JP2007024801A - Microscope controller and program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide microscope controller and program for easily achieving FRAP experiment that has living cells as the object and quantitative analysis of its experimental results. <P>SOLUTION: In the experiments and analyses, a computer 2 controls laser beam irradiation from a laser system 4 to make photobleaching on a partial area of a specimen in observation sample 108, while controlling a confocal laser scan type microscope body 1, to observe time variation in recovery of fluorescence on the area, where photobleaching in the specimen at this time has been carried out. Then diffusion coefficient of the specimen is computed based on the result of such observation for the specimen being a living cell. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microscope technique, and more particularly to a technique for acquiring and analyzing data by a FRAP (Fluorescence Recovery After Photobleaching) method performed using a confocal laser scanning microscope system.

  By measuring the fluorescence intensity of the photobleached region in the specimen and analyzing the recovery curve of the fluorescence intensity, the moving speed of the moving fluorescent molecule and the ratio of the moving molecule are obtained. FRAP (Fluorescence Recovery After Photobleaching: A fluorescence recovery method after photobleaching is known.

  In addition, the light from the laser light source is narrowed down to a minute spot by the objective lens, and the spot is scanned on the specimen, and the fluorescence and reflected light from the specimen are captured by the photodetector and converted into an electrical signal. A confocal laser scanning microscope that displays a specimen image on an image monitor based on the above is known.

  In such a confocal laser scanning microscope, an arbitrary region can be quickly specified using an acousto-optic modulation filter (AOTF), so that FRAP experiments can be performed even using living cells.

  In recent years, the use of fluorescent proteins such as GFP (Green Fluorescent Protein) and advances in microscopic techniques have made it possible to analyze protein behavior in cells relatively easily. For example, as described in Non-Patent Document 1 and Non-Patent Document 2, a technique for obtaining a diffusion rate (diffusion coefficient) of a fluorescent molecule by performing an FRAP experiment on a living cell, or movement in a fluorescent molecule Quantitative analysis techniques are also being established, such as a technique for obtaining the ratio of molecules that move (move) and molecules that do not move (move).

In addition, as described above, the AOTF is mounted on the confocal laser scanning microscope system, the system capable of independently scanning the bleach laser and the scan laser is developed, and further, a high-speed CCD (Charge Coupled) is developed. Development of a system in which a bleaching laser is added to an image acquisition system using a device (charge imaging device) has made it possible to perform FRAP experiments at high speed.
Hiroshi Kimura, “Intracellular dynamic analysis of fluorescently labeled proteins by photobleaching method 1—Setting conditions using fixed cells”, Experimental Medicine, Yodosha, August 1, 2004, Vol. 22, No. 12 , P. 1739-1745 Hiroshi Kimura, “Intracellular dynamic analysis of fluorescently labeled proteins by photobleaching method 2—Approach for measurement and quantitative analysis in living cells”, Experimental Medicine, Yodosha, September 1, 2004, No. 22 Volume 13, No. 13, p. 1851-1856

  In order to perform FRAP experiments using a laser scanning confocal microscope system and acquire data as described above, complicated operations such as specifying photo bleaching areas, setting photo bleaching conditions, and setting recovery observation times are performed. Therefore, the difficulty of the experiment is high.

  In addition, in order to obtain the molecular diffusion constant and Mobile / Immobile ratio as described above, in addition to performing complex measurements such as measuring the effective bleaching area (effective radius) and measuring the recovery time, quantitative Therefore, it is necessary to repeatedly perform the same measurement on a plurality of samples.

  The present invention has been made in view of the above-described problems, and a problem to be solved is to facilitate FRAP experiments on living cells and quantitative analysis of the experimental results. .

  A microscope control apparatus according to one aspect of the present invention includes a photobleaching execution control unit that controls laser beam irradiation to perform photobleaching on a partial region of a sample, and a confocal laser scanning microscope to control the sample. An observation control means for observing a temporal change in the recovery of fluorescence in the region subjected to photobleaching, and a diffusion coefficient calculating means for calculating a diffusion coefficient of the specimen based on a result of the observation on the specimen that is a living cell; , And the above-described problems are solved by this feature.

  The microscope control apparatus according to the present invention described above further includes an effective radius calculation means for calculating the effective radius of the laser beam used for the photo bleaching based on the result of the observation on the specimen that is a fixed cell, The diffusion coefficient calculating means may be configured to calculate the diffusion coefficient based on the effective radius.

