JP2009092795A - Method for adjusting lightness of image in imaging type microscope device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for adjusting lightness of an image in an imaging type microscope device capable of rapidly adjusting lightness of an image even when brightness is changed according to an object to be observed. <P>SOLUTION: A user sets the diameter of a pinhole 122 of an observation optical system to the maximum by using a low-powered objective 118b, so as to acquire a microscopic image, first. When the user switches the observation optical system in order to narrow down an observation surface after completing adjustment of brightness, the acquired microscopic image is stored as reference information showing the brightness of the image that is reference. An automatic adjusting means acquires the microscopic image after completing switching of the objective or the observation optical system, reads out the stored reference information on the brightness, compares it with the acquired information on brightness of the image, calculates a gain value of a photodetector 123, and sets the obtained gain value to the photodetector 123, thereby adjusting the brightness of the image after switching. The above processing is repeated until deviation from the reference information showing the brightness of the image is eliminated. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for adjusting the brightness of an image in an imaging microscope apparatus.

  An imaging microscope device that illuminates a sample with light from a light source, converts reflected light or fluorescence from the sample into an electrical signal by a photoelectric conversion device, and forms an image of the sample from the output of the photoelectric conversion device An example is a confocal microscope.

  A confocal microscope is an optical microscope that acquires a microscope image by measuring light from an observation object while two-dimensionally scanning spot light irradiated onto the observation object. For image acquisition, the magnification is adjusted by switching the objective lens according to the observation target, and the brightness of the image is adjusted by adjusting the light quantity value of the light source and the gain value of the light receiving element (photoelectric conversion device). To do. In addition, the pinhole, which is a feature of the confocal microscope, allows only the light from the focused sample surface to pass through. The other light is blocked by the pinhole wall, and the light from other than the focal plane is removed. This is an optical microscope capable of obtaining a clear image. By adjusting the Z direction position (optical axis direction position) of the focal plane, only a specific slice of the sample can be observed. can get.

  The confocal microscope requires adjustment of many observation optical systems as described above, and in order to adjust the change in the brightness of the image caused by switching of the optical parts such as the objective lens, In the confocal microscope, every time the magnification is changed, the brightness of the obtained image is adjusted by adjusting the level of the luminance information signal. For example, it is configured so that a luminance information signal is supplied to the image display device through a manually adjustable variable gain amplifier, and when the brightness of the image changes as the objective lens is switched, the gain of the variable gain amplifier is manually adjusted. By doing so, the image has been adjusted to an optimal brightness.

  As a basic operation method of the confocal microscope, the brightness of the microscope image is first adjusted while adjusting the focal plane with a large pinhole diameter. After adjustment, narrow down the observation surface while gradually reducing the pinhole diameter. By reducing the pinhole diameter, it is possible to obtain only light from a portion closer to the focal plane, and it is possible to obtain a focused image only on a specific plane, but by reducing the pinhole diameter, Since the amount of light obtained is reduced, the image becomes dark, and it is necessary to adjust the light amount of the light source and the sensitivity of the detector again.

  In such manual brightness adjustment, there is a problem that it takes time until an appropriate brightness is obtained. In addition, the adjustments required for switching the objective lens and pinhole are not only brightness, but also focus adjustment, so many adjustments must be made manually each time the objective lens is switched, which is very cumbersome. Was inviting the problem.

  Therefore, in order to be able to automate the adjustment of the brightness change of the image caused by the switching of the objective lens and the observation optical system, the optics corresponding to each of a plurality of combinations of the objective lens and the observation optical system. A function that stores characteristic information and reference information representing the brightness of the reference image, reads the information from the stored data for each observation condition, and sets the light quantity value of the light source and the gain value of the light receiving element. It has come to be mounted on a scanning optical microscope (for example, see Patent Document 1). A method of adjusting the gain of the light receiving element while observing an image (for example, see Patent Document 2) is also known.

JP 2005-156651 A JP 2000-321503 A

  However, image brightness adjustment in the above-described conventional scanning optical microscope has the following problems. When information on optical characteristics corresponding to each of a plurality of combinations of the objective lens and the observation optical system is not stored in the database, automatic adjustment cannot be performed, and manual adjustment must be performed. Therefore, it is necessary to manually adjust the brightness once for each observation condition and register it in the database.

