Fluorescence image analysis device

Abstract

The present invention relates to a fluorescence image analysis device. The fluorescence image analysis device stores a reference fluorescence sample image and a subject fluorescence sample image. The reference fluorescence sample image is an image obtained by irradiating a reference fluorescence sample whose relationship of in-plane fluorescence intensity is known with linearly polarized light and capturing a first predetermined polarization component of fluorescence from the reference fluorescence sample. The subject fluorescence sample image is an image obtained by irradiating a subject fluorescence sample with linearly polarized light and capturing a second predetermined polarization component of fluorescence from the subject fluorescence sample. The fluorescence image analysis device is configured to determine a correction coefficient for correcting unevenness in light intensity measured between pixels of a captured image based on the reference fluorescence sample image, and to correct the light intensity of pixels of the subject fluorescence sample image based on the correction coefficient.

Description

Technical Field

[0001] The invention relates to a fluorescence image analysis device. Background Art

[0002] Fluorescence image analysis is widely used to evaluate various states of various molecules. Fluorescence images are obtained by irradiating a fluorescently labeled sample with polarized light as excitation light and extracting a specific polarization component from the fluorescence. For example, the fluorescence depolarization method calculates the fluorescence polarization value based on the polarization component of the fluorescence parallel and perpendicular to the polarization direction of the excitation light, thereby analyzing the sample. The fluorescence polarization value (fluorescence anisotropy) teaches information about the molecule, such as size, shape, concentration, and viscosity. Summary of the invention

[0003] The fluorescence polarization measurement device used to acquire an image of polarized fluorescence may have polarization dependence. In other words, the fluorescence polarization measurement device may show different sensitivities to polarizations in different directions. In order to correct for this dependence, a G factor is calculated to be used as a correction factor in the calculation of the fluorescence polarization value. The G factor is expressed as the ratio of the polarization component of the fluorescence perpendicular to the polarization direction of the linearly polarized incident light relative to the polarization component of the fluorescence parallel to the polarization direction of the linearly polarized incident light.

[0004] However, fluorescence polarization measurement devices have various factors that change the fluorescence image; it is difficult to accurately measure fluorescence polarization using only the G factor.

[0005] One aspect of the present invention is a fluorescence image analysis device, which includes one or more processors and one or more storage devices. The one or more storage devices store a reference fluorescence sample image and a subject fluorescence sample image. The reference fluorescence sample image is an image obtained by irradiating a reference fluorescence sample whose relationship of in-plane fluorescence intensity is known with linearly polarized light and capturing a first predetermined polarization component of fluorescence from the reference fluorescence sample. The subject fluorescence sample image is an image obtained by irradiating the subject fluorescence sample with linearly polarized light and capturing a second predetermined polarization component of fluorescence from the subject fluorescence sample. The one or more processors are configured to determine a correction coefficient for correcting non-uniformity in light intensity measured between pixels of a captured image based on the reference fluorescence sample image, and to correct the light intensity of pixels of the subject fluorescence sample image based on the correction coefficient.

[0006] One aspect of the present invention enables more accurate measurement of fluorescence polarization.

[0007] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A schematic diagram shows an example of the structure of a fluorescence polarization measuring device included in the fluorescence polarization measuring system in Embodiment 1 and Embodiment 2;

[0009] Figure 2 An example of a logical structure of a fluorescence polarization measurement system is schematically shown;

[0010] Figure 3 Schematically shows an example of the structure of a fluorescence polarization measurement controller;

[0011] Figure 4 is a flow chart of an example of the operation of a fluorescence polarization measurement controller;

[0012] Figure 5 is a flow chart of an example of details of the steps of storing a reference fluorescent sample image;

[0013] Figure 6 schematically illustrates the relationship between a driving voltage for a liquid crystal cell and the intensity of light transmitted through the liquid crystal cell according to a device control program;

[0014] Figure 7 is a flow chart of an example of details of the steps of storing a subject fluorescent sample image;

