JP5714997B2 - Fluorescence microscope device - Google Patents

Description

  The present invention relates to a fluorescence microscope apparatus.

  2. Description of the Related Art Conventionally, in a microscope apparatus that acquires an image with an image sensor such as a CCD, the brightness of the entire image or a part of the image is measured, and the exposure time of the image sensor is determined based on the measured value ( For example, see Patent Documents 1 and 2.)

JP 2010-4203 A JP 2000-137168 A

  However, in the case of Patent Document 1, a part of the light guided from the microscope body to the image sensor is branched, and the branched light is measured. Therefore, there are inconveniences that the configuration becomes relatively large and that maintenance by the operator such as adjustment of the optical axis is required. Furthermore, when a fluorescent image is observed with the apparatus of Patent Document 1, since a bright fluorescent region and a dark background region are mixed in the photometric region, the photometric value is darker than the brightness of the fluorescent region. As a result, there is a problem that the imaging device is excessively exposed to the fluorescent region to be observed and the fluorescent region is unclearly displayed.

  On the other hand, the apparatus of Patent Document 2 appropriately adjusts the exposure time for the fluorescent region by measuring the spot region designated by the operator. However, for example, when a minute and complicated structure such as a cytoskeleton is the object of fluorescence observation, it is difficult to specify the spot area accurately so that only the fluorescence area is included, and the spot area includes the background area Will also be included. That is, as in Patent Document 1, there remains a problem that the image sensor is excessively exposed to the fluorescent region. In addition, there is an inconvenience that an operation by the operator is required.

  The present invention has been made in view of the above-described circumstances, and is a microscope capable of acquiring a clear fluorescent image whose exposure is optimally adjusted with respect to the fluorescent region without requiring the operator's effort. An object is to provide an apparatus.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source unit for irradiating excitation light to a stationary object, an imaging unit that pre-imaging and the imaging of the fluorescence that is generated from the subject by the irradiation of the excitation light, which is pre-captured by the image pickup unit A fluorescent region extracting unit that extracts a fluorescent region from a fluorescent image, an exposure time determining unit that determines an exposure time in the main imaging of the imaging unit based on the luminance of the fluorescent region extracted by the fluorescent region extracting unit, and the imaging A control unit that controls an exposure time and an imaging operation of the image capturing unit, and the control unit preliminarily captures the same field of view three times or more with the same exposure time to obtain three or more fluorescent images. the controlled to the imaging with an exposure time determined by the exposure time determination portion after the fluorescent region extraction unit, after the luminance between the three or more fluorescent images pre-shot by the imaging section To decrease continuously, and to provide a fluorescence microscope apparatus for extracting a region having a higher luminance predetermined threshold as the fluorescent region.

  According to the present invention, the imaging unit preliminarily captures the fluorescence excited by the excitation light irradiated to the subject from the light source unit a plurality of times with the same exposure time, and then exposes based on the plurality of preliminarily captured fluorescence images. Control is performed by the control unit so that the main imaging is performed with the exposure time determined by the time determination unit. The exposure time determination unit determines the exposure time of the main imaging based on the luminance of the fluorescent region extracted by the fluorescent region extraction unit from the preliminary captured fluorescent image. Therefore, a fluorescent image in which the exposure is adjusted with respect to the fluorescent region can be acquired by the main imaging without requiring the operator's effort.

  In this case, the fluorescence region extracted by the fluorescence region extraction unit is a region that is dimmed by the fading of fluorescence, and a region that exactly matches the structure of the observation target that generates fluorescence is extracted. As a result, the exposure time determined by the exposure time determination unit becomes an appropriate value for the fluorescent region, and in this imaging, a clear fluorescent image in which the exposure is optimally adjusted for the fluorescent region can be acquired. .

In the above invention, an image processing unit may be provided that removes the luminance of a region corresponding to a background region excluding the fluorescent region extracted by the fluorescent region extracting unit from the fluorescent image actually captured by the imaging unit. Good.
By doing so, it is possible to finally observe a fluorescent image in which the background region is displayed darker and the contrast between the fluorescent region and the background region is further adjusted.