  Further, in the above-described microscope control apparatus according to the present invention, a sample image acquisition unit that controls the confocal laser scanning microscope to acquire an image of the sample, and a sample image display unit that displays the image of the sample An area setting acquisition unit configured to acquire an area setting for display of the sample image, and the photo bleach control unit corresponds to the area acquired by the area setting acquisition unit. The control may be performed so that photobleaching is performed on the partial region.

  At this time, the area setting acquisition unit sets the first area to which photobleaching is performed in the specimen, the setting of the second area in which the specimen is represented in the display, and the specimen in the display. The setting of the third region that is not represented is acquired, and the diffusion coefficient calculation means calculates the fluorescence intensity of each of the first and second regions when the fluorescence intensity of the third region is used as a reference. The diffusion coefficient may be calculated based on the relative value.

  Further, in the microscope control apparatus according to the present invention described above, a setting request screen display means for displaying a setting request screen for the execution condition of the photo bleach and an input of the execution condition corresponding to the request on the setting request screen are acquired. An execution condition acquisition unit that performs the control so that the photo bleach execution control unit follows the execution condition acquired by the execution condition acquisition unit.

  The microscope control apparatus according to the present invention described above further includes mobile / in-mobile rate calculating means for calculating the mobile / in-mobile rate of the sample based on the result of the observation of the sample that is a living cell. It may be configured.

  Further, in the above-described microscope control apparatus according to the present invention, the microscope control device further includes a determination unit that determines whether or not the fluorescence of the region subjected to the photo bleaching has recovered and reached a plateau, and the observation control unit includes The control for the sample may be completed when it is determined that the fluorescence of the region subjected to the photo bleaching has reached a plateau.

  Further, the microscope control apparatus according to the present invention described above further includes a fluorescence intensity threshold value acquisition unit that acquires setting of a fluorescence intensity threshold value, and the photo bleach execution control unit is configured such that the fluorescence intensity of the partial region is You may comprise so that the control which completes photo bleaching may be performed when a threshold value is reached.

  In addition, the process of performing the photobleaching on the partial region in the specimen by controlling the irradiation of the laser light, and the recovery time of the fluorescence in the region of the specimen on which the photobleaching is performed by controlling the confocal laser scanning microscope A program for causing a computer to perform a process of observing a change and a process of calculating a diffusion coefficient of the sample based on the result of the observation of the sample that is a living cell is also related to the present invention. By executing the program, the computer causes the same effect as the above-described microscope control apparatus according to the present invention. As a result, the above-described problem is solved.

  According to the present invention, by configuring as described above, there is an effect that the FRAP experiment for living cells and the quantitative analysis of the experiment result can be easily performed.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows an outline of a system configuration of a confocal laser scanning microscope embodying the present invention.

  In the figure, a computer 2 is connected to the confocal laser scanning microscope main body 1. An operation panel 3, a laser device 4, and an image monitor 5 are connected to the computer 2.

  In the confocal laser scanning microscope 1, the laser light incident from the laser device 4 is reflected by the excitation dichroic mirror 102 and is incident on the scanning unit 104. The scanning unit 104 has a galvanometer mirror for scanning in the X-axis direction and a galvanometer mirror for scanning in the Y-axis direction, and causes the laser light from the laser device 4 to perform XY scanning according to a scanning control signal from the computer 2. At the same time, a scanning control end signal is output to the computer 2 every time one line is scanned by scanning in the X-axis direction. Further, the laser light scanned in the XY-axis direction passes through the objective lens 106 attached to the revolver 105, and is then irradiated as spot light onto the observation sample 108 placed on the stage 107.

  The light from the observation sample 108 (for example, reflected light or fluorescence generated from the observation sample 108) by the spot light is returned to the incident optical path, reflected by the mirror 103, and collected by the lens 109. A confocal aperture 110 is disposed at the condensing position of the lens 109, and a confocal optical system is formed. The wavelength of the light that has passed through the confocal aperture 110 is selected by the barrier filter 111, and only the light having the wavelength desired by the observer is input to the photoelectric converter 101 such as a photomultiplier. When the photoelectric converter 101 converts the input light into an analog signal, the analog signal is converted into digital data corresponding to the amount of light by the A / D input CH 2 b installed in the computer 2. This data is stored in the memory 2c, and is output to the image monitor 5 as image information as necessary.

  At least one of the revolver 105 and the stage 107 can be controlled in the Z direction (optical axis direction) by using an electric focusing unit that is not shown in the figure and is installed inside or outside the microscope. it can. The movement control in the Z direction is controlled by the computer 2.

  In addition to the keyboard, the operation panel 3 has a pointing device such as a trackball, a joystick, or a mouse. According to an instruction from the observer, various instructions are given to the computer 2, such as a laser beam scanning start command, An image input command or a sensitivity adjustment command of the photoelectric converter 101 is output.

