JP2003295064A - Laser scanning microscope system, system control method, and control program for making computer execute system control - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a laser scanning microscope system which can simply perform observation with a plurality of waveforms, such as observation of a desired multiply dyed specimen, a system control method, and a control program for operating a computer to execute system control. <P>SOLUTION: The laser scanning microscope system scans a specimen S with a laser beam, detects the observing light obtained from the specimen S by photodetectors 117 and 118 and forms two-dimensional images in accordance with their output signals. A plurality of different scanning conditions are inputted to the photodetectors 117 and 118 by using a GUI screen and a plurality of the inputted scanning conditions are successively set one by one. The scanning of the laser scanning microscope system is then executed and the data from the photodetectors 117 and 118 obtained under the individual scanning conditions are formed as a plurality of the images independent from each other corresponding to the respective scanning conditions. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser scanning microscope system, a system control method, and a control program that causes a computer to execute system control. More specifically, the present invention relates to a plurality of specimens labeled with a plurality of fluorescent dyes or fluorescent proteins. The present invention relates to a device that is excited by using an excitation wavelength and detects a plurality of fluorescences corresponding to each excitation wavelength.

[0002]

2. Description of the Related Art Conventionally, a laser scanning microscope has been used as a device for exciting a sample dyed multiple times with different fluorescent dyes at a plurality of excitation wavelengths and simultaneously detecting a plurality of fluorescences generated corresponding to these excitation wavelengths. Has been.

In such a laser scanning microscope, a plurality of photodetectors are prepared, and these detectors are selectively used for each of a plurality of excitation wavelengths (channels) for exciting a multi-stained sample, and one detector = Image data of a plurality of channels can be acquired by associating one channel, that is, a detector and a channel with one-to-one correspondence.

For example, Japanese Unexamined Patent Publication No. 2000-35400 discloses a system configuration in which four photodetectors are provided, and four-channel image data can be acquired by these photodetectors.

On the other hand, recently, it has been proposed to detect the detailed spectral characteristics of a laser scanning microscope or a specimen in which the number of photodetectors is reduced with respect to the number of channels for each excitation wavelength aiming at cost reduction. In the intended laser scanning microscope, in order to detect multiple wavelengths more than the number of detectors, it is possible to use one that can detect multiple wavelengths by using one detector. It has become.

[0006]

However, in the laser scanning microscope configured as described above, the spectral filter (dichroic mirror) and the barrier filter arranged in front of the detector are appropriately set for each wavelength to be detected. It is necessary to switch so that, and because the fluorescence intensity obtained differs depending on the target to be detected, the sensitivity adjustment of the detector is also
It is necessary to make settings that match the respective fluorescence intensities.

For this reason, in the prior art, first, focusing on the wavelength detection of a part of the multi-stained specimen, setting the filter for the detection and adjusting the sensitivity of the detector to obtain an image, and then , A series of operations such as setting the filter for detecting the remaining wavelengths of the multiple stained specimen and adjusting the sensitivity of the detector to acquire an image, and finally combining the two images acquired in this way by post-processing. Is executed.

Further, in order to execute such a series of operations, macro programming is also provided so that the switching operation of each filter and the adjustment operation of the sensitivity of the detector can be executed on a program. By using such macro programming, the user can pre-program using macro commands in accordance with his or her experiment, and by executing it, multiple users can observe multiple stained specimens of interest. I try to observe the wavelength.

However, in order to separately acquire images in this way and then combine these plural images, it is troublesome and extremely complicated work is required. In addition, even by the method of executing those series of operations by macro programming, it is necessary to select a macro command corresponding to the function to be adjusted, describe each command, and have knowledge about programming.
It is not easy to prepare a series of operations required for observation by programming.

The present invention has been made in view of the above circumstances, and provides a laser scanning microscope system capable of easily observing a plurality of wavelengths, a system control method, and a control program for causing a computer to execute the system control. The purpose is to

[0011]

The invention according to claim 1 is
A laser scanning microscope that scans a sample with laser light to detect observation light obtained from the sample with a photodetector, and forms a two-dimensional image based on an output signal from the photodetector, is used for the same light. A means for displaying an input screen for inputting a plurality of different scanning conditions to the detector, and the plurality of scanning conditions input using this setting screen are sequentially set one by one, and scanning is performed each time. Control means for performing
Image forming means for forming the data from the same photodetector obtained under each scanning condition as a plurality of independent images corresponding to each scanning condition.

According to a second aspect of the present invention, in the first aspect of the invention, the scanning conditions include a setting condition of a detection operation in the photodetector, a wavelength of laser light to be irradiated and a wavelength of observation light to be detected. And a switching state of the optical element to be determined.

