JP2014085599A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope capable of executing fast and accurate auto-focus processing.SOLUTION: A microscope generates observation image data by collecting light from a sample placed on a stage using an object lens and capturing an image of the sample using the collected light. The microscope includes: a light source which emits illumination light for illuminating a sample; an image sensor which has a plurality of pixels for receiving the light from the sample and captures an image of the sample; and a control unit which splits the plurality of pixels into a predefined number of pixel groups comprising adjacent pixels, provides control to automatically focus the sample image by using each split pixel group as one pixel, and provides exposure control for the image sensor in accordance with the number of pixels that constitute one pixel group.

Description

  The present invention relates to a microscope having a function of forming an image of a sample to be observed and capturing the image.

  2. Description of the Related Art In recent years, in the field of a microscope, an image pickup apparatus such as a digital camera having an autofocus (AF) function is mounted and an image captured by the image pickup apparatus is displayed on a display unit. In AF processing of an image pickup apparatus, a method (contrast AF) in which adjacent pixels in an image pickup device such as a CCD are compared and contrast calculation is performed, and a position where the contrast calculation value is maximized is used as a focus position (contrast AF) has been put into practical use .

  In contrast AF in microscopic observation, imaging is performed while changing the position of a focusing unit such as a stage that moves up and down by electric control (hereinafter referred to as Z position), and then Z position information is obtained from a control unit such as a CPU. A hill-climbing method is widely put into practical use in which the Z position information is acquired and registered as image position information, and the in-focus position where the contrast value is maximized is obtained from the result of contrast calculation. In contrast calculation in this hill-climbing method, calculation is generally performed by averaging the difference between adjacent pixels or the square of the difference between adjacent pixels over the entire pixel or a specified range.

  As an example of contrast AF, a technique for performing contrast AF while moving the stage from the AF start position at a fine pitch is disclosed (for example, see Patent Document 1). In this technique, in order to speed up contrast AF, a coarse focus operation with a large stage drive pitch (hereinafter referred to as “Rough scan”) and a fine focus operation with a small stage drive pitch (hereinafter referred to as “Fine scan”). The following two types of AF processing are performed. More specifically, in this technique, after finding a rough focus position by the Rough scan, an accurate focus position is found by the Fine scan. In this technique, the image data captured by the imaging device is acquired and the contrast calculation is performed, so the AF speed is determined by the imaging interval (hereinafter referred to as the frame rate). In recent years, image pickup devices tend to increase the number of pixels in order to improve the image quality of a microscope image, and the data transfer time also increases according to the number of pixels. For this reason, there is a problem that the frame rate cannot be increased and AF takes time.

  As a technique for shortening the AF processing time, there is a technique (binning function) in which a plurality of pixels of the image sensor are grouped together, the group of pixels is regarded as one pixel, the data size is reduced, and the frame rate is increased. Are known. In the binning function, since a plurality of pixels are regarded as one pixel, the light receiving area can be increased, and the sensitivity of the image sensor can be increased.

  However, when this binning function is used, the amount of received light per unit light receiving pixel increases and the luminance value increases. As a result, time is required for automatic exposure (AE) adjustment before and after execution of the binning function, and as a result, time may be required for AF processing.

  In order to solve the above-described problem of the AF processing time, a technique for determining the amount of illumination light and the exposure time according to the current frame rate is disclosed (for example, see Patent Document 2). According to this technique, AF processing can be performed quickly.

JP-A-10-197784 JP 2008-139796 A

By the way, in the case of a general imaging device, the imaging time interval needs to satisfy one of the following two conditions (A) and (B).
(A) Exposure time <imaging time interval and imaging data transfer time <exposure time (B) Imaging data transfer time <imaging time interval and exposure time <imaging data transfer time Here, FIG. FIG. 5 is a time chart showing output timing (condition (A)) of each signal of a conventional synchronization signal, camera shutter signal, and data transfer signal. 10 and 11 are time charts showing the output timing (condition (B)) of each signal of the conventional synchronization signal, camera shutter signal, and data transfer signal. 9 to 11, the frame synchronization signal corresponds to the imaging time interval, the output period of the camera shutter signal corresponds to the exposure time, and the output period of the data transfer signal corresponds to the data transfer time of the imaging data. The numbers in parentheses correspond to the respective frame numbers.