  The computer 2 controls the entire system shown in FIG. 1. In particular, when a scanning instruction command is input from the operation panel 3, the computer 2 outputs a scanning control signal to the scanning unit 104, and the photoelectric converter The analog signal of the observation sample 108 output from 101 is converted into digital data by the A / D input CH2b and transferred to the memory 2c, and an image represented by the data and a setting menu screen for various scanning conditions are displayed. The image is displayed on the image monitor 5. Furthermore, the computer 2 sets an applied voltage, an amplifier gain, an offset, and the like for the photoelectric converter 101 in accordance with a sensitivity adjustment command from the operation panel 3. Note that the control of the scanning unit 104 in the confocal laser microscope apparatus 1, the control of the incidence of laser light from the laser light source apparatus 4 to the confocal laser microscope apparatus 1, an image performed according to a predetermined input from the operation panel 3 Various controls such as output control of image information in the memory to the monitor 5 are realized by a CPU (central processing unit) provided in the calculation / processing unit 2a in the computer 2 executing the control program 2d. The

  Next, the analysis method described in Non-Patent Document 1 and Non-Patent Document 2 employed in the FRAP experiment performed using the above-described system configured as shown in FIG. 1 will be briefly described. explain.

  First, it is assumed that the breach characteristic by the laser beam shows a normal distribution characteristic. That is, as shown in FIG. 2A, it is assumed that the intensity distribution of the laser light is normally distributed around the irradiation point.

When this laser beam is used to perform a sufficiently short bleach on the sample, the intensity F _K (t) of the fluorescence emitted by the molecule when time t elapses after the bleaching is given by the following equation: It is known to be able to express.

In the above equation, (qP ₀ C ₀ / A) is the fluorescence intensity before bleaching or when a plateau is reached, K is bleach constant (constant determined by bleach experimental conditions), and τ _D is It is the diffusion time. The diffusion time τ _D is defined as follows:

Here, w is an effective radius, and represents a distance from the center of the irradiation point to a position where the intensity of the laser beam decreases to e ⁻² , as shown in FIG. D is a diffusion coefficient, which represents the speed of movement of molecules.

  By the way, among the above-mentioned [Equation 1] and [Equation 2], the bleach constant K and the effective radius w are fixed cells (the molecules cannot move) under the same bleach experimental conditions. Can be obtained from the change in the fluorescence intensity from the center of the bleach when the bleaching is carried out using as a sample.

  Percentage of molecules that were not bleached (not faded) in the molecule at a distance r from the bleach center, that is, in other words, relative fluorescence intensity (ratio of fluorescence intensity after bleaching to fluorescence intensity before bleaching) Cr is represented by the following equation.

In the above equation, I (r) is the fluorescence intensity at a position r away from the bleach center, and I (0) is the fluorescence intensity at the bleach center.

It can be expressed as. Therefore, the formula [3] is

It is expressed.
That is, bleaching is performed on a sample that is a fixed cell, and the relationship between the distance r and the relative fluorescence intensity Cr is observed and plotted. A general-purpose numerical graph between plot data obtained by bleaching on a plurality of samples (for example, about 10 to 20 cells) and a graph of [Expression 5] using bleach constant K and effective radius w as variables. Perform curve fitting analysis using analysis software. The bleach constant K and the effective radius w are obtained by obtaining the values of both variables when the two fit properly by this analysis.

Next, bleaching is performed on a sample that is a living cell, and the relationship between time t and fluorescence intensity F _K (t) is observed and plotted.
The data obtained by performing this bleaching on a plurality of samples (for example, about 10 to 20 cells), diffusion coefficient D, plateau P, and baseline (fluorescence intensity at t = 0) a 3 Curve fitting analysis using general-purpose numerical graph analysis software is performed with a graph of [Equation 1] using one of the variables as a variable (a graph of [Equation 1] to which the definition of [Equation 2] is applied).

At this time, as terms of Σ, for example, n = 0 to about 40 are expanded.
By this analysis, the values of variables when the graph of the formula [1] is appropriately fitted to the plot data are obtained, whereby the diffusion coefficient D, the plateau P, and the baseline a are obtained.

  The ratio of the moving molecule to the non-moving molecule in the fluorescent molecule (in the present application, this ratio is referred to as “Mobile / Immobile ratio”) is determined as follows. be able to. That is, the baseline a, the fluorescence intensity P when the plateau is reached, the fluorescence intensity of the living cells before bleaching, and the above ratio are in a relationship as shown in FIG. Therefore, the Mobile / Immobile ratio can be easily obtained from these fluorescence intensities.