[0015] Figure 8 is a flowchart of an example of details of the steps of analyzing a subject fluorescent sample image;

[0016] Fig. 9 The location of the ROI is shown;

[0017] Fig.10 is a graph of the uncorrected light intensity measured from the subject fluorescent sample (when the liquid crystal cell is turned on);

[0018] Fig.11 is a graph of the light intensity measured from a reference fluorescent sample (when the liquid crystal cell is turned on);

[0019] Fig.12 is a graph of the result after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample;

[0020] Fig.13 is a graph of the uncorrected light intensity measured from the subject fluorescent sample (when the liquid crystal cell is off);

[0021] Fig.14 is a graph of the light intensity measured from a reference fluorescent sample (when the liquid crystal cell is off);

[0022] Fig.15 is a graph of the result after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample;

[0023] Fig.16 An example of a temporal change in a voltage applied to a liquid crystal cell in Embodiment 2 is shown;

[0024] Fig.17 Schematically shows that Fig.16 FIG. 1 is a graph showing a change in the intensity of fluorescent light transmitted through the liquid crystal cell when a voltage is applied to the liquid crystal cell; ...

[0025] Fig.18 is a flowchart of an example of details of the step of storing a reference fluorescent sample image in Embodiment 2;

[0026] Fig.19 is a flowchart of an example of details of the step of storing the target fluorescent sample image in Embodiment 2;

[0027] Fig. 20 This is a flowchart showing an example of details of the step of analyzing the target fluorescent sample image in Embodiment 2;

[0028] Fig.21 is a graph of uncorrected light intensity measured from a subject fluorescent sample during time period D1;

[0029] Fig. 22 is a graph of light intensity measured from a reference fluorescent sample during time period D1;

[0030] Fig.23 is a graph of the result after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample;

[0031] Fig.24A Schematically shows an example of the structure of a unit for containing a liquid sample;

[0032] Fig. 24B Shown along Fig.24A The cross-sectional structure of the unit taken along line BB in FIG.

[0033] Fig.25 The location of the ROI is shown;

[0034] Fig.26 It is shown according to the reference Figures 4 to 8 a graph of polarization values ​​before and after correction using the described method;

[0035] Fig. 27 It is shown according to the reference Figures 16 to 20 a graph of polarization values ​​before and after correction using the described method;

[0036] Fig.28 Another structural example of a fluorescence polarization measuring device is schematically shown;

[0037] Fig.29 An example of the logical structure of the fluorescence polarization measurement system in Embodiment 3 is schematically shown;

[0038] Fig.30 is a flowchart of details of the step of storing a reference fluorescent sample image in Embodiment 3;

[0039] Fig.31 is a flowchart of details of the step of storing the target fluorescent sample image in Embodiment 3;

[0040] Fig.32 Provides an average value of uncorrected light intensity and an average value of corrected light intensity measured by the polarization camera from the object fluorescence samples filled in the five channels of the microchannel unit in Embodiment 3;

[0041] Fig.33 Provides the standard deviation (σ) of the uncorrected light intensity and the standard deviation (σ) of the corrected light intensity measured by the polarization camera from the object fluorescence sample filled in the five channels of the microchannel unit in Embodiment 3;

[0042] Fig.34 Provided are the dispersion (σ / average) of the uncorrected light intensity and the dispersion (σ / average) of the corrected light intensity measured by the polarization camera from the object fluorescent sample filled in the five channels of the microchannel unit in Embodiment 3;

[0043] Fig.35 Another structural example of a fluorescence polarization measuring device is schematically shown;

[0044] Fig.36 An example of the logical structure of the fluorescence polarization measurement system in Embodiment 4 is schematically shown;

[0045] Fig.37 is a flowchart of an example of details of the step of storing a reference fluorescent sample image in Embodiment 4;

[0046] Fig.38 is a flowchart of an example of details of the step of storing the target fluorescent sample image in Embodiment 4;

[0047] Fig.39 providing an average value of uncorrected light intensities and an average value of corrected light intensities measured from the object fluorescent samples filled in the nine channels of the microchannel unit in Embodiment 4;