  In Patent Document 2, in the case of the condition (A) in which the imaging time interval is restricted by the exposure time, light source control and exposure time control according to the frame rate are effective.

  However, if the amount of light received is large and the data transfer time is longer than the camera shutter time (exposure time) (B), even if the ratio between the exposure time and the imaging time interval is different at the same frame rate, this difference Is taken into consideration, and light source control and exposure time control according to the frame rate are performed. As a result, a difference occurs in the obtained luminance value depending on the ratio between the exposure time and the imaging time interval, and there is a problem in that AF processing cannot be performed with an appropriate luminance value.

  The present invention has been made in view of the above, and an object of the present invention is to provide a microscope capable of executing a quick and accurate autofocus process.

  In order to solve the above-described problems and achieve the object, a microscope according to the present invention condenses light from a sample placed on a stage by an objective lens, and the above-described light is collected based on the collected light. A microscope that generates image data for observation by taking an image of a sample, and includes a light source that emits illumination light that illuminates the sample, and a plurality of pixels that receive light from the sample, An image sensor that captures a sample image and the plurality of pixels are divided into a predetermined number of pixel groups including adjacent pixels, and the sample image is automatically regarded as one pixel. And a control unit that performs focusing control and performs exposure control of the imaging device in accordance with the number of pixels constituting the pixel group.

  The microscope according to the present invention is characterized in that, in the above invention, the control unit performs exposure control by changing either an exposure time of the image sensor or brightness of the light source.

  In the microscope according to the present invention, in the above invention, the control unit divides the exposure time before focusing or the brightness of the light source before focusing by the number of pixels constituting the pixel group. The focusing is performed using the quotient obtained by the division.

  The microscope according to the present invention is characterized in that, in the above invention, the control unit determines whether to perform exposure control according to an observation method set in the microscope.

  In the microscope according to the present invention as set forth in the invention described above, the control unit determines whether to perform exposure control according to the magnification of the objective lens.

  In the above invention, the microscope according to the present invention is an optical filter provided between the light source and the sample and capable of adjusting the brightness of the light source, and an optical diaphragm for adjusting the diaphragm of the illumination light. The control unit performs exposure control by changing any one of the exposure time of the image sensor, the brightness of the light source, the optical filter, and the optical diaphragm.

  According to the present invention, a light source that emits illumination light for illuminating a sample, a plurality of pixels that receive light from the sample, an imaging element that captures an image of the sample, and a predetermined number of the plurality of pixels And a control unit that performs autofocusing that automatically focuses the sample image by regarding the divided pixel as one pixel, and the control unit captures an image according to a predetermined number. Since the exposure control of the element is performed, there is an effect that a quick and accurate autofocus process can be executed.

FIG. 1 is a schematic diagram illustrating an overall configuration of a microscope according to the first embodiment of the present invention. FIG. 2 is a flowchart showing AF processing performed by the microscope according to the first embodiment of the present invention. FIG. 3 is a flowchart showing a focusing process performed by the microscope according to the first embodiment of the present invention. FIG. 4 is a diagram for explaining a focusing process performed by the microscope according to the first embodiment of the present invention. FIG. 5 is a schematic diagram illustrating an overall configuration of a microscope according to the second embodiment of the present invention. FIG. 6 is a diagram illustrating an example of data stored in the storage unit of the microscope according to the second embodiment of the present invention. FIG. 7 is a flowchart showing AF processing performed by the microscope according to the second embodiment of the present invention. FIG. 8 is a diagram illustrating an example of data stored in the storage unit of the microscope according to the modification of the second embodiment of the present invention. FIG. 9 is a time chart showing the output timing of each signal of the conventional synchronization signal, camera shutter signal, and data transfer signal. FIG. 10 is a time chart showing the output timing of each signal of the conventional synchronization signal, camera shutter signal, and data transfer signal. FIG. 11 is a time chart showing the output timing of each signal of the conventional synchronization signal, camera shutter signal, and data transfer signal.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment. The drawings referred to in the following description only schematically show the shape, size, and positional relationship so that the contents of the present invention can be understood. That is, the present invention is not limited only to the shape, size, and positional relationship illustrated in each drawing.