The invention according to claim 3 is the invention according to claim 1 or 2.
In the invention described above, a plurality of the photodetectors are provided.

According to a fourth aspect of the invention, in the invention according to any one of the first to third aspects, the scanning condition input from the input screen can be set more than the number of the photodetectors. I am trying.

According to a fifth aspect of the present invention, a sample is scanned with a laser beam, observation light obtained from the sample is detected by a photodetector, and a two-dimensional image is formed based on an output signal from the photodetector. A method for controlling a laser scanning microscope system to be formed, which displays an input screen for inputting a plurality of different scanning conditions for the same photodetector,
The plurality of scanning conditions input using this setting screen are sequentially set one by one, and scanning is performed each time, and the data from the same photodetector obtained under each scanning condition is used for each scanning condition. It is characterized in that it is formed as a plurality of independent images corresponding to.

According to a sixth aspect of the present invention, a sample is scanned with a laser beam, observation light obtained from the sample is detected by a photodetector, and a two-dimensional image is formed based on an output signal from the photodetector. A control program that causes a computer to execute control of a laser scanning microscope system to be formed, which displays an input screen for inputting a plurality of different scanning conditions to the same photodetector, and a setting screen for this. The scanning conditions input by using the above are sequentially set one by one, and scanning is performed each time, and the data from the same photodetector obtained under each scanning condition is corresponded to each scanning condition. It is characterized in that it is formed as a plurality of images independent of each other.

According to the present invention, by setting the conditions necessary for scanning for image acquisition on the input screen prepared in advance, a plurality of image acquisitions that are not limited by the actual number of photodetectors can be performed. The conditions can be set, and the desired multiple-stained specimen can be easily observed with a short preparation time.

Further, according to the present invention, a plurality of photodetectors are caused to perform scanning based on a plurality of setting conditions,
Separate channel data obtained by each scan can be acquired as one image.

Further, according to the present invention, it is possible to set an independent adjustment for each wavelength detection, and it is possible to make it look as if a detector is prepared for each wavelength to be detected.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 shows a system configuration of a laser scanning microscope according to a first embodiment of the present invention.

In this case, the laser scanning microscope system according to the first embodiment comprises a laser scanning microscope main body 100 and a computer system 200.

The laser scanning microscope main body 100 has a control unit 101, which outputs a scanning control signal to the scanning unit 102 when a scanning instruction command is input from the computer system 200. To do. Further, the control unit 101 is connected to laser light sources 103, 104, 105, and these laser light sources 103, 104, 10 are connected.
5. Select the laser to use for scanning from 5. in this case,
The laser light source 103 includes an Ar laser and a laser light source 104.
Includes a HeNe-Green laser and a laser source 1
A HeNe-Red laser is used for each.

The laser beams emitted from the laser light sources 103, 104 and 105 selected by the control unit 101 are combined into dichroic mirrors 106 and 10 for synthesis.
7. The light is collected by the mirror 108 and input to the scanning unit 102 via the mirror 109.

The scanning unit 102 has an X-scan galvano mirror and a Y-scan galvano mirror, which are not shown, and drives the X-scan galvano mirror and the Y-scan galvano mirror based on a scanning control signal from the control unit 101. The laser light input through the mirror 109 is XY-scanned as spot light on the sample S mounted on the stage 111 through the objective lens 110.

Fluorescence or reflected light generated from the sample S returns to the reverse of the incident optical path by the spot light scanned on the sample S in XY scanning. Of these, the fluorescent component is reflected by the spectral filter 112 and input to the spectral filter 113.

The spectral filter 113 selects a long wavelength and a short wavelength for the fluorescent component, and the fluorescent light of each wavelength is input to the barrier filter 115 and the barrier filter 116 via the mirror 114. Then, the wavelengths are further selected by the barrier filters 115 and 116 and input to the two photodetectors 117 and 118.

Then, it is converted into an electric signal by the photodetectors 117 and 118 and input to the control unit 101.

Here, the spectral filter 113 and the barrier filters 115 and 116 having various characteristics are mounted on, for example, a rotating turret, and the turret is rotated according to an instruction from the control unit 101, so that the optical path is changed. It can be selectively switched with.

The control unit 101 transfers the electric signals from the photodetectors 117 and 118 as image information (image data) of the sample S to the computer system 200 via the connection interface 119.