[0048] Fig.40providing a standard deviation (σ) of uncorrected light intensity and a standard deviation (σ) of corrected light intensity measured from the object fluorescent sample filled in the 9 channels of the microchannel unit in Embodiment 4; and

[0049] Fig.41 The dispersion (σ / average value) of the uncorrected light intensity and the dispersion (σ / average value) of the corrected light intensity measured from the object fluorescent samples filled in the nine channels of the microchannel unit in Embodiment 4 are provided. DETAILED DESCRIPTION

[0050] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It should be noted that these embodiments are merely examples for implementing the present invention and are not intended to limit the technical scope of the present invention.

[0051] The fluorescence image analysis device disclosed herein corrects the in-plane light intensity distribution in the fluorescence image. The fluorescence image is taken by a fluorescence polarization measurement device. The fluorescence polarization measurement device may include several factors that change the fluorescence image.

[0052] For example, when the fluorescence polarization measurement device includes a dichroic mirror, the dichroic mirror may bias the in-plane light intensity in the fluorescence image. That is, a fluorescence image of a sample with uniform in-plane light intensity may exhibit light intensity biased in the plane. Another example of a factor is the light intensity distribution of the excitation light. When the light intensity distribution of the excitation light is uneven, the in-plane light intensity of the fluorescence image may change. That is, a fluorescence image of a sample with uniform in-plane light intensity may exhibit uneven light intensity in the plane.

[0053] The fluorescent image analysis device disclosed herein determines a correction value for the non-uniform measured light intensity of pixels of a captured image based on a reference fluorescent sample image, and corrects the light intensity value of the pixels in the object fluorescent sample image. As a result, the value of the light intensity of the object fluorescent sample image that changes due to instrumental reasons can be appropriately corrected.

[0054] Implementation Method 1

[0055] Device structure

[0056] Figure 1 The structure example of the fluorescence polarization measuring device included in the fluorescence polarization measuring system is schematically shown. As will be described later, the fluorescence polarization measuring system includes the fluorescence polarization measuring device and a fluorescence polarization measuring controller for controlling the fluorescence polarization measuring device and analyzing a fluorescence image captured by the fluorescence polarization measuring device.

[0057] exist Figure 1In the illustrated structural example, the fluorescence polarization measuring device 10 includes a light source 101, a condenser lens 103, an aperture 105, a collimator 107, a polarization adjuster 109, an excitation filter 111, and a dichroic mirror 113. The fluorescence polarization measuring device 10 also includes an objective lens 115, a workbench 117, an emission filter 119, a polarization adjuster 121, an imaging lens 123, and an imaging device 125.

[0058] The sample 151 is placed on the workbench 117. The light source 101 may be an LED; the light source 101 outputs excitation light of a wavelength that excites fluorescence in the sample 151 (e.g., blue light with a central wavelength of 470 nm). The excitation light from the light source 101 is collected by a condenser lens 103 and passes through an aperture 105. The aperture 105 reduces external light.

[0059] The excitation light having passed through the aperture 105 is converted into parallel light by the collimator 107 and enters the polarization adjuster 109. The polarization adjuster 109 may be a polarization plate, a polarization beam splitter, or a liquid crystal cell. In the example described below, the polarization adjuster 109 is a polarization plate. The polarization adjuster 109 transmits linearly polarized light in a specific direction. The linearly polarized excitation light from the polarization adjuster 109 passes through the excitation filter 111 and is reflected toward the objective lens 115 by the dichroic mirror 113. The excitation filter 111 is used to select a wavelength range including the wavelength of the excitation light and reduce light different from the excitation light from the polarization adjuster 109.