  In addition, the above brightness adjustment seems to be effective for industrial samples such as metals, but in a biological microscope, even if the observation optical system is the same, the observation target is different or the sample is a fluorescent sample. The brightness changes depending on the discoloration. Moreover, since the brightness of cells changes with time, an observation image acquired using a value set by automatic adjustment before is not always appropriate brightness. Therefore, it is necessary to adjust the brightness including not only the observation optical system but also the observation target.

  The present invention has been made in view of such circumstances, and adjustment of image brightness in an imaging microscope apparatus capable of quickly adjusting the brightness of an image even when the brightness changes depending on an observation target. It is an object to provide a method.

A first means for solving the above-described problem is that a sample is illuminated with light from a light source, reflected light or fluorescence from the sample is converted into an electrical signal by a photoelectric conversion device, and an output of the photoelectric conversion device is used. In the imaging microscope apparatus for forming the image of the sample, the brightness of the captured image is adjusted, and after setting the optical system according to the initial conditions, the brightness of the image is controlled by the image brightness control apparatus. Is adjusted to an appropriate value, and the lightness I (0) of the image at that time and the operation amount G (0) given to the image lightness control device are stored, and then one of the optical elements of the optical system is A method for adjusting the brightness of an image in an imaging microscope apparatus, wherein the brightness of the image is automatically adjusted by the following step A every time switching is performed.
Process A
(1) When one of the optical elements of the optical system is switched and an appropriate image is acquired, the brightness I (1) of the image is acquired, and the brightness I (1) of the image and I A step of completing the step A when the difference (0) falls within a predetermined threshold value, and performing the following step B, which is a sub-step of the step A, when the difference does not fall within the predetermined threshold value.
Process B
(2) Using the optical characteristic parameter α _cal (1) stored corresponding to the switched optical element, the manipulated variable G (1) given to the image brightness control device is obtained by the following equation (1). And giving the image brightness control device as an operation amount.
G (1) = I (0) · G (0) / (α _cal (1) · I (1)) (1)
(3) When the difference between the brightness I (k) and I (0) of the image changed as a result of the process of (2) or (4) falls within a predetermined threshold value, the process B is terminated. A new optical characteristic parameter α _cal (k) according to the following equation (2), assuming that k = k + 1. However, the initial value of k is 2.
α _cal (k) = I (k) · G (k-1) / I (k-1) · G (k-2) (2)
(4) An operation amount G (k) to be given to the image brightness control device is obtained by the following equation (1) (where k is a value in step (3)), and an operation amount is given to the image brightness control device. Giving and incrementing the value of k by one.
G (k) = I (0) · G (0) / (α _cal (k) · I (1)) (3)
(5) A step of repeating the steps (3) and (4).

The second means for solving the above problem is that the difference between the changed brightness I (k) and I ₀ in the step (3) in the first means falls within a predetermined threshold. When the process B is completed, the value of α _cal (k) obtained at that time is replaced with the value of the optical characteristic parameter α _cal (1) stored corresponding to the switched optical element. The method further includes a step of adjusting the brightness of the image in the imaging microscope apparatus.

  A third means for solving the problem is the first means or the second means, wherein the imaging microscope apparatus is a confocal microscope.

  A fourth means for solving the problem is any one of the first to third means, wherein the image brightness control device adjusts a gain of the photoelectric conversion device. It is characterized by.

  A fifth means for solving the problem is any one of the first to fourth means, wherein the image brightness control device is a device for adjusting the brightness of the light source. It is a feature.

  According to the present invention, it is possible to provide a method for adjusting the brightness of an image in an imaging microscope apparatus capable of quickly adjusting the brightness of an image even when the brightness changes depending on an observation target.

  Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic configuration diagram of a confocal microscope using an image brightness adjustment method in an imaging microscope apparatus according to an embodiment of the present invention. This microscope system includes a microscope main body 101, a CPU 102, an operation unit 103, a memory 104, and a monitor 105.

  The microscope body 101 includes a light source 111, a dichroic mirror 112, a two-dimensional scanning mechanism 114, a pupil projection lens 115, imaging lenses 116 and 121, an objective lens holding revolver 117, a mirror 120, a pinhole 122, and a light receiving element 123.