The computer system 200 includes a CPU 2
01, storage medium 202, monitor 203, keyboard 20
4 and a mouse 205, a GUI (Graphical User) for inputting scanning conditions for acquiring an image with a fluorescent reagent on the monitor 203.
In addition to having a function of displaying an interface screen, it has an image forming function of forming data from the photodetectors 117 and 118 input via the control unit 101 as a plurality of images corresponding to each scanning condition.
Further, the image information is transferred to the storage medium 202 and displayed on the monitor 203.

In this case, the storage medium 202 has a memory section for storing the conditions necessary for scanning by the scanning unit 102, in addition to the memory section for storing the image information.

Next, with respect to the laser scanning microscope system thus constructed, triple staining (fluorescent dye: FI)
An example of acquiring an image of the sample S that has been subjected to TC, PI, Cy5) will be described.

The observer first looks at the computer system 2
From 00, the scanning conditions for acquiring an image by the fluorescent reagent of FITC and PI are input. Specifically, the scanning condition is input using the GUI screen as shown in FIG. 2 on the monitor 203 of the computer system 200.

In this case, the first set group number is 1 (group No. 1), and the types of various filters actually used as optical elements for determining the wavelength of the laser light to be irradiated and the wavelength of the observation light to be detected are respectively set. Enter in the setting part of. Here, the DM is set in the spectral filter 112.
488/543/633, SDM on spectral filter 113
560, BA 505 to the barrier filters 115 and 116
525 and BA560 are input respectively.

Similarly, using the GUI screen shown in FIG. 2, the type of laser light source actually used to excite the FITC and PI is input to each set portion. Here, the laser light source 103 is an Ar laser, and the laser light source 1
A HeNe-Green laser is inputted to the No. 04.

In this case, the FIT is detected by the photodetector 117.
C is detected as the channel CH1, and the photodetector 118 detects PI as the channel CH2.

Next, using the GUI screen shown in FIG. 3, the sensitivity of these photodetectors 117 and 118 is set as a setting condition for the detection operation of the photodetectors 117 and 118. Here, for the photodetector 117, as shown in A in the figure, the FITC corresponding to the channel CH1 is input, and the high voltage (PM) is set as the sensitivity setting value.
T), gain (Gain), offset (offse)
Enter each value of t). Similarly, as to the photodetector 118, as shown in B in the figure, PI corresponding to the channel CH2 is input, and each of high sensitivity (PMT), gain (Gain), and offset (offset) is set as a sensitivity setting value. Enter the value of.

These pieces of input information are stored in the storage medium 202 of the computer system 200 as scanning conditions for group No1.

Next, the scanning conditions for acquiring an image with the Cy5 fluorescent reagent are input. Also in this case, using the GUI screen as shown in FIG. 4 on the monitor 203 of the computer system 200, the wavelength of the laser light to be irradiated and the wavelength of the observation light to be detected are determined by setting the group number to 2 (groove No2). The types of various filters actually used as the optical elements to be input are input to the respective setting parts. Here, DM488 / 54 is added to the spectral filter 112.
3/633, Glass is input to the spectral filter 113, and BA660 is input to the barrier filter 116.

Similarly, using the GUI screen shown in FIG. 4, the type of the laser light source used for exciting Cy5 is input to the set portion. Here, the laser light source 105
The HeNe-Red laser is input to.

In this case, the photodetector 118 causes
Cy5 is detected as the channel CH3.

Next, using the GUI screen shown in FIG. 3, the sensitivity of these photodetectors 118 is set as a setting condition for the detection operation in the photodetectors 118. Here, with respect to the photodetector 118, Cy5 corresponding to the channel CH3 is input as shown by C in the figure, and high sensitivity (PMT) and gain (Gai) are set as sensitivity setting values.
n) and the offset (offset) are input.

These pieces of input information are also stored in the storage medium 202 of the computer system 200 as scanning conditions for group No2.

Next, when the observer gives an instruction to start scanning from a state in which the scanning conditions for each group have been input, the computer system 200 reads the scanning conditions for the groove No. 1 stored in the storage medium 202, In the control unit 101, the sensitivity setting values of the photodetectors 117 and 118 are set together with the information of the spectral filter 112, the spectral filter 113, the barrier filters 115 and 116, the types of the laser light sources 103 and 104.

When the setting of these pieces of information is completed, the computer system 200 sends a scan start instruction to the control unit 101.

