JP2011247656A - Fluorescence detection device and fluorescence microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence detection device and fluorescence microscope that are capable of correcting a fading based on a spatial position in the field of view.SOLUTION: The fluorescence detection device and fluorescence microscope are configured to detect a fluorescent image of a sample generated by emitting excitation light to a sample dyed with fluoresce using an image detector. The fluorescent detection device is provided with means for correcting a fluorescent fading of the sample image for each of the positions of the sample image detected by the image detector in a position.

Description

  The present invention relates to a fluorescence detection device and a fluorescence microscope, and more particularly to a fading correction of a fluorescently stained sample.

  For example, as a powerful technique for photographing cells with a microscope, a specific portion of a sample is fluorescently stained with a fluorescent indicator, such as staining of cells with GFP (Green Fluorescent Proteins), and laser light is applied to the fluorescently stained sample. A method has been established in which two-dimensional intensity information of fluorescence generated by a sample is detected as an image.

  However, one of the weak points of this method is a phenomenon called fluorescent fading. This is a phenomenon in which when the intensity of a laser that excites a fluorescent substance is high, the structure of the fluorescent substance is modified, the fluorescent intensity is weakened, and the color fades.

  In observation with a fluorescence microscope, it is important to accurately measure the amount of fluorescence of a sample. This is because it is desired to quantify how much the fluorescent substance is distributed in which part of the cell. However, when there is a fading phenomenon, the amount of fluorescence decreases, and only a measurement value lower than the original measurement amount can be obtained.

  In general, the amount of fading depends on the amount of laser irradiation and shows a non-linear property with respect to the intensity. Even with the same integral amount, the fading is stronger when the strong light is applied for a short time than when the weak light is applied for a long time. Have.

  In order to perform accurate photometry, for example, as described in Patent Document 1, there is an idea of quantifying and correcting fading. FIG. 6 is an example of fading characteristics of the fluorescent dye described in Patent Document 1. In FIG. 6, Tl is the time required to scan the sample from the scan start wavelength to the scan end wavelength, and Ti is the time at the i-th scan, and the luminance value at that time is expressed as F (Ti). Δt is a time required to scan the sample once.

  Patent Document 1 proposes a technique of measuring discoloration due to laser irradiation and performing correction depending on the integrated irradiation amount based on the measurement result. In the technique described in Patent Document 1, fading is handled as a function of the integrated irradiation amount (integration of irradiation time).

JP-A-2005-214728

  However, when taking a two-dimensional or three-dimensional image, spatially, only irradiation at a certain fixed position serving as a reference point in the field of view is considered, and the amount of irradiation varies depending on the position in the field of view. The effect is not considered.

  Actually, the amount of fading differs depending on the illumination light source and the peripheral light reduction (shading) of the illumination optical system, because the amount of irradiation differs near the optical axis in the field of view and at the edge of the field of view. Since the effect is not taken into account, there is a problem in that the effect of causing a different fading at each position in the field of view forming the image cannot be corrected.

  The present invention solves these problems, and its purpose is to treat the fading of fluorescence as a function related to the spatial position in the field of view, thereby to be based on the spatial position in the field of view. An object is to realize a fluorescence detection apparatus and a fluorescence microscope capable of performing fading correction.

The invention of claim 1 which achieves such a problem,
In a fluorescence detection apparatus configured to detect a fluorescence image generated by the sample by irradiating excitation light to the fluorescently stained sample with an image detector,
Means is provided for quantifying and correcting fluorescence fading according to the position of the sample image detected by the image detector.

The invention of claim 2
In a fluorescence microscope configured to detect a fluorescent image generated by the sample by irradiating the fluorescently stained sample with a laser beam with an image detector,
Means is provided for quantifying and correcting fluorescence fading according to the position of the sample image detected by the image detector.

The invention of claim 3 is the fluorescence microscope according to claim 2,
The fluorescence microscope is configured as a confocal microscope.

  According to the fluorescence detection device and the fluorescence microscope configured as described above, the fluorescence fading correction can be performed based on the irradiation amount and the spatial position.