(Embodiment 1)
FIG. 1 is a schematic diagram illustrating an overall configuration of a microscope according to the first embodiment of the present invention. The microscope 1 shown in FIG. 1 has an autofocus (AF) function, and includes a microscope mount 2, a stage 3, a light projection tube 4, an imaging device 5, a control device 6, and a display device 7.

  The microscope mount 2 includes a stage support portion 21 that fixes and supports the stage 3, a light projection tube support portion 22 that supports the light projection tube 4 so as to move up and down, and a drive motor that controls the vertical movement of the light projection tube 4. And a control unit 23.

  The drive motor control unit 23 moves the light projecting tube 4 and the imaging device 5 up and down by driving a drive motor 45 included in the light projecting tube 4 based on the motor drive amount sent from the control device 6.

  The light projecting tube 4 includes an imaging optical system 41, a light source 42, a light source control unit 43, a half mirror 44, and a drive motor 45. The light projecting tube 4 holds the objective lens 8.

  The light (illumination light) emitted from the light source 42 is reflected by the half mirror 44 and applied to the sample Sp placed on the stage 3 via the objective lens 8. On the other hand, the light reflected by the sample Sp passes from the objective lens 8 via the half mirror 44, passes through the imaging optical system 41, is condensed by the image sensor 51, and is photoelectrically converted into an electric signal.

  The drive motor 45 has a function of a drive unit that drives the light projecting tube 4 and the imaging device 5. Hereinafter, the light projecting tube 4 and the imaging device 5 are collectively referred to as a “head portion”.

  The imaging device 5 includes an imaging device 51 such as a CCD or a CMOS, a signal processing unit 52 that performs signal processing such as A / D conversion on a signal acquired by the imaging device 51, and the imaging device 5 (the imaging device 51 and the signal). And an imaging control unit 53 that controls the operation of the processing unit 52).

  The control device 6 includes an input unit 61, a contrast calculation unit 62, a storage unit 63, and a control unit 64. The contrast calculation unit 62 receives image data from the imaging device 5 and performs contrast calculation for calculating a contrast value of the image data.

  The storage unit 63 stores a program (application software) that controls the operation of the microscope 1. In addition, the storage unit 63 stores information related to the imaging process (frame rate, exposure time (shutter speed), brightness of the light source 42, gain, etc.), and information related to the AF process (binning number (n × n), etc.). Remember. The storage unit 63 is realized by a semiconductor memory such as a flash memory, a RAM, and a ROM, a recording medium such as an HDD, an MO, a CD-R, and a DVD-R, and a driving device that drives the recording medium.

  The control unit 64 is configured using a CPU or the like, and comprehensively controls the operation of the microscope 1 as a whole. The control unit 64 performs autofocus for automatically focusing the image of the sample Sp. The control unit 64 writes and stores the maximum value of the calculation result by the contrast calculation unit 62 and the Z coordinate when the maximum value is obtained in the storage unit 63. The control unit 64 transmits an instruction signal for adjusting the brightness of the light source 42 to the light source control unit 43.

  The display device 7 includes a monitor screen for displaying a microscope image and the like, and a microscope operation GUI touch panel. A touch panel is laminated | stacked on a monitor screen, and receives the input signal according to the contact position from the outside.

  In the microscope 1 having the above configuration, when an instruction to perform autofocus (AF) processing is input to the input unit 61 via the touch panel, AF processing is executed under the control of the control unit 64. FIG. 2 is a flowchart illustrating AF processing performed by the microscope 1 according to the first embodiment.

  First, when an instruction to perform autofocus (AF) processing is input, the control unit 64 turns on the binning function (step S101). At this time, the control unit 64 refers to the storage unit 63 and acquires the binning number (n × n) for the binning function.