In the aspect described above, the fluorescent region extraction luminance decreases over time, and, in a Turkey to extract region having a higher luminance predetermined threshold as the fluorescent area, is pre-shot In addition, it is possible to remove a region where luminance is reduced due to noise or a region where autofluorescence is reduced between a plurality of fluorescent images from the fluorescent region, and to extract the fluorescent region more accurately.

Moreover, in the said invention, it is good also as providing the light-shielding means which interrupts | blocks the said excitation light with respect to the said to-be-photographed object when the said imaging part is not imaging.
In this way, it is possible to prevent unnecessary discoloration of the fluorescent light by stopping the irradiation of the illumination light to the subject except during the imaging, and a clear fluorescent image can be obtained every time the imaging is repeated.

  According to the present invention, there is an effect that it is possible to acquire a clear fluorescent image in which the exposure is optimally adjusted with respect to the fluorescent region without requiring the labor of the operator.

1 is an overall configuration diagram of a fluorescence microscope apparatus according to an embodiment of the present invention. It is a flowchart explaining the operating method of the fluorescence microscope apparatus of FIG. It is a flowchart explaining the modification of the operating method of the fluorescence microscope apparatus of FIG.

Hereinafter, a fluorescence microscope apparatus 100 according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, a microscope apparatus 100 according to this embodiment includes a microscope main body (hereinafter referred to as a main body) 10 and an imaging unit (imaging unit) that is connected to a camera port 7 of the main body 10 and includes an imaging device 21. ) 20 and a control unit 30 for controlling the operation of the imaging unit 20.

  The main body 10 includes a stage 1 on which a sample (subject) A whose observation target is fluorescently labeled is placed, a light source unit 2 that emits illumination light, an objective lens 3 that is disposed to face the sample A, and an eyepiece. A lens 4 and an optical path 5 that guides illumination light and fluorescence generated from the sample A are provided. Although an upright optical microscope is illustrated as the main body 10 in FIG. 1, an inverted optical microscope or a scanning optical microscope may be used.

The stage 1 can be moved up and down along the optical axis direction of the objective lens 3 by the rotation of the handle 6. The sample A is, for example, a cell fixed in advance, and the observation target is maintained in a stationary state.
The objective lens 3 collects excitation light (described later) incident from the optical path 5 on the sample A and also collects fluorescence from the sample A.

  The light source unit 2 includes a light source 2a such as a xenon lamp or a mercury lamp that emits white illumination light, and a shutter (light shielding unit) 2b that opens or closes an optical path between the light source 2a and the optical path 5 by opening and closing. It has. The shutter 2b is controlled by the control unit 35, which will be described later, to an open state when the image sensor 21 captures an image, and to a closed state when the image sensor 21 does not capture an image. By irradiating the sample A with the excitation light only at the time of imaging as described above, unnecessary fading of fluorescence is prevented, and a sufficiently clear fluorescence image can be obtained every time imaging is repeated.

  The optical path 5 extracts the excitation light from the illumination light from the light source unit 2 and reflects the excitation light extracted by the excitation light filter 5a in the direction along the optical axis of the objective lens 3 and the objective. A dichroic mirror 5b that transmits the fluorescence collected by the lens 3 and a beam splitter 5c that branches the fluorescence transmitted through the dichroic mirror 5b to the camera port 7 and the eyepiece 4 are provided. Reference numeral 5d denotes an absorption filter that cuts excitation light out of light transmitted through the dichroic mirror 5b.

  The imaging unit 20 images the fluorescence branched in the direction of the camera port 7 by the beam splitter 5c by the imaging element 21. The image sensor 21 outputs the captured fluorescent image to the storage unit 31 included in the control unit 30.

  As will be described later, the control unit 30 includes a storage unit 31 that stores first and second fluorescent images captured in the preliminary imaging mode at least temporarily, and a maximum luminance that detects the maximum luminance Imax from the first fluorescent image. The exposure time of the image sensor 21 is determined based on the detection unit 32, the fluorescent region extracting unit 33 that extracts the fluorescent region from the first and second fluorescent images, and the luminance of the fluorescent region extracted by the fluorescent region extracting unit 33. An exposure time determining unit 34 for controlling the exposure time and the imaging operation of the image sensor 21 in the preliminary imaging mode and the main imaging mode.