It is a block diagram which shows the principal part of one Example of this invention. It is a circuit diagram which shows the other Example of this invention. It is a flowchart which shows the flow of the operation | movement of the fading correction based on this invention. It is a block diagram which shows the specific example of the image processing system using the fading correction based on this invention. It is a block diagram which shows the specific example of the confocal microscope provided with the fading correction function based on this invention. It is an example of the fading characteristic of a fluorescent dye.

  Hereinafter, the present invention will be described with reference to the drawings. FIG. 1 is a block diagram showing the main part of one embodiment of the present invention. In FIG. 1, a fluorescence information detection unit 10 detects various information related to fluorescence for each position of an image of a sample 30 detected by an image detector 20, such as a CCD or CMOS, and corrects the fluorescence fading. Field-of-view position information storage unit 11, position-specific fluorescence intensity information acquisition unit 12, position-specific fluorescence intensity information storage unit 13, position-specific discoloration amount calculation unit 14, fading coefficient storage unit 15, position-specific fluorescence intensity The information correction unit 16 is configured.

  The in-view position information storage unit 11 stores position information in the view corresponding to the pixels of the image detector 20. In many cases, the fluorescence fading in an actual apparatus can be described as a function symmetric with respect to the optical axis. Therefore, it is not necessary to have a fading coefficient for every pixel of the image detector 20, and for example, it may have a fading coefficient as a function of the position of the optical axis and the distance from the optical axis. That is, position information in the field of view may be provided as a function of only the radius R in polar coordinates of the radius R and the angle θ.

  The position-specific fluorescence intensity information acquisition unit 12 stores the two-dimensional image data based on the position information in the field of view stored in the position information storage unit 11 in the field of view and the surface image of the sample 30 detected by the image detector 20. The fluorescence intensity information is acquired in a format, and the acquired fluorescence intensity information is stored in the position-specific fluorescence intensity information storage unit 13.

  The position-specific fade amount calculation unit 14 calculates the fluorescence fade amount for each position (pixel) of the two-dimensional data acquired by the position-specific fluorescence intensity information acquisition unit 12. Note that these calculation results may be combined into a function of only the radius R as a fading amount with respect to the distance from the optical axis.

  The fading coefficient storage unit 15 stores a fading coefficient based on the calculation result of the position-specific fading amount calculation unit 14 in the form of two-dimensional image data. In storing the fading coefficient, a plurality of coefficients may be provided in some cases, or as a function of the radius R in polar coordinates as described above.

  The position-specific fluorescence intensity information correction unit 16 obtains the ratio of the fading as a function of the position and time (past exposure amount) using the obtained coefficient, and obtains the fluorescence intensity before the fading by correcting with the ratio of the fading. .

The operation of FIG. 1 will be described in detail.
The amount of effective fluorescent substance capable of emitting fluorescence is expressed as g (r). Here, g represents GFP (green fluorescent protein), and the variable r represents the radial direction of two-dimensional polar coordinates with the optical axis in the field of view as the origin. In general, the angle direction θ is also a variable, but only r is shown here for simplicity. In addition, although you may describe by a two-dimensional orthogonal coordinate, in order to demonstrate a concept here, it shows with a polar coordinate. The amber color is a decrease in fluorescent material having the ability to emit light, and can be expressed by the following equation (1).

Here, a is a positive constant depending on the fluorescent material, B is the intensity of the incident light quantity, and fi (r) is normalized to represent the peripheral dimming of the illumination (excitation) optical system (the value is 1 on the optical axis). It is a function. In order to represent a non-linear fading with respect to the amount of incident light, it is assumed here that n> = 1 assuming that “power” is a power function type of n. In general, different functional forms are possible. "Fi" of the function fi (r) represents the f lat-field of i ncoming ray . What is understood from this equation (1) is that since there is a spatial distribution of excitation light intensity, the fading also varies depending on the position r of the space.

  Solving equation (1) gives equation (2) with A as the integration constant.

  For simplicity, it is assumed that the peripheral dimming fi (r) of the illumination optical system is represented by a two-dimensional Gaussian function. In order to explain the concept of the present invention, a function that can be calculated analytically will be described. However, in practice, an arbitrary function form may be numerically calculated.

