JP4844157B2 - Scanning microscope - Google Patents

Description

  The present invention relates to a scanning microscope.

2. Description of the Related Art Conventionally, a scanning microscope is known that scans excitation light two-dimensionally on a sample and detects fluorescence from the sample through a pinhole. In this scanning microscope, since the fluorescence emitted from other than the focal plane can be removed by the pinhole, an image of a thin cross section in the optical axis direction of the sample can be measured. Accordingly, it is possible to acquire a plurality of images by shifting the position of the sample in the optical axis direction and measure the three-dimensional shape of the sample (see, for example, Patent Document 1).
Japanese Patent Laid-Open No. 11-295051

  However, a biological sample such as a cell has a problem that the measurement range in the optical axis direction must be set every time measurement is performed because the three-dimensional shape of the cell changes with time.

  The present invention has been made in view of the above problems, and even when the three-dimensional shape of a sample changes with time, an image of the sample can be acquired in a range where the sample exists in the optical axis direction. An object is to provide a scanning microscope.

  In order to solve the above-described problems, the present invention provides an objective lens, a stage on which a sample is placed, scanning means for scanning illumination light on the sample, and a pinhole disposed at a position conjugate with the sample. In the scanning microscope having the detector for detecting the sample through the pinhole, the objective lens and the stage are moved relative to each other, and as a result of being driven by the drive means, the objective An image generating means for acquiring a signal from the detector at an arbitrary position in the optical axis direction of the lens and generating image data of the sample, and a gaze to be watched by the sample based on the image data from the image generating means A gaze area setting means for setting an area, and a reference value setting means for setting a reference value for determining the presence or absence of the sample based on image data of the gaze area set by the gaze area setting means And the drive means relatively drives the objective lens and the stage, the image generation means acquires a plurality of the image data at different positions in the optical axis direction, and the gaze area image data and the Provided is a scanning microscope characterized by comprising a control means for stopping and controlling the driving means when it is determined that the sample does not exist by comparing with a reference value set by a reference value setting means To do.

  ADVANTAGE OF THE INVENTION According to this invention, even when the three-dimensional shape of a sample changes temporally, the scanning microscope which can acquire the image of a sample in the range in which a sample exists in an optical axis direction can be provided.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a schematic configuration diagram of a scanning microscope according to an embodiment of the present invention.

  In FIG. 1, the laser light emitted from the laser light source 1 is reflected by the dichroic mirror 2 in the direction of the objective lens 4 and applied to a two-dimensional scanner 3 that scans a sample (cells cultured in a petri dish) 5 two-dimensionally. Incident. Laser light that is two-dimensionally scanned by the two-dimensional scanner 3 enters the objective lens 4 and is focused on the sample 5 placed on a stage (stage that moves in the XYZ directions) 6. The laser light condensed on the sample 5 excites the fluorescent dye contained in the sample 5 in advance, and the fluorescent light is emitted from the sample 5. Any one of the objective lens 4 and the stage 6 may be driven as long as it can move in the optical axis direction relatively.

  The fluorescent light emitted from the sample 5 is collected by the objective lens 4, descanned by the two-dimensional scanner 3, and enters the dichroic mirror 2. The fluorescent light incident on the dichroic mirror 2 passes through the dichroic mirror 2 and enters a pinhole 7 disposed at a position conjugate with the sample 5. Since the pinhole 7 is disposed at a position conjugate with the focal position of the objective lens 4, the fluorescent light excited at a position before or behind the focal position of the focused laser beam as a position on the optical axis. Only the fluorescent light excited at the focal position is allowed to pass through.

  The fluorescent light that has passed through the pinhole 7 enters the photomultiplier tube 8. The photomultiplier tube 8 converts the received light intensity into an electrical signal and sends it to the computer 9. The computer 9 A / D converts the received electrical signal into digital data. By arranging this digital data two-dimensionally in synchronization with the scanning of the two-dimensional scanner 3, two-dimensional image data having only information on the focal position can be acquired in the computer 9.