  If it is assumed that the bleaching characteristic by the laser exhibits a circular disk characteristic as shown in FIG. 2B, the diffusion coefficient D can be calculated by the following equation.

In the above equation, w is an effective radius, and t _1/2 is the time until the fluorescence intensity after bleaching is restored by half of the fluorescence intensity at the plateau, as shown in FIG. It is.

  To perform the FRAP experiment based on the analysis method described above using the above-described system configured as shown in FIG. That is, first, an experiment using the FRAP method is performed on fixed cells, and the effective radius w and the bleach constant K are obtained. Hereinafter, this processing is referred to as “preparation processing”. Thereafter, an experiment by the FRAP method is performed on living cells (unfixed cells), and the diffusion in the living cells is obtained from the data obtained by this experiment and the effective radius w and bleach constant K obtained previously. Determine constants, fluorescence recovery time, and Mobile / Immobile ratio. Hereinafter, this processing is referred to as “actual measurement processing”.

  The operator performs instructions by a GUI (Graphical User Interface) function in a wizard format (interactive format) as shown in FIG. 4 or FIG. 8 described later, that is, an operation necessary for each processing step. By following the instruction by the function of proceeding with the experiment while requesting the operator, the experiment can be performed along the flow of the preparation process and the actual measurement process, and the necessary measurement result can be obtained.

  4 will be briefly described. First, the screen (1-1) is a screen that prompts the user to set the observation sample 108 including the specimen that is a fixed cell on the stage 107 of the confocal laser scanning microscope 1. . When the operator sets the observation sample 108 on the stage 107 in accordance with the instruction on this screen, operates the operation panel 3 and presses the button labeled “Next” on the screen, the image monitor 5 The display is switched to the screen of (2-1).

  The screen of (2-1) is a setting screen for laser beam scanning conditions that is performed in order to obtain an image of the observation sample 108 once. The operator selects the scanning direction (selects unidirectional scanning or bidirectional scanning), scanning speed, aspect ratio and size of the acquired image, acquisition range of the image in the microscope field of view, and Z-direction position of the observation sample 108 This screen is used to switch between a scanning operation that repeats scanning with changing the scanning operation and a scanning operation that repeats scanning at the same position in the Z direction at predetermined time intervals, and settings for conditions for repeating scanning in each scanning operation. Do. Then, when the operation panel 3 is operated and the button labeled “Next” on the screen is pressed, an operation of acquiring an image of the observation sample 108 is started, and the observation sample 108 as shown in (2-2) is started. The image is displayed in a separate window on the image monitor 5, and the screen of (2-1) is switched to the screen of (3-1).

  The screen (3-1) is a setting screen for setting a photo bleach condition for the observation sample 108. The operator performs setting of the scanning speed of the laser beam for the photo bleaching, the scanning time interval, the number of times of scanning, the intensity, and the range of various regions for the image of the observation sample 108 on this screen. Here, instead of setting the ranges of the various areas on the screen of (3-1), the operation panel 3 is operated to display the observation sample 108 being displayed as in the image of (3-2). This may be done by drawing a line specifying the range of the image. In this example, three areas are set: a bleach area, a cell area, and a background area. Here, the bleaching region is a region where laser light for photobleaching is irradiated, and the cell region is a region where a cell as a specimen is represented in an image (that is, an outline of the outer shape of the cell). Yes, the background area is an area that does not include cells in the image (ie, the background portion of the image).

  After performing the above settings, when the operator operates the operation panel 3 and presses the button labeled “Next” on the screen (3-1), the FRAP experiment is started. When this experiment is started, the image monitor 5 displays an image of the observation sample 108 during the experiment as shown in (4-1) and the above-mentioned as shown in (4-2). A graph showing temporal changes in fluorescence intensity in each region is also displayed.

  Next, processing performed by the computer 2 in order to proceed with the FRAP method experiment using the GUI function described above will be described. The following processing is realized by a CPU (central processing unit) provided in the calculation / processing unit 2a in the computer 2 executing the control program 2d.

First, FIG. 5 will be described. This figure is a flowchart showing the contents of the FRAP experiment process.
When the process of FIG. 5 is started, first, a preparation process button and an actual measurement process button (not shown) are displayed on the image monitor 5. Here, in S101, a process of determining whether or not the pressed operation on these buttons performed by the operator operating the operation panel 3 is detected and whether or not the detected pressed operation is performed on the preparation process button is performed. At this time, when the detected pressing operation is for the preparation process button (when the determination result is Yes), it is assumed that the execution instruction for the preparation process has been acquired from the operator, and the process proceeds to S102. On the other hand, when the detected pressing operation is for the actual measurement process button (when the determination result is No), it is considered that the execution instruction for the actual measurement process has been acquired from the operator, and the process proceeds to S104.