When the fluorescent substance has a distribution as shown in Equation (3), the actually observed image is obtained by integrating the peripheral light reduction fi (r) of the illumination optical system and the peripheral light reduction fo (r) of the imaging optical system. If the peripheral attenuation fo (r) of the imaging optical system is also expressed by a two-dimensional Gaussian function, the following equation (4) is obtained. It should be noted that, "fo" of the function fo (r) represents the f lat-field of o utgoing ray .

  FIG. 2 is an example of a light quantity change characteristic on the imaging surface of the image detector 20, where the horizontal axis indicates the position (arbitrary unit) of the imaging surface, and the vertical axis indicates the observed light quantity (arbitrary unit). In a two-dimensional image of 1000 × 1000 pixels, n = 1, peripheral light attenuation σi = 500 of the observation optical system, peripheral light attenuation σo = 500 of the excitation optical system, and fading by one exposure on the optical axis of r = 0 Is a plot of equation (4) with 3% (a = 0.03).

  The four graphs A to D show changes in the amount of light according to the number of times of shooting. From the top, graph A is observed at the 0th time, graph B is at the 10th time, graph C is at the 100th time, and graph D is observed at the 200th time. Represents the change in the amount of light emitted. In graphs A and B, the amount of light in the vicinity of the optical axis is higher than that in the vicinity, but in this portion, the color is also fading, fading rapidly with the number of shootings, and eventually the surrounding portion appears brighter. That is, in the 100th exposure of the graph C, the periphery starts to become brighter, and in the 200th exposure of the graph D, the periphery becomes brighter than the vicinity of the optical axis.

  The flat field is a function of position and time. In order to correct such fading, it is necessary to correct not only time (integrated dose) but also as a function of position depending on the illumination and optical system. It is clear.

  In actual photographing, flat field correction may be performed on the photographed raw image using equation (4).

FIG. 3 is a flowchart showing the flow of the operation for fading correction according to the present invention.
First, image data before correction is read (step S1).
Subsequently, the past irradiation amount data is acquired for the read image (step S2). Past dose data is calculated or read from a recorded database.

Next, each pixel of the image is scanned (step S3).
Then, for each position (pixel) of the image, a flat field (amount of light reduction) is calculated as a function of the past irradiation amount data acquired in step S2 and the position in the image (step S4).

The fluorescence intensity of the corresponding pixel is divided by the flat field (light reduction amount) calculated in step S4, and fading correction is performed (step S5).
When scanning for each pixel of the image is completed (step S6), the corrected image data is stored in a predetermined storage unit and used for the next calculation (step S7).

  Here, parameters such as a, n, σi, and σo in equation (4) are determined by the cell type, fluorescent material type, staining method, observation wavelength, optical system properties (settings), and the like. The reference data is acquired under the same conditions, and these parameters can be determined by fitting by the least square method or the like from the state of dimming. If it is difficult to obtain reference data in advance, it may be estimated from actual data.

  FIG. 4 is a block diagram showing a specific example of an image processing system using fading correction based on the present invention. In FIG. 4, the camera used as the image detector 20 takes a fluorescently stained image of the sample 30 via the confocal microscope 40 and inputs the image output data to the image acquisition PC 50.

  A plurality of PCs as a client including the fluorescence information detection unit 10, a server 70, a storage 80, and the like are connected to the PC 50 for image acquisition via the switch 60 and the network NW.

  In such a configuration, the fluorescence-stained image data of the sample 30 taken by the camera 20 and taken into the PC 50 is sequentially stored in the storage 80 via the server 70.

  The PC 10 as a client reads desired image data to be image-processed from the storage 80 via the server 70 and stores the image processing result in the storage 80 via the server 70 as necessary.

  As a result, the image data stored in the storage 80 can be shared by a plurality of PCs 10 as clients, and the efficiency of image processing can be improved as a whole system.

  FIG. 5 is a block diagram showing a specific example of a confocal microscope as a fluorescence microscope having a fading correction function based on the present invention. In FIG. 5, the output light of the laser light source 41 is irradiated to the sample 30 that is fluorescently stained with the fluorescent indicator through the confocal scanner 42 and the objective lens 43.