  After acquiring the two-dimensional image data, the computer 9 transmits to the stage 6 a command for moving a certain amount in the optical axis direction. In response to this command, the stage 6 moves a certain amount in the optical axis direction. After the movement is completed, two-dimensional image data having different focal positions can be acquired by acquiring two-dimensional image data.

  In this way, by repeating operations such as “moving the stage 6 by a certain amount in the optical axis direction” and “performing two-dimensional image data acquisition”, a plurality of two-dimensional image data having different focal positions can be acquired. it can. The focal position of the laser light emitted from the objective lens 4 is unchanged. When the optical axis position of the stage 6 changes, the focal position on the sample 5 changes relatively. As a result, each acquired two-dimensional image data becomes two-dimensional image data having information on the focal position corresponding to the position of each sample 5.

  The user acquires a preview image as will be described later, and the computer 9 obtains a minimum contrast value for the acquired two-dimensional image data by a method described later. When the contrast value obtained after acquiring the main image is below the minimum contrast value set by the user, the computer 9 “moves the stage 6 by a certain amount in the optical axis direction”, “acquires two-dimensional image data acquisition”. The repeat process of “perform” is stopped, and the acquisition of a plurality of pieces of two-dimensional image data is ended.

  Each two-dimensional image data acquired in this way is stored in the memory of the computer 9. Further, the acquired or stored two-dimensional image data can be processed by the computer 9 and the fluorescence image can be observed by displaying a two-dimensional image or a three-dimensional image on the monitor 10. In this way, the inverted scanning microscope 100 is configured.

  In the scanning microscope according to the present embodiment, the dichroic mirror 2 has a characteristic of reflecting laser light and transmitting fluorescent light, but the dichroic mirror 2 is a set of a dichroic mirror and a fluorescent filter. It is also possible to use a filter set. Further, the number of laser light sources 1 is not limited to one, and a laser light source having a plurality of wavelengths can be configured to be switched to the optical axis of the illumination optical system.

  Next, an example of a method for calculating the contrast value from the acquired two-dimensional image data will be shown. 2A shows an example of the vertical contrast calculation filter, FIG. 2B shows the horizontal contrast calculation filter, and FIG. 2C shows the Laplacian filter.

  Let the pixel of interest in the two-dimensional image data acquired by the above-mentioned measurement be n. A vertical direction contrast calculation filter shown in FIG. 2A is multiplied by a matrix operation for a pixel in the vicinity of the target pixel n. The result is set as a vertical contrast value Vn of the pixel of interest n.

  Similarly, the horizontal contrast value Vh of the target pixel n is obtained by multiplying the horizontal contrast calculation filter shown in FIG.

  Subsequently, a value obtained by squaring Vn and Vh is set as the contrast value of the target pixel n.

  Contrast values are calculated for all pixels (eg, m), and the m contrast values are summed. A value obtained by dividing the total contrast value by the number of all pixels (m) is the contrast value of the two-dimensional image data.

  Further, the contrast value is divided by a value obtained by doubling the square of the maximum value that the image data can take (for example, 255 when the digital data is 8 bits), and normalized as data from 0 to 1. In this way, it is possible to cope with a case where the number of bits of digital data is different (for example, 10 bits).

  In the present embodiment, the case where the vertical direction contrast calculation filter and the horizontal direction contrast calculation filter are used for calculating the contrast value has been described. However, it is also possible to calculate using the Laplacian filter shown in FIG. . When using the Laplacian filter, the Laplacian filter is multiplied by a matrix operation for the pixel of interest n of the acquired two-dimensional image data and its neighboring pixels, and the result squared is used as the edge value En of the pixel of interest n. Can be handled by dividing the square of all the number of pixels (m) by four times. Note that a filter other than the above-described vertical direction contrast calculation filter, horizontal direction contrast calculation filter, or Laplacian filter may be used as a filter used for calculating the contrast value.