In S102, a preparation process is performed. Details of this processing will be described later.
When the preparation process is completed, a process of displaying an actual measurement process button (not shown) on the image monitor 5 is performed in S103. Then, a process of determining whether or not a pressing operation on this button, which is performed by the operator operating the operation panel 3, is detected. Here, when a pressing operation on the actual measurement process button is detected (when the determination result is Yes), it is assumed that an instruction to execute the actual measurement process is acquired from the operator, and the process proceeds to S104. On the other hand, when the pressing operation on the actual measurement processing button is not detected (when the determination result is No), the processing in FIG. 5 is terminated.

In S104, an actual measurement process is performed, and thereafter the process of FIG. 5 is terminated. Details of the actual measurement process will be described later.
The above processing is the FRAP experiment processing.

Next, FIG. 6 will be described. This figure is a flowchart showing the processing contents of the preparation processing in S102 of FIG.
When the process of FIG. 6 is started, a screen as shown in (1-1) of FIG. Here, when the operator sets an observation sample 108 including a specimen that is a fixed cell on the stage 107 in accordance with an instruction on this screen and performs a predetermined operation on the operation panel 3, the display on the image monitor 5 is (2 -1) screen is displayed. When the operator operates the operation panel 3 to make a predetermined setting while this screen is displayed, an operation for acquiring an image of the observation sample 108 is started, and the observation sample 108 as in (2-2) is started. Are displayed on the image monitor 5 in a separate window.

  Here, the operator sets bleaching execution conditions. First, a spot breach button (not shown) and a ROI (Region Of Interest) breach button are displayed on the image monitor 5, and in S201, pressing operations on these buttons performed by the operator operating the operation panel 3 are detected. Then, processing for determining whether or not the detected pressing operation is for the spot breach button is performed. At this time, when the detected pressing operation is for the spot breach button (when the determination result is Yes), an instruction to calculate the effective radius based on the result of the spot breach is acquired from the operator. Assuming that the process proceeds to S202. On the other hand, when the detected pressing operation is for the ROI breach button (when the determination result is No), it is considered that an instruction to calculate the effective radius based on the result of the ROI breach is acquired from the operator. , The process proceeds to S203.

  At this time, the setting request screen for the bleaching execution condition (3-1) is displayed on the image monitor 5 together with the image (2-2) in FIG. The operator makes a predetermined setting for these displays on the image monitor 5.

  In S202, processing for acquiring the above-described setting of the position of the bleaching area as the “bleaching position” among the above settings made by the operator is performed, and thereafter, the process proceeds to S204.

In S <b> 203, a process of acquiring the position of the breach area described above as the “ROI position” among the above settings made by the operator is performed.
In S204, processing for measuring the luminance of each area before photo bleaching is performed.

  In S205, processing for obtaining settings of laser conditions (laser scanning speed, laser beam intensity, etc.) among the settings made by the operator on the screen of (3-1) in FIG. In S206, processing for acquiring the setting of the breach time among the settings is performed. Note that these settings are stored in the memory 2c of the computer 2.

  In S207, the laser beam is controlled according to the various conditions acquired as described above, and photobleaching is performed on the observation sample 108, and predetermined data (luminance of each pixel of the image of the observation sample 108 after photobleaching) is obtained. The measurement is performed by the photoelectric converter 101, and the process of observing the temporal change of the fluorescence recovery in each region described above is performed.

  In S208, a process for determining whether or not the measurement for all the observation samples 108 as the experiment target has been completed is performed. When it is determined that the measurement has been completed (when the determination result is Yes), the process proceeds to S209. . On the other hand, when it is determined to be incomplete (when the determination result is No), the process returns to S201, and the above-described measurement process for the observation sample 108 that has not been measured is performed again.

  In S209, a process for calculating the effective radius of the laser beam used for the photo bleaching is performed based on the observation result obtained as described above, and in a subsequent S210, a process for calculating the bleach constant is performed. These calculation processes are performed as follows.

  First, the relationship between the distance r from the center of the bleaching position and the relative fluorescence intensity Cr described above at the position of the distance in the observation sample 108 is obtained for each observation sample 108 from the luminance value measured by execution of photo bleaching. . In addition, according to the nonpatent literature 2 mentioned above, this relationship is calculated | required as follows.

First, in the image of the observation sample 108, the average luminance of the pixels in each region described above is calculated. Here, the luminance of the pixel in the cell region is I _total and the luminance of the pixel in the background region is I _BG . In the breach region, the luminance of a pixel located concentrically at a distance r from the center of the breach position is I _r .