  The storage unit 31 stores the first fluorescent image and the second fluorescent image captured by the image sensor 21 in the preliminary imaging mode in an identifiable manner by attaching different tags. When a first fluorescent image is newly input from the image sensor 21, the storage unit 31 deletes the first fluorescent image stored so far and replaces it with a new first fluorescent image. Update the fluorescence image.

  The maximum luminance detection unit 32 reads the first fluorescent image stored in the storage unit 31 and detects the maximum luminance Imax among the luminances of the read first fluorescent image. The maximum luminance detection unit 32 outputs the detected maximum luminance Imax to the control unit 35. The maximum luminance detection unit 32 detects the maximum luminance Imax and outputs it to the control unit 35 every time the storage unit 31 updates the first fluorescent image.

  The fluorescence region extraction unit 33 reads the first fluorescence image and the second fluorescence image from the storage unit 31, compares the luminance of corresponding pixels of these images, and compares the second fluorescence image with the second fluorescence image rather than the first fluorescence image. A pixel group having a lower luminance in the fluorescent image is extracted as a fluorescent region. The first fluorescent image and the second fluorescent image are images captured under the same imaging conditions as will be described later. That is, the fluorescence region extraction unit 33 extracts a region that has been dimmed due to fluorescence fading as a fluorescence region. As the luminance of each pixel in the fluorescent region, the luminance in the second fluorescent image is adopted.

  Here, the fluorescent region extracting unit 33 has a predetermined threshold for the luminance of the fluorescent image, extracts a pixel group having a luminance equal to or higher than the predetermined threshold among the dimmed regions as a fluorescent region, and is less than the predetermined threshold A pixel group having luminance may be excluded from the fluorescent region. By doing in this way, the area | region where the brightness | luminance decreased by the weak auto-fluorescence generated from noise other than an observation object is excluded from a fluorescence area | region. As a result, a region that more accurately matches the structure of the observation target can be extracted as a fluorescent region.

  The exposure time determination unit 34 detects the maximum fluorescence luminance Fmax among the luminances of the pixel group constituting the fluorescent region extracted by the fluorescent region extraction unit 33. Then, the exposure time determination unit 34 determines a second exposure time t2 for main imaging based on the detected maximum fluorescence luminance Fmax. The second exposure time t2 is calculated from a relational expression of t2 = k × Isat / Fmax × t1, for example. k is a coefficient greater than 0 and equal to or less than 1, and is set to 0.9, for example. The exposure time determination unit 34 outputs the determined second exposure time t2 to the control unit 35.

  In the preliminary imaging mode, the control unit 35 causes the imaging device 21 to capture the first fluorescent image with the initially set first exposure time t1, and then the maximum luminance Imax input from the maximum luminance detecting unit 32 is predetermined. The imaging device 21 is made to repeat imaging while resetting the first exposure time t1 until a value within the range is reached.

  Specifically, when the maximum luminance Imax is the saturation luminance Isat corresponding to the saturation output voltage of the image sensor 21, the first exposure time t1 is reset to half. On the other hand, when the maximum luminance Imax is smaller than half of the saturation luminance Isat, the first exposure time t1 is reset to a time longer by a predetermined time Δt, for example, a time t1. The control unit 35 repeats the resetting of the first exposure time t1 in this way, and the first exposure time in which the maximum luminance Imax in the fluorescent image is in a range that is not less than half the saturation luminance Isat and smaller than the saturation luminance Isat. t1 is determined. That is, the first exposure time t <b> 1 is determined such that the exposure amount of the strongest fluorescent portion of the observation target is within half or more of the dynamic range of the image sensor 21.

  As a result of repeating the resetting of the first exposure time t1, the control unit 35 determines that the first exposure time t1 when the first exposure time t1 is greater than a predetermined maximum time tmax, for example, 1 second. In place of the resetting, the pixels of the image sensor 21 are binned, and the number of bins is determined such that the exposure amount of the strongest fluorescent part of the observation target falls within half or more of the dynamic range of the image sensor 21.