  In addition, by setting the range of image data for which the contrast value is calculated by the computer 9, it is possible to reduce the amount of calculation of the contrast value and to increase the processing speed. Furthermore, when there are a plurality of cells in the screen, it is possible to acquire a plurality of two-dimensional image data by paying attention to only a specific cell.

  In addition, in order to remove the influence that the contrast value close to zero among the pixels corresponding to the background, or the data where the contrast value is saturated and overexposed has an influence on the calculated contrast value (preview time, etc.) A histogram analysis of luminance values is performed on the acquired two-dimensional image data, and a threshold value A of a background pixel and a threshold value B of a saturated pixel are determined using a histogram analysis as shown in FIG.

  In FIG. 3, the horizontal axis indicates the luminance value of the pixel from 0 to 255. The vertical axis indicates the appearance frequency of the luminance value.

  In the case of the scanning microscope 100 according to the embodiment of the present invention, since the background of the fluorescent image is dark (the pixel data has a small value and a value close to sero), for example, the pixel used for the contrast calculation is determined. Therefore, the pixel data is not used because it is regarded as background pixel data whose luminance value is lower than the lower limit A. In the example of FIG. 3, an example in which a value of luminance value 18 is set from 0 to 255 is shown. Similarly, as pixels that are not used for contrast calculation, pixels that have over-saturated data and pixels that exceed the upper limit B are set. In the example of FIG. 3, an example in which a value of luminance value 230 is set from 0 to 255 is shown. The lower limit value A and the upper limit value B may be input as fixed values from the computer 9, or may be automatically set using a predetermined threshold value by analyzing the histogram of FIG.

  In the calculation of the contrast value, the influence of dark background data and overexposure saturation data can be eliminated by calculating using only pixel data between the lower limit value A and the upper limit value B in FIG. That is, data that is less than the lower limit value A and exceeds the upper limit value B is not used for the calculation of the contrast value, and the contrast value is not added to the calculated number of pixels.

  By performing the above processing steps at regular time intervals, it is possible to perform so-called time lapse measurement in which the sample 5 such as a cell is measured at predetermined time intervals. At this time, even if the three-dimensional shape of the cell changes with time, the stage 6 is moved in the optical axis direction as long as the contrast value does not fall below the lower limit A, that is, as long as the image contrast information exists. Continue to acquire 2D image data.

  Thus, in the scanning microscope 100 according to the embodiment of the present invention, the lower limit value A and the upper limit value B of the luminance value are set from the two-dimensional image data of the preview image before the measurement of the sample 5 is started, and the range thereof The minimum contrast value (reference value) for determining the presence or absence of the sample 5 can be calculated based on the image data. Even if the three-dimensional shape of the sample 5 changes during the subsequent long-time measurement, the presence or absence of the sample 5 is determined by comparing the contrast value of the two-dimensional image data acquired sequentially with the minimum contrast value of the sample 5. Thus, it is possible to determine whether or not to continue the acquisition of the two-dimensional image data by moving the stage 6 in the optical axis direction. Therefore, it is possible to follow a change in the three-dimensional shape of a sample such as a cell and obtain a three-dimensional shape having an optimum thickness as two-dimensional image data.

  Note that the above-described processing is an example, and when processing an image, there is image data that is neither background nor saturated in the image using another means for extracting a feature value such as a contrast value or an edge value. By determining whether or not, the same effect can be obtained.

  FIG. 4 shows a time-lapse image acquisition flowchart of the scanning microscope 100 according to the embodiment of the present invention. Hereinafter, each step will be described.

(Step S01)
Place the sample 5 on the stage 6 of the scanning microscope 100, set the objective lens 4 at the observation position, irradiate the sample 5 with excitation light, record the fluorescence image of the sample 5 as a preview image, and observe it on the monitor 10 To do. The preview image may be a single image on a predetermined Z axis or a plurality of images on a plurality of Z axes.