In this case, fluorescence intensity _{net Non} I _r of the fluorescence intensity _{net Non} I _total and bleach region of a cell region when relative to the fluorescence intensity of the background area, according to Non-Patent Document 2 described supra, obtained by the following equation.

The fluorescence intensity _{net Non} I _total and _{net Non} I _r, determined for before and after bleaching.
Here, the average of the fluorescence intensity of the cell area before bleaching is defined as I _total (before), the average of the fluorescence intensity of the cell area after bleaching is defined as I _total (after), and the average of the fluorescence intensity of the bleach area before bleaching is defined as Assuming that I _r (before) and the average of the fluorescence intensity in the bleached region after bleaching are I _r (after), according to the non-patent document 2 mentioned above, the relative fluorescence intensity Cr is obtained by the following equation.

Therefore, by using this [Equation 8], it is possible to obtain the value of the relative fluorescence intensity Cr at the position r from the center of the breach position in the observation sample 108 from the photobleach measurement result for the observation sample 108. it can. Here, a general-purpose numerical graph analysis software is executed, and the relationship between the distance r and the relative fluorescence intensity Cr obtained in this way, the effective radius w and the bleach constant K are used as variables. Curve fitting analysis is performed with this graph. The effective radius w and the bleach constant K are obtained by obtaining the values of both variables when the two fit appropriately by this analysis.

  The final values of the effective radius w and the bleach constant K are obtained by performing the above calculation on all the observed samples 108 and calculating the average of the results obtained for each of the observed samples 108.

Returning to the description of FIG. 6, in S211, a process of storing the result obtained as described above in the memory 2c of the computer 2 is performed, and thereafter, the process of FIG. 6 ends.
Next, FIG. 7 will be described. This figure is a flowchart showing the processing contents of the actual measurement processing in S104 of FIG.

  First, in S301, processing for reading information (laser conditions, bleaching time, effective radius w, bleaching constant K value, etc.) stored in the memory 2c of the computer 2 in the above-described preparation processing is performed.

  Here, the image monitor 5 has the same screen as (1-1) in FIG. 4 in the above-described preparation process as shown in FIG. 8 (1), that is, each part of the confocal laser scanning microscope 1. An image of the observation sample 108 acquired by the computer 2 is displayed. Here, when the operator sets the observation sample 108, which is a living cell, on the stage 107 in accordance with an instruction on this screen and performs a predetermined operation on the operation panel 3, an operation for acquiring an image of the observation sample 108 is started. The display on the image monitor 5 displays an image of the observation sample 108 as shown in (2) of FIG.

  The operator creates an ROI for these images on the image monitor 5, that is, sets three areas, a bleach area, a cell area, and a background area. (3) in FIG. 8 shows how the ROI is created.

In S302, a process for acquiring these ROI creation results is performed.
In S303, photobleaching is performed on the observation sample 108 in accordance with the process for the experiment, that is, the various conditions acquired in the process of S301, and predetermined data (the luminance of each pixel in the image of the observation sample 108 after photobleaching). ) Is measured to observe the temporal change in the recovery of the fluorescence intensity.

  In S304, a process for determining whether or not the fluorescence intensity has reached a plateau by this observation is performed. In this determination process, for example, the fluorescence intensity is observed every predetermined time, and if the difference between the fluorescence intensity value observed last time and the fluorescence intensity value observed this time is equal to or less than a predetermined value, the fluorescence intensity is It is determined that the plateau has been reached. Alternatively, the fluorescence intensity is observed every predetermined time, and the average of the fluorescence intensity values observed at the most recent predetermined number (for example, 5 times) and the previous predetermined number of times (for example, 5 times) are observed. A difference from the average of the fluorescence intensity values may be obtained, and when the difference becomes a predetermined value or less, it may be determined that the fluorescence intensity has reached a plateau.

  Only when it is determined in the determination process of S304 that the fluorescence intensity has reached a plateau (only when the determination result is Yes), the temporal change in the recovery of the fluorescence intensity for the observation sample 108 that is the current observation target And the process proceeds to S305. The determination process of S304 makes it possible to acquire data without excess or deficiency, reduce the storage capacity of the memory 2c consumed for holding the data, and quantitatively measure the fluorescence recovery time. It becomes.

  In S305, a process for determining whether or not the above-described experiment has been completed for all of the prepared observation samples 108 (for example, about 10 to 20 cells) is performed, and when it is determined that all the experiments have been completed (the determination result is Yes) ), The process proceeds to S306. On the other hand, when it is determined that the untested observation sample 108 remains (when the determination result is No), the process returns to S302 and the above-described process is repeated.