  Specifically, the control unit 35 adds the luminances of a predetermined number of pixels adjacent to the image sensor 21, for example, four pixels adjacent in the vertical direction and the horizontal direction, and sets the plurality of pixels as one pixel. It is considered. The control unit 35 gradually increases the number of pixels to be binned (binning number) until the maximum luminance Imax is within a predetermined range among the added luminances. Thus, by providing an upper limit for the first exposure time t1, it is possible to prevent an increase in noise and image blur due to an excessively long exposure time.

Next, the control unit 35 causes the image sensor 21 to pre-capture the second fluorescent image with the determined first exposure time t1 and the number of binning, and ends the pre-imaging mode. Accordingly, the storage unit 31 stores the first and second fluorescent images having the same first exposure time t1, binning number, and field of view, which are used by the fluorescent region extracting unit 33 to extract the fluorescent region.
Next, the control unit 35 shifts to the main imaging mode, and after the second exposure time is determined by the exposure time determination unit 34, the image sensor 21 is set with the second exposure time t2 and the binning number determined by the preliminary imaging. Let's take a real image.

Next, the operation of the fluorescence microscope apparatus 100 configured as described above will be described with reference to FIG.
The fluorescence microscope apparatus 100 according to the present embodiment starts preliminary imaging when, for example, an operator instructs the start of the preliminary imaging mode from an input unit (not shown).

  First, the control unit 35 causes the imaging device 21 to preliminarily image fluorescence with the first exposure time t1 (step S1), and the maximum luminance Imax in the fluorescent image detected by the maximum luminance detection unit 32 (step S2) is saturated. If it is (YES in step S3), the first exposure time t1 is shortened to half (step S4), and if the maximum luminance Imax is smaller than half the saturation luminance Isat (NO in step S5), the first exposure The time t1 is extended by the time Δt (step S7), and the first fluorescent image is again preliminarily captured at the reset first exposure time t1 (step S1). Thereby, the first exposure time t1 at which the observation target is imaged sufficiently clearly in the first fluorescence image is determined.

  When the first exposure time t1 becomes longer than the predetermined maximum time tmax due to the extension of the first exposure time t1 (YES in step S6), the control unit 35 first reset the first exposure. The time t1 is maintained, the pixels of the image sensor 21 are binned (step S8), and the number of binning in which the observation target is imaged sufficiently clearly in the first fluorescent image is determined. The control unit 35 acquires the second fluorescent image by preliminarily capturing the fluorescence again with the determined first exposure time t1 and the number of binning, and ends the preliminary imaging mode (step S9).

  Next, the fluorescent region extraction unit 33 extracts a region that is dimmed between two fluorescent images preliminarily imaged under the same imaging condition as a fluorescent region (step S10). The exposure time determination unit 34 detects the maximum fluorescence brightness Fmax of the extracted fluorescence region (step S11), and determines the second exposure time t2 of the main imaging based on the maximum fluorescence brightness Fmax (step S12). The control unit 35 causes the image sensor 21 to perform the main imaging in the main imaging mode at the second exposure time t2 input from the exposure time determination unit 34 (step S13). The actually captured fluorescent image is output to the display unit 40. Thereby, the operator can observe the final fluorescence image whose exposure is adjusted.

  As described above, according to the present embodiment, a fluorescent region is extracted from two fluorescent images that have been preliminarily imaged based on the fading of fluorescence, and the second exposure time t2 of the main imaging with respect to the extracted fluorescent region. Is determined. Since the extracted fluorescent region is a region that exactly matches the structure of the observation target, the second exposure time t2 is determined to an appropriate value with respect to the fluorescent region, and fluorescence from the observation target is optimally exposed. An image can be taken. In addition, it is possible to optimize the exposure of the image sensor 21 with only a simple operation without requiring a special operation by the operator.

  In the present embodiment, the exposure time determination unit 34 determines the second exposure time t2 based on the maximum fluorescence brightness Fmax in the extracted fluorescence region, but instead of this, the fluorescence region The second exposure time t2 may be determined on the basis of the average value of the luminance of the pixel group that constitutes.

