspection range. The detection condition expected by the user including the secondary peak can be set in advance, and the peak is detected from the I-Z graph based on the condition. Then, it is judged whether or not a peak matching the detection condition of the observer can be detected (S301), and if it cannot be detected, the observer is notified by a dialog display that the peak detection has failed (S302). ), I
-Request the observer to input the expected focus position by the Z graph. When it is determined in S301 that a peak matching the detection condition of the observer has been detected, or in S303, the operation panel 3
If the input of the expected focus position is confirmed, the command to move the stage to the designated expected focus position is output (S215). Subsequent operations in S216 and thereafter perform the same processing as the operation content in FIG. 2 described above.

In this state, if a command to start scanning the observation sample 108 is output from the operation panel 3 to the control unit 2, it becomes possible to perform scanning at the in-focus position expected by the observer.
The expected image is obtained. (Supplementary Note 1) The observation sample on the stage is scanned with a spot in which the laser light is narrowed down, and the light from the observation sample is condensed by the spot, and a pin fall is arranged at a position where the light is condensed. A control method used in a confocal laser scanning microscope for acquiring an observation image of the observation sample based on light passing through, wherein the observation sample is scanned in a certain direction with respect to the confocal laser scanning microscope. The first scanning method in which the X-axis scanning line is repeatedly scanned in parallel to the observation sample in the Y-axis direction orthogonal to the scanning line, and the X-axis scanning line is set at the maximum scanning speed in the scanning method. A control method characterized by selectively executing one of the second scanning methods of thinning out in the Y-axis direction and controlling the diameter of the pinfall according to the selected scanning method. . (Additional remark 2) Further, the control method according to additional remark 1, wherein the laser light intensity is adjusted according to the size of the diameter of the pin fall. (Additional remark 3) Further, the information of the observation sample is obtained by executing the second scanning method, and the focus is adjusted to the in-focus position determined by the observer based on the information. The described control method.

[0029]

As described above, according to the present invention, the observation target area can be quickly captured, the acquired observation image is optimized, and the discoloration of the observation sample due to unnecessary irradiation with the laser light is also suppressed. Can be prevented.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram in first and second embodiments.

FIG. 2 is a flowchart in the first embodiment.

FIG. 3 is an IZ graph created in the first embodiment.

FIG. 4 is a flowchart in the second embodiment.

FIG. 5 is an IZ graph created in the second embodiment.

[Explanation of symbols]

1 Confocal laser scanning microscope body 2 control unit 2a Change amount setting section 2b Calculation and processing unit 3 Operation panel 4 memory 5 image monitor 101 laser light source 102 mirror 103 dichroic mirror 104 scanning unit 105 Revolver 106 Objective lens 107 stages 108 observation sample 109 lens 110 confocal aperture 111 Photodetector 112 Stage position detector

Claims (5)

[Claims] 1. A spot on which a laser beam is narrowed is scanned on an observation sample on a stage, light from the observation sample by the spot is condensed, and a pin fall is arranged at the condensed position. A confocal laser scanning microscope apparatus for acquiring an observation image of the observation sample based on light passing through, wherein an X-axis scanning line scanned in a fixed direction of the observation sample is parallel to the observation sample. A scan line is repeatedly scanned in the Y-axis direction orthogonal to the scan line, and the X-axis scan line is set to Y with the maximum scan speed in the scan system.
Acquisition means for two observation images of the second scanning method thinned out in the axial direction, and the acquisition means to selectively execute either of the first and second scanning methods, and the selected scanning method A confocal laser scanning microscope apparatus, comprising: a control unit that adjusts the diameter of the pinfall according to the above. 2. The confocal laser scanning microscope apparatus according to claim 1, wherein the control means has a function of adjusting the laser light intensity according to the size of the diameter of the pin fall. 3. An observation sample on a stage is made to scan a spot in which a laser beam is narrowed down, the light from the observation sample is condensed by the spot, and a pin fall is arranged at a position where the light is condensed. It is used for a control device of a confocal laser scanning microscope that acquires an observation image of the observation sample based on light passing through a fall, and the constant of the observation sample with respect to the confocal laser scanning microscope. A first scanning method in which an X-axis scanning line scanned in a direction is repeatedly scanned in a Y-axis direction parallel to the observation sample and orthogonal to the scanning line; and the X-axis with the maximum scanning speed in the scanning method. It is possible to selectively execute one of the second scanning methods in which the scanning lines are thinned out in the Y-axis direction, and control the diameter of the pinfall according to the selected scanning method. A control program for causing the control unit. 4. The control program according to claim 3, further comprising the step of adjusting the laser light intensity according to the diameter of the pin fall. 5. The control device further comprises: acquiring information of an observation sample by executing the second scanning method and focusing on a focus position determined by an observer based on the information. The control program according to claim 3, which is executed.