Subsequently, the control unit 64 refers to the storage unit 63 and acquires the currently set exposure time (step S102). When acquiring the binning number (n × n) and the currently set exposure time T ₁ , the control unit 64 determines whether or not T ₁ / (n × n)> T ₀ (step S103). . Here, T ₀ is the minimum exposure time, which is the shortest exposure time that can be set in the microscope 1.

When the control unit 64 determines that T ₁ / (n × n)> T ₀ is satisfied (step S103: Yes), the control unit 64 proceeds to step S104 and sets the exposure time to T ₁ / (n × n). .

In contrast, when the control unit 64 determines that T ₁ / (n × n) <T ₀ (step S103: No), the control unit 64 proceeds to step S105, and sets the brightness B ₀ of the currently set light source. Acquire and set the brightness of the light source to B ₀ / (n × n).

  When the exposure time or the light source brightness is reset in step S104 or step S105, the control unit 64 refers to the storage unit 63 and acquires the currently set frame rate (step S106).

  After acquiring the frame rate, the control unit 64 performs a focusing process described later (step S107). Thereafter, when the focusing process is completed, the control unit 64 turns off the binning function (step S108), returns to the exposure time before setting or the brightness of the light source in step S104 or step S105, and ends the process (step S109). ).

  Here, the focusing process in step S107 of FIG. 2 will be described with reference to FIGS. FIG. 3 is a flowchart illustrating the focusing process performed by the microscope according to the first embodiment. FIG. 4 is a diagram for explaining a focusing process performed by the microscope according to the first embodiment.

  The control unit 64 sends a signal to the drive motor control unit 23 to drive the drive motor 45 so that the head unit moves to a predetermined start position (step S201). For example, the head unit moves a distance of AF range / 2 toward the Near direction by driving the drive motor 45.

  Next, the control unit 64 outputs an instruction signal for driving the drive motor 45 at a predetermined coarse search pitch to the drive motor control unit 23 (step S202). The head unit moves in the Far direction at a rough search pitch by driving of the drive motor 45.

  Subsequently, the control unit 64 acquires image data captured by the imaging device 5 (step S203) and performs contrast calculation (step S204). Thereby, the contrast curve CA by the rough search is obtained. At this time, the control unit 64 acquires image data using the frame rate acquired in step S106 described above.

Thereafter, the control unit 64 determines whether or not a search end condition is satisfied (step S205). The conditional expression for the search end condition is given by the following expression (1).
Contrast calculation peak value × 1/2> current contrast value (1)
Note that another value smaller than 1 may be applied instead of 1/2 of the left side. Further, instead of applying the expression (1) as the search end condition, for example, the search may be ended when the value of the contrast calculation becomes small a predetermined number of times after the peak value is exceeded.

  When the search condition is satisfied in step S205 (step S205: Yes), the control unit 64 proceeds to step S206. On the other hand, when the search condition is not satisfied in step S205 (step S205: No), the control unit 64 returns to step S202.

  In step S206, the control unit 64 calculates a fine search start position that is a position returned by a predetermined distance Dp in the Near direction from the detected peak position (detected peak position) (step S206).

  Subsequently, the control unit 64 sends a signal to the drive motor control unit 23 to drive the drive motor 45 to move the head unit to the fine search start position calculated in step S206 (step S207).

  Thereafter, the control unit 64 transmits a signal to the drive motor control unit 23 to drive the drive motor 45 at a predetermined fine search pitch (step S208). Thereafter, the control unit 64 acquires image data captured by the imaging device 5 (step S209), and performs contrast calculation (step S210). Thereby, a contrast curve CB by fine search is obtained. At this time, the control unit 64 acquires image data using the frame rate acquired in step S106 described above.

  Subsequently, the control unit 64 determines whether or not the search end condition of the above formula (1) is satisfied (step S211). As a result of the determination, if the search end condition is satisfied (step S211: Yes), the head unit is moved to the detection peak position (step S212), and the series of focusing processes is ended. On the other hand, when the search condition is not satisfied in step S211 (step S211: No), the control unit 64 returns to step S208.