  The sample 30 emits fluorescence when irradiated with laser light. The fluorescent image generated by the sample 30 is input to the spectroscopic optical system 44 via the objective lens 43 and the confocal scanner 42 and is dispersed.

  The spectral output of the spectroscopic optical system 44 is input to the two-dimensional image detector 45 and converted into an electric signal, the electric signal is input to the A / D converter 46 and converted into digital data, and the digital data is input to the PC 47. A fading correction process is performed.

  The PC 47 is provided with a CPU 47a, a data memory 47b, a position-specific discoloration amount calculation program 47c for calculating a discoloration amount for each position in the field of view, a position-specific discoloration amount correction program 47d for correcting the discoloration amount for each position in the field of view, and the like. It has been.

  The PC 47 calculates a fading amount for each position in the field of view of the digital data converted and input from the A / D converter 46 based on the position-specific color fading calculation program 47c, and based on the position-specific color fading correction program 47d The fading amount for each position in the field of view of the digital data converted and input from the / D converter 46 is corrected.

  Then, the display device 48 displays a two-dimensional image in which the fading amount for each position in the field of view is corrected by the PC 47 in this way.

  For example, when observing a living cell (live cell) for a long time, the cell moves in the field of view with time. That is, the discoloration of a certain cell at a certain time depends on the peripheral dimming of illumination at the position where the cell has moved in the past.

  Therefore, by using the confocal microscope configured as shown in FIG. 5, by tracking the cell movement position with time and integrating the fading according to the history, the fading of the cell can be estimated more accurately, and more Accurate observation is possible.

  In the above-described embodiment, the photographed image is temporarily stored, and the fading amount correction is performed at the subsequent data analysis stage. However, the fading amount correction may be performed simultaneously with photographing. For example, using a calculation block such as a CPU, DSP, FPGA or the like provided in a camera casing or an image acquisition board, a fading amount correction calculation is performed immediately after shooting, and the corrected image is stored immediately after shooting. You may make it display on a display apparatus.

  In the above embodiment, the intensity of illumination for exciting the sample fluorescently stained with the fluorescent indicator is constant, but it may be changed according to the passage of time (number of shots). In that case, the function form of an appropriate correction function corresponding to the equation (4) may be obtained analytically or numerically.

  The excitation light is not limited to laser light, and may be a combination of a mercury lamp and a monochromatic filter, for example.

  Furthermore, in the above-described embodiment, an example in which a confocal microscope is used as a fluorescence microscope and a photographing target is a cell has been described. However, the present invention is not limited to this, and various types of discoloration may occur in a photographing target due to irradiation for photographing. It can also be applied to shooting of a shooting target.

  As described above, according to the present invention, it is possible to realize a fluorescence detection apparatus and a fluorescence microscope that can perform fading correction based on an irradiation amount and a spatial position.

DESCRIPTION OF SYMBOLS 10 Fluorescence information detection part 11 Position information storage part in a visual field 12 Fluorescence intensity information acquisition part according to position 13 Fluorescence intensity information storage part according to position 14 Fading amount calculation part according to position 15 Fading coefficient storage part 16 Fluorescence intensity information correction part according to position 20 Image Detector (camera)
30 Sample 40 Confocal microscope 41 Laser light source 42 Confocal scanner 43 Objective lens 44 Spectroscopic optical system 45 Two-dimensional image detector 46 A / D converter 47 PC
47a CPU
47b Data memory 47c Discoloration amount calculation program for each position 47d Discoloration amount correction program for each position 48 Display device

Claims (3)

In a fluorescence detection apparatus configured to detect a fluorescence image generated by the sample by irradiating excitation light to the fluorescently stained sample with an image detector,
A fluorescence detection apparatus comprising means for quantifying and correcting fluorescence discoloration for each position of the sample image detected by the image detector. In a fluorescence microscope configured to detect a fluorescent image generated by the sample by irradiating the fluorescently stained sample with a laser beam with an image detector,
A fluorescence microscope comprising means for quantifying and correcting fluorescence discoloration for each position of the sample image detected by the image detector.   The fluorescence microscope according to claim 2, wherein the fluorescence microscope is configured as a confocal microscope.

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