In S306, a process for calculating the diffusion constant D is performed. That is, average plot data showing the relationship between time t and fluorescence intensity F _K (t) obtained by bleaching a plurality of observation samples 108 that are living cells, diffusion coefficient D, plateau P, And curve fitting analysis processing by general-purpose numerical graph analysis software between the graph of [Formula 1] with the baseline a as a variable (graph of [Formula 1] applying the definition of [Formula 2]) Execute. At this time, as the term of Σ, for example, n = 0 to about 40 are expanded. And by this analysis, the diffusion coefficient D plateau P and the baseline a are obtained by obtaining the values of the variables when the graph of the [Equation 1] is appropriately fitted to the plot data. When the plateau intensity (qP ₀ C ₀ / A) can be clearly identified from the plot data of the fluorescence intensity with time, the plateau intensity may be obtained directly from the plot data.

In S307, a process for calculating the fluorescence recovery time is performed. This processing is obtained by obtaining the time to reach the plateau from the average plot data indicating the relationship between the time t and the fluorescence intensity F _K (t).

In S308, processing for calculating the Mobile / Immobile rate is performed. In this process, the fluorescence intensity a when t = 0 and the fluorescence intensity P when the plateau is reached are obtained based on the relationship between the moving molecules and the non-moving molecules shown in FIG. Alternatively, the average plot data indicating the relationship between the time t and the fluorescence intensity F _K (t) may be obtained based on the relationship shown in FIG.

  In S309, the diffusion constant, the fluorescence recovery time, and the Mobile / Immobile rate obtained as described above are output on the image monitor 5 and output, and then the processing of FIG. 7 ends. .

  The system shown in FIG. 1 is an experiment of the FRAP method using the GUI function by performing the processing of FIGS. 5, 6, and 7 described above by the CPU provided in the calculation / processing unit 2 a in the computer 2. Is possible.

  In the above-described embodiment, the breach characteristic by the laser has the normal distribution characteristic shown in FIG. 2A. However, the breach characteristic by the laser has a circular characteristic as shown in FIG. When it can be regarded as exhibiting disk characteristics, the calculation process of the diffusion coefficient D can be simplified by using the formula [6].

  In the above-described embodiment, the photo bleaching is executed according to the setting of the photo bleaching time acquired in the process of S206 in FIG. 6, but instead, the bleaching set by the operator is executed. Acquisition brightness value (fluorescence intensity threshold) setting is acquired, bleaching is performed until the acquired brightness value is reached, and control processing is performed to complete the bleaching when the set brightness value is reached. May be. This also allows observation of fluorescence recovery to be started immediately after completion of photobleaching, and measurement of the fluorescence recovery time can be performed with high accuracy.

Further, instead of the operator setting the reached luminance value, an average value of the background area may be calculated and this value may be automatically set as the reached luminance value.
In the above-described embodiment, the observation sample 108 is replaced one by one in the repetition of the processing from S302 to S305 in FIG. 7, but instead, a plurality of cells that are the observation sample 108 are registered in advance. In addition, the ROI creation for each cell is performed collectively, and in the process of S302, the creation result of the ROI is acquired at once, and the multipoint time lapse system combined with the electric XY stage device is used together. The processing in FIG. 7 can be changed so that the FRAP experiment and analysis are automatically repeated.

  As described above, when the diffusion constant of a fluorescent molecule is measured using the FRAP method, it is necessary to carry out a complicated experiment and a task of decoding a complicated calculation formula. According to the present embodiment, since a series of work flow for the FRAP experiment proceeds with the wizard-type GUI function, the operator can perform the work according to the instructions displayed on the screen. Proceeding makes it possible to easily perform accurate experiments and quantitatively calculate diffusion constants.

  By the way, the processing shown in the flowcharts in FIGS. 5, 6, and 7 is performed by a standard computer, that is, a CPU that controls each component by executing a control program, a ROM, a RAM, and a magnetic memory. And a storage unit used as a work area or a storage area for various data when the CPU executes the control program, and various types corresponding to operations by the user. An input unit from which data is acquired, an output unit that presents various data on a display and notifies the user, and an I / F unit that provides an interface function for data exchange with other devices, It is possible to have a computer equipped. For this purpose, a control program for causing the CPU to perform these processes is created and recorded on a computer-readable recording medium, and the program is read from the recording medium to the computer and executed by the CPU. That's fine.