  According to the first embodiment described above, in the AF process, the focusing process is performed using the binning function, and the exposure time or the brightness of the light source is reduced according to the number of binning. An increase in contrast value (brightness value) when turned on can be suppressed, and quick and accurate autofocus processing can be executed.

  In the first embodiment described above, the exposure time and the brightness of the light source have been described as the subject of exposure control. However, in addition to the exposure time (shutter speed) and the light source brightness (gain), the optical for controlling the amount of light. Examples include filter insertion / extraction, optical aperture control, and the like, and at least one of them is applied. In addition, the optical filter which controls light quantity selects the filter from which performance differs according to the number of binning.

  Further, in the first embodiment described above, it has been described that the binning number is divided and reduced with respect to the set exposure time or the brightness of the light source. However, the exposure time or the light source according to the binning number is reduced. As long as the brightness is reduced, for example, it is also possible to apply a predetermined coefficient after dividing the number of binning.

(Embodiment 2)
FIG. 5 is a schematic diagram showing an overall configuration of a microscope 1a according to the second embodiment of the present invention. In addition, the same code | symbol is attached | subjected to the component same as the structure demonstrated in FIG. The microscope 1a according to the second embodiment includes a control device 6a including a storage unit 63a and a control unit 64a instead of the control device 6 described above.

  The storage unit 63a stores a program (application software) that controls the operation of the microscope 1a. The storage unit 63a also stores information related to the imaging process (frame rate, exposure time (shutter speed), brightness of the light source 42, gain, etc.), and information related to the AF process (binning number (n × n), etc.). In addition, exposure control data 631 is stored.

  FIG. 6 is a diagram illustrating an example of data stored in the storage unit 63a of the microscope 1a according to the second embodiment. The exposure control data 631 stored in the storage unit 63a is, for example, a table shown in FIG. 6, and is associated with information on whether or not to perform exposure control for the observation method. In the table of FIG. 6, when the observation method is a bright field observation method (BF) and a differential interference observation method (DIC), exposure control is performed, and the observation method is a dark field observation method. In the case of (DF: Dark Field microscopy) and simple polarization observation method (PO: Polarizing microscope), the association is made such that exposure control is not performed.

  The control unit 64a refers to the above-described table, determines whether or not to perform exposure control during the AF process, and executes the AF process. FIG. 7 is a flowchart illustrating AF processing performed by the microscope 1a according to the second embodiment.

  First, when an instruction to perform autofocus (AF) processing is input, the control unit 64a turns on the binning function (step S301). At this time, the control unit 64a refers to the storage unit 63a to obtain the binning number (n × n) related to the binning function.

  Subsequently, the control unit 64a refers to the storage unit 63a and acquires the currently set exposure time (step S302). When the control unit 64a acquires the binning number (n × n) and the exposure time, the control unit 64a acquires the on / off information of the exposure control setting for the current observation method from the table of the storage unit 63a, and turns it on. / Off is determined (step S303).

When the exposure control setting for the current observation method is on (step S303: No), the control unit 64a determines that exposure control is performed with reference to the exposure control data 631 stored in the storage unit 63a, and the process proceeds to step S304. To do. Thereafter, the control unit 64a determines whether or not T ₁ / (n × n)> T ₀ is satisfied (step S304). Here, the minimum exposure time is the shortest exposure time that can be set in the microscope 1a.

When the control unit 64a determines that T ₁ / (n × n)> T ₀ holds (step S304: Yes), the control unit 64a proceeds to step S305 and sets the exposure time to (currently set exposure time) / ( n × n).

On the other hand, when the control unit 64a determines that T ₁ / (n × n) <T ₀ (step S304: No), the control unit 64a proceeds to step S306 to change the brightness of the light source to B ₀ / (n Xn).

  On the other hand, in step S303, when the exposure control setting for the current observation method is off (step S303: Yes), the control unit 64a does not perform exposure control with reference to the exposure control data 631 stored in the storage unit 63a. Determination is made, and the process proceeds to step S307.

  When the control unit 64a determines in step S305 or step S306 that the exposure time or the light source brightness is reset or in step S303 that exposure control is not performed, the frame rate currently set with reference to the storage unit 63a is determined. Is acquired (step S307).