  A plurality of objective lenses 118a, 118b, and 118c are attached to the revolver 117. Here, three objective lenses are shown for convenience, but the number of objective lenses attached to the revolver 117 is generally arbitrary. The observation object in the microscope main body 101 is a sample 119, and the light source 111 is a light source for illuminating the sample 119. For example, a laser light source is used.

  In this case, as the observation optical system, a confocal optical system that irradiates the sample 119 with illumination light from the light source 111 and guides the light from the sample 119 to the light receiving element 123 is provided. The light receiving unit of the confocal optical system includes an imaging lens 121, a pinhole 122, and a light receiving element 123. The pinhole 122 is disposed at a position optically conjugate with the imaging position on the sample 119.

  The illumination light emitted from the light source 111 passes through the dichroic mirror 112 and is guided to the two-dimensional scanning mechanism 114. The two-dimensional scanning mechanism 114 scans the spot light in the XY direction on the sample 119 by two-dimensionally scanning the illumination light. As the two-dimensional scanning mechanism 114, for example, a galvanometer mirror is used.

  The illumination light that is XY scanned by the two-dimensional scanning mechanism 114 passes through the pupil projection lens 115 and the imaging lens 116 and is guided to the objective lens 118b. The objective lens is switched by rotating the revolver 117 and changing the objective lens arranged on the optical path of the illumination light. The revolver 117 may be rotated electrically or manually.

  At this time, the illumination light XY-scanned by the two-dimensional scanning mechanism 114 is configured to always pass through the pupil position of the objective lens 118b. The illumination light that has passed through the objective lens 118b is condensed on the sample 119 by the objective lens 118b to become spot light, and the sample 119 is scanned in the XY direction.

  Light from the sample 119 by illumination light is guided to the objective lens 118b, the imaging lens 116, the pupil projection lens 115, and the two-dimensional scanning mechanism 114, converted into non-scanning light, and reaches the dichroic mirror 112. At this time, light is guided by the dichroic mirror 112 toward the light receiving element 123 rather than toward the light source 111.

  The light guided to the light receiving element 123 is only light having a certain wavelength or more in the dichroic mirror 112. The light is guided to the light receiving unit of the confocal optical system including the imaging lens 121, the pinhole 122, and the light receiving element 123.

  The light receiving element 123 is an element that detects luminance information of light, converts it into an electrical signal, and outputs it. As these light receiving elements, for example, a photomultiplier, a photodiode or the like is used. In the case of a photomultiplier, the gain of an electrical signal to be output is determined by adjusting sensitivity according to the applied voltage. In the case of a photodiode, the gain of an electric signal to be output is determined by adjusting a built-in amplifier circuit.

  The electrical signal output from the light receiving element 123 is converted into image data by data processing, and is displayed on the screen of the monitor 105 as a microscope image. At this time, the brightness of the displayed microscope image is determined by the magnitude of the electrical signal output from the light receiving element 123.

  The CPU 102 and the memory 104 correspond to, for example, an information processing apparatus (computer) such as a personal computer, and the memory 104 includes a ROM. The CPU 102 executes a control program using the memory 104 to control the microscope main body 101 and perform processing necessary for image brightness adjustment. As a result, the revolver 117 and the light receiving elements 123 and 124 are controlled by the CPU 102.

  Programs and data used by the CPU 102 can be loaded into the memory 104 via any computer-readable recording medium such as an HDD. Also, these programs and data can be loaded into the memory 104 from an external device via a communication network. In this case, the external device generates a carrier signal for carrying the program and data, and transmits the carrier signal to the confocal microscope via the communication network.

  The operation unit 103 is, for example, an input device such as a keyboard, a pointing device such as a mouse, or a touch panel, and inputs instructions and information from the operator to the CPU 102 via a GUI (not shown). The monitor 105 is a display device that displays a microscope image, and displays a GUI operation screen together with the image.

  2 and 3 are flowcharts showing an outline of an example of an image brightness adjustment method in the imaging microscope apparatus according to the embodiment of the present invention. The operation procedure of the operator and the control processing procedure by the CPU 102 will be described with reference to the flowcharts of FIGS. The operator first sets the sample 119 on the microscope main body 101, activates the microscope system, and starts scanning (step S1). Next, an objective lens and an observation optical system used for observation are set to an initial state (S2). As a basic usage, select a low-magnification objective lens and select the maximum pinhole diameter.