  As a recording medium that can be read by the computer, the recorded control program can be inserted into, for example, a storage device such as a ROM or a hard disk device provided as an internal or external accessory device in the computer, or a medium driving device provided in the computer. A portable recording medium such as a flexible disk, MO (magneto-optical disk), CD-ROM, DVD-ROM, or the like from which the control program recorded can be read out can be used.

  Further, the recording medium may be a storage device provided in a computer system functioning as a program server connected to a computer via a communication line. In this case, a transmission signal obtained by modulating a carrier wave with a data signal representing a control program is transmitted from the program server to a computer through a communication line as a transmission medium, and the computer demodulates the received transmission signal. By replaying the control program, the control program can be executed by the CPU of the computer.

  As mentioned above, although embodiment of this invention was described, this invention is not limited to each embodiment mentioned above, A various improvement and change are possible within the range which does not deviate from the summary of this invention.

It is a figure which shows the outline of the system configuration | structure of the confocal laser scanning microscope which implements this invention. It is a figure which shows the example of the bleach characteristic by a laser beam. It is a figure which shows the example of the fluorescence recovery curve after photo bleaching. It is a figure which shows the example of a display on an image monitor (the 1). It is a flowchart which shows the processing content of a FRAP experiment process. It is a flowchart which shows the processing content of a preparation process. It is a flowchart which shows the processing content of the measurement process. It is a figure which shows the example of a display on an image monitor (the 2).

Explanation of symbols

1 Confocal Laser Scanning Microscope Body 2 Computer 2a Calculation / Processing Unit 2b A / D Input CH
2c memory 2d control program 3 operation panel 4 laser device 5 image monitor 101 photoelectric converter 102 excitation dichroic mirror 103 mirror 104 scanning unit 105 revolver 106 objective lens 107 stage 108 observation sample 109 lens 110 confocal aperture 111 barrier filter

Claims (9)

Photobleaching execution control means for performing photobleaching on a partial region of the specimen by controlling the irradiation of the laser beam;
An observation control means for controlling a confocal laser scanning microscope to observe a temporal change in the recovery of fluorescence in the photobleached region in the specimen;
A diffusion coefficient calculating means for calculating a diffusion coefficient of the sample based on a result of the observation on the sample that is a living cell;
A microscope control apparatus comprising: Based on the result of the observation on the specimen that is a fixed cell, further has an effective radius calculation means for calculating the effective radius of the laser beam used for the photobleaching,
The diffusion coefficient calculating means calculates the diffusion coefficient based on the effective radius;
The microscope control apparatus according to claim 1. A specimen image acquisition means for controlling the confocal laser scanning microscope to acquire an image of the specimen;
A specimen image display means for displaying an image of the specimen;
Area setting acquisition means for acquiring setting of an area made for display of the image of the specimen;
Further comprising
The photo bleach control means performs the control such that photo bleach is performed on the partial area corresponding to the area acquired by the area setting acquisition means.
The microscope control apparatus according to claim 1. The area setting acquisition means sets a first area to which photobleaching is performed in the specimen, sets a second area in which the specimen is represented in the display, and does not represent the specimen in the display. Get the settings for the three areas,
The diffusion coefficient calculating means calculates the diffusion coefficient based on a relative value of the fluorescence intensity of each of the first and second areas when the fluorescence intensity of the third area is used as a reference.
The microscope control apparatus according to claim 3. A setting request screen display means for displaying a setting request screen for execution conditions of the photo bleach;
Execution condition acquisition means for acquiring an input of the execution condition corresponding to the request on the setting request screen;
Further comprising
The photo bleach execution control means performs the control according to the execution condition acquired by the execution condition acquisition means.
The microscope control apparatus according to claim 1.   The microscope control apparatus according to claim 1, further comprising a mobile / in-mobile rate calculating unit that calculates a mobile / in-mobile rate of the sample based on a result of the observation of the sample that is a living cell. A determination means for determining whether the fluorescence of the region subjected to the photo bleaching has recovered and reached a plateau;
The observation control means completes the control for the specimen when it is determined that the fluorescence of the region subjected to the photo bleaching has reached a plateau.
The microscope control apparatus according to claim 1. A fluorescence intensity threshold acquisition means for acquiring the setting of the fluorescence intensity threshold;
The photo bleach execution control means performs control to complete photo bleach when the fluorescence intensity of the partial region reaches the threshold value.
The microscope control apparatus according to claim 1. A process of performing photobleaching on a partial region of the specimen by controlling the irradiation of the laser beam; and
A process of controlling a confocal laser scanning microscope to observe a temporal change in the recovery of fluorescence in the photobleached region in the specimen;
Processing for calculating the diffusion coefficient of the sample based on the result of the observation on the sample that is a living cell;
A program that causes a computer to perform