  After acquiring the frame rate, the control unit 64a performs the above-described focusing process (step S308). Thereafter, when the focusing process is completed, the control unit 64a turns off the binning function (step S309), returns to the exposure time before setting in step S305 or step S306 or the brightness of the light source, and ends the process (step S310). ). If the exposure control is not performed, the exposure time or the brightness of the light source is not changed, or if a set value is set, the set value is changed.

  According to the second embodiment described above, in the AF process, the focusing process is performed using the binning function, and the exposure time or the brightness of the light source is reduced according to the number of binning. An increase in contrast value (brightness value) when turned on can be suppressed, and quick and accurate autofocus processing can be executed.

  Further, according to the second embodiment described above, since the AF process is performed by determining whether or not the exposure control is performed by the observation method, the scattered light is received as in the dark field observation method. In the case of an observation method with a small S / N, it is possible to execute autofocus processing while suppressing the influence of noise.

  FIG. 8 is a diagram illustrating an example of data stored in the storage unit 63a of the microscope 1a according to the modification of the second embodiment. In Embodiment 2 described above, whether or not exposure control is performed according to the observation method is associated. However, in each observation method, exposure control is performed according to the magnification of the objective lens 8 to be used. Also good. For example, in the table shown in FIG. 8, when the observation method is DF, exposure control is not performed regardless of the magnification. However, when the observation method is BF, exposure control is performed when the magnification is × 1 to × 4, and the magnification is In the case of × 16, it is set not to perform exposure control. Thus, in the same observation method, it may be possible to set whether or not to perform exposure control according to the S / N according to the magnification.

  In addition to this modification, the necessity of exposure control may be set according to the sample.

  In the above-described embodiment, an erecting microscope has been described as an example. For example, an inverted microscope that performs AF processing to capture a sample image of a sample, an imaging function that captures a sample, and an image are displayed. The present invention can also be applied to an imaging apparatus having a display function to be performed, such as a video microscope.

  As described above, the microscope according to the present invention is useful for executing a quick and accurate autofocus process.

DESCRIPTION OF SYMBOLS 1,1a Microscope 2 Microscope mount 3 Stage 4 Projection tube 5 Imaging device 6, 6a Control device 7 Display device 8 Objective lens 21 Stage support part 22 Projection tube support part 23 Drive motor control part 41 Imaging optical system 42 Light source 43 Light source control unit 44 Half mirror 45 Drive motor 51 Imaging element 52 Signal processing unit 53 Imaging control unit 61 Input unit 62 Contrast calculation unit 63, 63a Storage unit 64, 64a Control unit 631 Exposure control data Sp sample

Claims (6)

A microscope that collects light from a sample placed on a stage by an objective lens and generates image data for observation by capturing an image of the sample based on the collected light,
A light source that emits illumination light for illuminating the sample;
An image sensor that has a plurality of pixels that receive light from the sample, and that captures an image of the sample;
The plurality of pixels are divided into a predetermined number of pixel groups including adjacent pixels, the divided pixel group is regarded as one pixel, and control for automatically focusing the image of the sample is performed. A control unit that performs exposure control of the image sensor according to the number of pixels constituting the group;
A microscope comprising:   The microscope according to claim 1, wherein the control unit performs exposure control by changing either an exposure time of the image sensor or a brightness of the light source.   The control unit divides the exposure time before focusing or the brightness of the light source before focusing by the number of pixels constituting the pixel group, and uses the quotient obtained by the division. The microscope according to claim 2, wherein focusing is performed.   The microscope according to claim 1, wherein the control unit determines whether or not to perform exposure control according to an observation method set in the microscope.   The microscope according to claim 4, wherein the control unit determines whether to perform exposure control according to a magnification of the objective lens. An optical filter provided between the light source and the sample and capable of adjusting the brightness of the light source;
An optical diaphragm for adjusting the diaphragm of the illumination light;
Further comprising
The microscope according to claim 1, wherein the control unit performs exposure control by changing any one of an exposure time of the imaging element, brightness of a light source, the optical filter, and the optical diaphragm.

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