In this state, the laser beams emitted from the laser light sources 103 and 104 are combined by the combining dichroic mirrors 106 and 107, input to the scanning unit 102 via the mirror 109, and the scanning control from the control unit 101. Based on the signal, the objective lens 11
The sample S on the stage 111 is XY scanned as spot light through 0. The fluorescence emitted from the sample S returns to the reverse of the incident optical path by the spot light scanned in the XY direction on the sample S, and the fluorescence component is reflected by the spectral filter 112 and input to the spectral filter 113, and the barrier filter. The wavelengths are selected at 115 and 116 and input to the photodetectors 117 and 118.
It is converted into an electric signal at 18, and the control unit 10
Sent to 1. The control unit 101 converts an electric signal from the photodetector 117 into image information of the channel CH1 and also converts an electric signal from the photodetector 118 into image information of the channel CH2, and a computer via the connection interface 119. Transfer to system 200. The computer system 200 generates a plurality of images corresponding to the scanning conditions by the image forming function and stores the images in the storage medium 202.

Next, the computer system 200 reads out the scanning conditions of the groove No. 2 stored in the storage medium 202, and with respect to the control unit 101, information of the spectral filter 112, the spectral filter 113, the barrier filter 116, and the laser light source 105. And the sensitivity setting value of the photodetector 118 are set.

When the setting of these pieces of information is completed, the computer system 200 sends a scanning start instruction to the control unit 101, and the electric signal from the photodetector 118 is transmitted to the image information of the channel CH3 by the same operation as described above. As the connection interface 11
The image is captured through the image capturing unit 9, and is generated as an image corresponding to the scanning condition by the image forming function, and is stored in the storage medium 202.
In this case, the storage medium 202 stores the image information in an area different from the image information acquired under the scanning conditions of the groove No1.

In this way, a plurality of images for each channel (here, channel CH1: FITC, channel CH2: PI, channel CH3: Cy5) are treated as one image data such as an image acquired by one scanning. To be acquired.

When performing dynamic observation on the sample S, it is necessary to acquire the image at each Z position while shifting the position of the stage 111 in the Z direction. Group No. 1 and Group No. 2
The image acquisition at and may be repeated for each Z position.

The same applies to the case where the sample S is observed over time.
The image acquisition for o1 and groove No2 may be repeated.

Therefore, in this way, as the conditions necessary for scanning for image acquisition by the fluorescent reagent, selection of the laser light sources 103, 104, 105 required for wavelength detection, the spectral filters 112, 113, and the barrier filter. 11
By inputting the combination of various filters 5 and 116 and the setting of the detection sensitivity of the photodetectors 117 and 118 on the GUI screen prepared in advance, the number of the photodetectors 117 and 118 is limited. Since it is possible to set conditions for multi-channel image acquisition that is not performed, the experimenter must not perform complicated operations until the final image acquisition, use complicated macro commands, and execute programming. Without simply setting the conditions for each wavelength detection, you can easily perform multiple wavelength observation such as observation of the target multiple stained specimen exceeding the number of photodetectors in a short preparation time. it can.

Since the settings required for each scan can be easily confirmed on the GUI screen, setting mistakes can be prevented.

Furthermore, since a system composed of only a minimum number of photodetectors can observe images of multiple wavelengths on a multi-stained sample, a system of lower cost can be realized.

(Second Embodiment) Next, the second embodiment of the present invention will be described.
An embodiment will be described.

In this case, the system configuration of the laser scanning microscope according to the second embodiment is similar to that of FIG. 1 described above, and therefore the figure is incorporated herein by reference.

By the way, in the first embodiment, the observer first groups the respective wavelength detections in the triple-stained specimen into groups, and actually performs excitation in accordance with the fluorescent reagent used for observation. The setting of each group of various filters actually used as an optical element for determining the laser light source used and the wavelength of the observation light to be detected is manually performed individually. For example, when observing FITC and PI, the spectral filter 1 is set as the group No1.
12 to DM488 / 543/633, spectral filter 11
3 for SDM 560, B for barrier filters 115 and 116
FITC image information on channel CH1 and PI on channel CH2 using A505-525 and BA560
To obtain the image information of and to observe Cy5,
As the group No. 2, DM48 is added to the spectral filter 112.
8/543/633, Glass on spectral filter 113
s, BA660 is used for the barrier filter 116 to acquire the image information of Cy5 on the channel CH3.

Therefore, in the second embodiment, combinations of laser light sources and various filters for each group for these fluorescent reagents are stored in the storage medium 202 of the computer system 200 as table data and database files. And

As a result, the observer can see the FITC, PI,
When the name of the reagent to be observed such as Cy5 is selected, the computer system 200 observes the FITC and PI based on the stored contents of the storage medium 202 in the group No1.
As a result, the filter set stored in them is automatically set, and the filter set stored in them is automatically set as the group No. 2 for the remaining observations of Cy5.