(Step S02)
The user determines a gaze cell while viewing the preview image recorded in step S01 on the monitor 10. In addition, the user moves the stage 6 in the XYZ directions while looking at the preview image of the real-time fluorescent image, searches for a desired cell, and determines a cell to be observed from which two-dimensional image data is acquired.

(Step S03)
When the cell to be observed is determined, the user designates the contrast value calculation position so as to surround the target cell on the window in which the preview image is displayed. That is, the gaze area is set. The gaze area may be set automatically by automatically recognizing cells on the computer 9 side.

(Step S04)
Histogram analysis is performed on the displayed preview image, and the analysis result is displayed on the monitor 10 as a graph as shown in FIG.

(Step S05)
The user sets the lower limit value A and the upper limit value B for calculating the contrast value of the sample 5 on the histogram displayed on the monitor 10 using the input means of the computer 9.

(Step S06)
The user moves the stage 6 in the direction of the optical axis, and finishes the acquisition of the two-dimensional image data. A desired minimum contrast value (a reference value for ending the acquisition of the two-dimensional image data when the value becomes equal to or less than this value, for example, 0. Is set by the input means of the computer 9. That is, the user sets the scanning microscope 100 to the preview mode (mode switching can be performed by a switch (not shown)). The scanning microscope 100 is controlled by the computer 9 and displays a preview image of the sample 5 on the monitor 10. The user moves the stage 6 in the XYZ directions while looking at the preview image, and searches for a position where a desired image can be obtained. The minimum contrast value of the sample 5 is obtained from the gaze area by setting a gaze area where the user gazes from one of the preview images. Further, the user determines a minimum contrast value for ending the acquisition operation when acquiring the main image.

(Step S07)
A maximum moving range in the optical axis direction (Z direction) for moving the stage 6 is set. The values to be set are the Z0 position (start position) at which the acquisition of the two-dimensional image data is started, the feed interval (ΔZ) of the stage 6, and the maximum movement position Zm of the stage 6 (the stage 6 can be moved beyond this). It is a value to avoid). The start position Z0 is set near the upper surface of the cell (a position far from the objective lens 4), and the stage 6 is fed from the upper surface of the cell toward the lower surface (a position close to the objective lens 4). As a result, there is no possibility that the objective lens 4 collides with the sample 5 during the measurement.

(Step S08)
Set the time-lapse setting conditions. That is, the two-dimensional image data acquisition time interval and the number of acquired images are set from the input means of the computer 9 (GUI pointer or keyboard of the monitor 10).

(Step S09)
The start button for starting the time lapse for acquiring this image is turned on. The user starts acquiring 2D image data.

(Step S10)
Get the time lapse setting condition.

(Step S11)
The stage 6 is moved to the set acquisition start position Z0.

(Step S12)
XYZ acquisition (three-dimensional acquisition) is started.

(Step S13)
First, two-dimensional image data is acquired.

(Step S14)
A contrast value is calculated from the acquired two-dimensional image data.

(Step S15)
It is determined whether or not the calculated contrast value is smaller than the minimum contrast value input in (Step S06). When the contrast value is smaller than the minimum contrast value, the process jumps to (step S18) and the two-dimensional image data acquisition is completed. If the contrast value is equal to or greater than the minimum contrast value, step S16 is executed.

(Step S16)
The stage 6 is moved by the feed interval ΔZ value and set to the next target position.

(Step S17)
It is determined whether the stage position (Z value) after moving the stage 6 exceeds the maximum movement position Zm. When the Z position of the stage 6 exceeds the maximum movement position Zm, the process jumps to (Step S18) and the two-dimensional image data acquisition is completed. If the stage 6 is within the maximum movement position Zm, the process jumps to (Step S13) (Step S13) and the subsequent steps are executed.