  According to the fluorescent region extraction method by the fluorescent region extraction unit 33, even when the observation target has a fine and complicated structure, a region that exactly matches the structure of the observation target is extracted as the fluorescent region, and the observation target It is prevented that a background area that does not contain the light is extracted together with the fluorescent area. Therefore, even if the second exposure time t2 is determined based on the average value of the brightness of the entire extracted fluorescent region, the second exposure time t2 does not become excessive with respect to the fluorescent region, and the optimum exposure is achieved. Real imaging can be performed.

  In the present embodiment, an image processing unit (not shown) that corrects the luminance of the background region other than the fluorescent region to a sufficiently small value in the fluorescent image that has been actually captured may be provided. Specifically, as shown in FIG. 3, the fluorescent region is extracted (step S10) and the background region other than the fluorescent region is extracted (step S14). And the brightness | luminance of the area | region corresponding to a background area | region among the fluorescence images actually imaged is removed to a sufficiently small value, Preferably it is 0 (step S15). In this way, the background generated by the fluorescence generated from other than the observation target and the electrical noise of the image sensor 21 is removed, and a fluorescent image with a better contrast between the fluorescent region and the background region is finally obtained. Can be observed.

In the present embodiment, the fluorescence region is extracted by comparing two fluorescence images that have been preliminarily imaged, but instead, three or more fluorescence images are preliminarily imaged, An area where the luminance continuously decreases may be extracted as a fluorescent area.
By doing in this way, the influence of the fluctuation | variation of the brightness | luminance of each pixel by the noise etc. of the image pick-up element 21 can be reduced, and a fluorescence area | region can be extracted more correctly.

  In the present embodiment, the main imaging may be repeated a plurality of times. In this case, the exposure time for the second and subsequent main imaging may be determined based on the maximum fluorescence brightness Fmax of the fluorescence image that was captured immediately before, so there is no need to perform preliminary imaging again.

DESCRIPTION OF SYMBOLS 1 Stage 2 Light source part 2a Light source 2b Shutter (light-shielding means)
DESCRIPTION OF SYMBOLS 3 Objective lens 4 Eyepiece 5 Optical path 5a Excitation light filter 5b Dichroic mirror 5c Beam splitter 5d Absorption filter 6 Handle 7 Camera port 10 Microscope main body 20 Imaging unit (imaging part)
DESCRIPTION OF SYMBOLS 21 Image pick-up element 30 Control unit 31 Memory | storage part 32 Maximum brightness | luminance detection part 33 Fluorescence area extraction part 34 Exposure time determination part 35 Control part 40 Display part 100 Fluorescence microscope apparatus A Sample (subject)

Claims (3)

A light source unit that emits excitation light to a stationary subject;
An imaging unit that performs preliminary imaging and main imaging of fluorescence generated from the subject by irradiation of the excitation light; and
A fluorescence region extraction unit that extracts a fluorescence region from a fluorescence image preliminarily captured by the imaging unit;
An exposure time determining unit that determines an exposure time in the main imaging of the imaging unit based on the luminance of the fluorescent region extracted by the fluorescent region extracting unit;
A control unit for controlling an exposure time and an imaging operation of the imaging unit,
The control unit captures the imaging unit at the exposure time determined by the exposure time determination unit after acquiring the three or more fluorescent images by performing preliminary imaging of the same field of view three times or more with the same exposure time. To control and
The fluorescent region extracting unit is configured to determine a region having a luminance that continuously decreases between the three or more fluorescent images preliminarily captured by the imaging unit and has a luminance equal to or higher than a predetermined threshold value. Fluorescence microscope device that extracts as a fluorescent region.   The fluorescence microscope according to claim 1, further comprising: an image processing unit that removes luminance of a region corresponding to a background region excluding the fluorescent region extracted by the fluorescent region extracting unit from the fluorescent image actually captured by the imaging unit. apparatus. Fluorescence microscope apparatus according to claim 1 or claim 2 comprising a light-blocking means for blocking the excitation light to the subject when the imaging unit is not captured.

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