  Then, various settings such as focus adjustment and zoom adjustment of the microscope image are performed, and the microscope image to be observed is displayed on the monitor 105 (step S3). Next, the operator adjusts the brightness of the image (step S4). This adjustment is manually performed via the operation unit 103. The brightness value adjusted by the operation unit 103 is converted to the gain of the currently selected observation optical system (photoelectric conversion device) via the CPU 102 and set in the light receiving element. Then, the brightness I (0) of the image at the time when the adjustment is completed and the gain G (0) that is the operation amount of the light receiving element at that time are stored in the memory as reference values (step S5).

  After the microscope image is adjusted to the optimum brightness, the operator switches one of the optical elements that constitute the objective lens or the observation optical system (step S6). Then, the CPU 102 executes a process A that is a step of adjusting the brightness of the image under the new imaging condition. Details of the process A will be described later (step S7).

  Steps S6 and S7 are repeated until switching of all necessary optical elements is completed. When it is determined in step S8 that all switching has been completed, all processing is terminated.

  Hereinafter, an outline of the processing A will be described with reference to FIG. Process A is performed each time one of the optical elements is switched. First, the operator performs various settings such as focus adjustment and zoom adjustment of the microscope image, and displays the microscope image to be observed on the monitor 105 (step S11). When this adjustment is completed, the CPU 102 acquires the brightness I (1) of the image displayed on the monitor 105 (step S12).

Then, a comparison is made as to whether or not the difference between the image brightness I (1) and the reference image brightness I (0) is less than or equal to a threshold value (step S13). The process proceeds to step S8 If the threshold value is exceeded, the process proceeds to step S14 of the process B, and the stored optical parameter α _cal (1) is used for the image brightness control device (in this example, the light receiving element). Recalculate the gain that is the amount of operation to give.

Here, the optical parameter α _cal corresponds to an adjustment coefficient for each optical element when determining the gain, and a standard value is stored in the memory, and this value is used as an initial value. If not stored, 1 is used as the initial value.

The new gain G (1) is obtained by the following equation (1).
G (1) = I (0) · G (0) / (α _cal (1) · I (1)) (1)
Then, G (1) is given to the light receiving element as a gain which is an operation amount. As a result, the brightness of the image changes to I (k) (in this case, k = 2). The brightness of this image is acquired (step S15).

  Then, it is compared whether or not the difference between the image brightness I (k) and the reference image brightness I (0) is equal to or less than the threshold (step S16). The process proceeds to step S8 If the threshold is exceeded, the process proceeds to step S17 to update the optical parameter.

The new optical parameter α _cal (k) is obtained by the following equation (2).
α _cal (k) = I (k) · G (k-1) / I (k-1) · G (k-2) (2)
However, in this case, k = 2.

When the new optical parameter α _cal (k) is determined, the process proceeds to step S18, and the gain that is the operation amount of the optical element is obtained from the following equation (3) by the following equation (3).
G (k) = I (0) · G (0) / (α _cal (k) · I (1)) (3)
However, k is the same as the value in step S17, and in this case k = 2. This G (k) is given to the light receiving element as a gain which is an operation amount. As a result, the brightness of the image changes to I (k + 1) (in this case, k = 2). Here, the value of k is incremented by 1.

  Thereafter, returning to step S15, the calculation is repeated. At this time, in step S15, step S16, step S17, and S18, each time the value of k is repeated, the value is incremented by 1, and the initial value of k is 2.

  By performing the processing shown in FIGS. 2 and 3, even if the optical element is switched, the brightness of the image is kept within a certain range every time the switching is performed, and the operator can initially set the final adjustment even after the final adjustment. It is kept within a certain range of brightness from the set brightness.

When shifting from step S16 to step S8, the value stored in the memory corresponding to the optical element may be updated with the value of α _cal (k) obtained at that time.

  In the claims and the present specification, examples of the “image brightness control device” include a photoelectric device, a one-dimensional, two-dimensional imaging device, a light source intensity adjustment device, and the like. Is the operation amount. Further, examples of the optical element of the optical system that can be switched include an objective lens, a pinhole, and a filter.