(Step S18)
XYZ acquisition ends.

(Step S19)
It is determined whether or not the time-lapse shooting acquisition number has been reached. If the number of shots has been reached (step S22), the time lapse acquisition is terminated, and the acquisition of two-dimensional image data is also terminated. If the number of shots has not been reached, (step S20) is executed.

(Step S20)
Wait for the acquisition time interval set in (Step S08).

(Step S21)
It is determined whether the waiting time has reached the acquisition time interval. When the acquisition time interval is reached, the process jumps to (Step S11) and the subsequent steps are executed. If not, (step S20) and (step S21) are repeated.

(Step S22)
End time-lapse shooting.

  Through the above flow, the three-dimensional image data of the sample 5 is acquired by the computer 9 and stored in the memory. Thereafter, various analyzes are possible based on the acquired three-dimensional image data.

  Note that the maximum acquisition range in the Z direction of the two-dimensional image data is set in advance, the stage 6 moves to the maximum value of the maximum acquisition range in the Z direction, and the contrast value does not fall below the reference value (minimum contrast value). Even in this case, the acquisition of the next two-dimensional image data is forcibly interrupted. By doing so, it is possible to prevent an unexpected increase in the two-dimensional image data acquisition range.

  In the above-described embodiment, an example of acquiring a fluorescence image has been described. However, the present invention is not limited to the fluorescence image, and can be applied to other observation methods such as phase observation and DIC observation.

  ADVANTAGE OF THE INVENTION According to this invention, the scanning microscope which enables acquisition of the three-dimensional image data corresponding to the change of the three-dimensional structure of the cell which acquires an image is realizable.

  The above-described embodiment is merely an example, and is not limited to the above-described configuration and shape, and can be appropriately modified and changed within the scope of the present invention.

1 is a schematic configuration diagram of a scanning microscope according to an embodiment of the present invention. An example of a filter used for the contrast value calculation is shown. (A) shows an example of a vertical contrast calculation filter, (b) shows a horizontal contrast calculation filter, and (c) shows a Laplacian filter. The example of the histogram used for contrast value calculation, and the setting example of a lower limit and an upper limit are shown. 3 shows a time-lapse image acquisition flowchart of a scanning microscope according to an embodiment of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Dichroic mirror 3 Two-dimensional scanner 4 Objective lens 5 Sample 6 Stage 7 Pinhole 8 Photomultiplier tube 9 Computer 10 Monitor 100 Scanning microscope

Claims (4)

An objective lens;
A stage on which a sample is placed;
Scanning means for scanning the sample with illumination light;
A pinhole disposed at a position conjugate with the sample;
In a scanning microscope having a detector for detecting the sample through the pinhole,
Driving means for relatively moving the objective lens and the stage;
As a result of being driven by the driving means, an image generating means for acquiring a signal from the detector at an arbitrary position in the optical axis direction of the objective lens and generating image data of the sample;
Based on the image data from the image generation means, gaze area setting means for setting a gaze area to be watched of the sample,
Reference value setting means for setting a reference value for determining the presence or absence of the sample based on the image data of the gaze area set by the gaze area setting means;
The drive means relatively drives the objective lens and the stage, the image generation means acquires a plurality of the image data at different positions in the optical axis direction, and the gaze area image data and the reference value A scanning microscope comprising: a control unit that controls to stop the driving unit when it is determined that the sample does not exist by comparing with a reference value set by a setting unit. Scanning microscope according to claim 1, characterized in that to set the minimum value of the contrast value calculated based on the image data of the region of interest manually via the reference value setting means. Region for calculating the contrast value, based on the Luminance data of the image data of the attention area, the scanning microscope according to claim 2, wherein the fixation region setting means sets. It said drive means, towards a position close to a position farther from the objective lens of said sample, according to any one of claims 1 3, characterized by the step movement at a predetermined interval in the optical axis direction Scanning microscope.

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