From this state, if the sensitivity of the photodetectors 117 and 118 is set for each group and then the start of scanning is instructed, in the same manner as described in the first embodiment, the FITC channel CH1 is used. Image information, channel CH
2 can acquire the image information of PI and the channel CH3 can acquire the image information of Cy5.

Therefore, in this way, the observer can obtain the image information of each reagent simply by setting the name of the reagent to be observed and setting the sensitivity of each photodetector. Furthermore, the desired multiple-stained specimen can be observed more easily with a short preparation time.

In the above-described embodiment, the case where two photodetectors are used has been described, but the number of photodetectors is not limited to two, and at least one photodetector is used. Good.

In addition, the present invention is not limited to the above-described embodiment, and can be variously modified at the stage of carrying out the invention without departing from the spirit of the invention.

Furthermore, the above-described embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, the problem described in the section of the problem to be solved by the invention can be solved, and it is described in the section of the effect of the invention. In the case where the effect described above is obtained, a configuration in which this constituent element is deleted can be extracted as an invention.

[0066]

As described above, according to the present invention, a laser scanning microscope system, a system control method and a system control computer capable of easily performing a multi-wavelength observation such as an observation of a target multi-stained specimen are performed by a computer. It is possible to provide a control program to be executed by.

[Brief description of drawings]

FIG. 1 is a diagram showing a system configuration of a laser scanning microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a display example of a GUI screen used in the first embodiment.

FIG. 3 is a diagram showing a display example of a GUI screen used in the first embodiment.

FIG. 4 is a diagram showing a display example of a GUI screen used in the first embodiment.

[Explanation of symbols]

100 ... Laser scanning microscope body 101 ... Control unit 102 ... Scanning unit 103, 104, 105 ... Laser light source 106.107 ... Dichroic mirror for synthesis 108, 109 ... Mirror 110 ... Objective lens 111 ... Stage 112, 113 ... Spectral filter 114 ... Mirror 115, 16 ... Barrier filter 117, 118 ... Photodetector 119 ... Connection interface 200 ... Computer system 201 ... CPU 202 ... Storage medium 203 ... Monitor 204 ... keyboard 205 ... mouse

Continued front page    F-term (reference) 2G043 AA03 BA16 DA02 EA01 FA02                       GA02 GB19 HA02 HA03 HA09                       JA02 KA02 KA09 LA03 LA05                       NA05 NA06 NA13                 2H052 AA07 AA09 AB24 AC04 AC14                       AC15 AC34 AD34 AF02 AF14

Claims (6)

[Claims] 1. A laser scanning microscope which scans a sample with laser light, detects observation light obtained from the sample with a photodetector, and forms a two-dimensional image based on an output signal from the photodetector. , A means for displaying an input screen for inputting a plurality of different scanning conditions to the same photodetector, and the plurality of scanning conditions input using this setting screen are sequentially set one by one. And control means for performing scanning each time, and image forming means for forming data from the same photodetector obtained under each scanning condition as a plurality of independent images corresponding to each scanning condition. A laser scanning microscope system having. 2. The scanning condition includes a setting condition of a detection operation in the photodetector, and a switching state of an optical element that determines a wavelength of laser light to be irradiated and a wavelength of observation light to be detected. The laser scanning microscope system according to claim 1. 3. The laser scanning microscope system according to claim 1, further comprising a plurality of the photodetectors. 4. The laser scanning microscope system according to claim 1, wherein the scanning conditions input from the input screen can be set more than the number of the photodetectors. 5. A laser scanning microscope that scans a sample with laser light, detects observation light obtained from the sample with a photodetector, and forms a two-dimensional image based on an output signal from the photodetector. A method of controlling the system, wherein an input screen for inputting a plurality of different scanning conditions to the same photodetector is displayed, and the plurality of scanning conditions input using this setting screen are displayed. One by one is set sequentially, and scanning is performed each time, and the data from the same photodetector obtained under each scanning condition is formed as a plurality of independent images corresponding to each scanning condition. Laser scanning microscope system control method. 6. A laser scanning microscope which scans a sample with laser light, detects observation light obtained from the sample with a photodetector, and forms a two-dimensional image based on an output signal from the photodetector. A control program that causes a computer to execute system control. An input screen for inputting a plurality of different scanning conditions to the same photodetector is displayed, and input is performed using this setting screen. The plurality of scanning conditions are sequentially set one by one, and scanning is performed each time, and the data from the same photodetector obtained under each scanning condition is converted into a plurality of independent data corresponding to each scanning condition. A control program that causes a computer to control a laser scanning microscope system characterized by being formed as an image.