  Although the above description has been given by taking a confocal microscope as an example, the present invention is not limited to a confocal microscope, and a sample is illuminated with light from a light source, and reflected light or fluorescence from the sample is subjected to photoelectric conversion. The present invention can be applied to an imaging microscope apparatus that converts an electrical signal by an apparatus and forms an image of the sample from the output of the photoelectric conversion apparatus.

It is a schematic block diagram of the confocal microscope which uses the adjustment method of the brightness of the image in the imaging type microscope apparatus which is embodiment of this invention. It is a flowchart which shows the outline | summary of the example of the adjustment method of the brightness of the image in the imaging microscope apparatus which is embodiment of this invention. It is a flowchart which shows the outline | summary of the example of the adjustment method of the brightness of the image in the imaging microscope apparatus which is embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 ... Microscope main body, 102 ... CPU, 103 ... Operation part, 104 ... Memory, 105 ... Monitor, 111 ... Light source, 112 ... Dichroic mirror, 114 ... Two-dimensional scanning mechanism, 115 ... Pupil projection lens, 116 ... Imaging lens, 117: Revolver, 118a, 118b, 118c ... Objective lens, 119 ... Sample, 120 ... Mirror, 121 ... Imaging lens, 122 ... Pinhole, 123 ... Light receiving element

Claims (5)

In an imaging microscope device that illuminates a sample with light from a light source, converts reflected light or fluorescence from the sample into an electrical signal by a photoelectric conversion device, and forms an image of the sample from the output of the photoelectric conversion device The method of adjusting the brightness of the captured image, after setting the optical system according to the initial conditions, adjusting the brightness of the image to an appropriate one by the image brightness control device, the image of the image at that time The lightness I (0) and the operation amount G (0) given to the image lightness control device are stored, and thereafter, each time one of the optical elements of the optical system is switched, the image A is A method for adjusting the brightness of an image in an imaging microscope apparatus, wherein the brightness of the image is automatically adjusted.
Process A
(1) When one of the optical elements of the optical system is switched and an appropriate image is acquired, the brightness I (1) of the image is acquired, and the brightness I (1) of the image and I A step of completing the step A when the difference (0) falls within a predetermined threshold value, and performing the following step B, which is a sub-step of the step A, when the difference does not fall within the predetermined threshold value.
Process B
(2) Using the optical characteristic parameter α _cal (1) stored corresponding to the switched optical element, the manipulated variable G (1) given to the image brightness control device is obtained by the following equation (1). And giving the image brightness control device as an operation amount.
G (1) = I (0) · G (0) / (α _cal (1) · I (1)) (1)
(3) When the difference between the brightness I (k) and I (0) of the image changed as a result of the process of (2) or (4) falls within a predetermined threshold value, the process B is terminated. A new optical characteristic parameter α _cal (k) according to the following equation (2), assuming that k = k + 1. However, the initial value of k is 2.
α _cal (k) = I (k) · G (k-1) / I (k-1) · G (k-2) (2)
(4) An operation amount G (k) to be given to the image brightness control device is obtained by the following equation (1) (where k is a value in step (3)), and an operation amount is given to the image brightness control device. Giving and incrementing the value of k by one.
G (k) = I (0) · G (0) / (α _cal (k) · I (1)) (3)
(5) A step of repeating the steps (3) and (4). The difference between the changed brightness I (k) and I ₀ of the image in the step (3) of the image brightness adjustment method in the imaging microscope apparatus according to claim 1 falls within a predetermined threshold. A step of replacing the value of the optical characteristic parameter α _cal (1) stored corresponding to the switched optical element with the value of α _cal (k) obtained at the time of ending the process B The method for adjusting the brightness of an image in an imaging microscope apparatus, further comprising:   The method for adjusting the brightness of an image in the imaging microscope apparatus according to claim 1, wherein the imaging microscope apparatus is a confocal microscope.   The image lightness control device according to any one of claims 1 to 3, wherein the image lightness control device is a device that adjusts a gain of the photoelectric conversion device. Method.   The image brightness control method according to any one of claims 1 to 4, wherein the image brightness control device is a device that adjusts the brightness of the light source. .

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