[0060] The objective lens 115 focuses the linearly polarized excitation light reflected by the dichroic mirror 113 on the sample 151 on the workbench 117. The sample 151 emits fluorescence (e.g., green light) having a specific wavelength in response to the linearly polarized excitation light from the objective lens 115. The fluorescence is converted into parallel light by the objective lens 115, and passes through the dichroic mirror 113 and the emission filter 119. The dichroic mirror 113 selectively transmits light within a predetermined wavelength range including the fluorescence from the sample 151, and reflects other light. The emission filter 119 is used to select a wavelength range including the wavelength of the fluorescence from the sample 151, and reduce light other than the fluorescence.

[0061] The fluorescence that has passed through the emission filter 119 enters the polarization regulator 121. The polarization regulator 121 may be a polarization plate, a polarization beam splitter, or a liquid crystal cell. The polarization regulator may also be a polarization filter in a polarization camera. A polarization camera is an imaging device that includes a polarization filter on a sensor to obtain polarization information of an object. The polarization regulator 121 in the example described below is a liquid crystal cell that is controlled by an applied voltage. The polarization regulator 121 transmits only a specific linear polarization component of the fluorescence. Specifically, the polarization regulator 121 transmits light that is linearly polarized in a direction parallel to or perpendicular to the polarization direction of the excitation light.

[0062] The linearly polarized fluorescence that has passed through the polarization adjuster 121 is imaged on an imaging plane of an imaging device 125 by an imaging lens 123. An example of the imaging device 125 includes a CMOS image sensor. The imaging device 125 has a plurality of pixels to generate image data according to the light intensity of the fluorescence and transmits the generated data to a fluorescence polarization measurement controller ( Figure 1 not shown).

[0063] The fluorescence polarization measuring device 10 may have Figure 1 For example, the fluorescence polarization measuring device 10 can remove the dichroic mirror 113 by obliquely irradiating the sample with the excitation light. The aperture 105, the excitation filter 111 and the emission filter 119 are optional. Figure 1 In addition to or instead of the lenses, the fluorescence polarization measurement device 10 may include one or more lenses.

[0064] Figure 2 FIG. 1 schematically shows an example of a logical structure of a fluorescence polarization measurement system. The fluorescence polarization measurement system 1 includes a fluorescence polarization measurement device 10, a fluorescence polarization measurement controller 20, and a D / A converter (DAC) 30. The fluorescence polarization measurement device 10 refers to Figure 1 The fluorescence polarization measurement controller 20 controls the fluorescence polarization measurement device 10 and analyzes a fluorescence image captured by the fluorescence polarization measurement device 10. Specifically, the fluorescence polarization measurement controller 20 controls the light source 101, the liquid crystal unit (polarization adjuster) 121, and the imaging device 125 of the fluorescence polarization measurement device 10.

[0065] For example, the fluorescence polarization measurement controller 20 keeps the light source 101 turned on to irradiate the sample with excitation light during measurement. The fluorescence polarization measurement controller 20 controls the voltage to be applied to the liquid crystal cell 121 using the D / A converter 30. By controlling the voltage applied to the liquid crystal cell 121, the polarization component of the fluorescence to be transmitted through the liquid crystal cell 121 can be controlled.

[0066] The liquid crystal cell 121 includes two transparent substrates facing each other, transparent electrodes provided on the opposite surfaces of the substrates, a liquid crystal material encapsulated between the substrates, and a polarizing plate provided on the outer surface on the imaging device side (emission side or downstream side). The liquid crystal cell 121 can be configured as desired as long as the liquid crystal cell 121 can adjust the transmittable polarization component of the fluorescence.

[0067] In addition to the voltage to be applied to the liquid crystal cell 121, the fluorescence polarization measurement controller 20 controls the exposure time (imaging time) of the imaging device 125 or the start time of imaging and the duration of imaging to obtain image data of the desired polarization component of the fluorescence. The fluorescence polarization measurement controller 20 also analyzes the acquired image data. As will be described later, the fluorescence polarization measurement controller 20 corrects the value measured from the sample to be analyzed (object sample) based on the image of the reference sample. This operation corrects the image changed by the device structure of the fluorescence polarization measurement device 10 to achieve more accurate measurement.

[0068] Figure 3 There is schematically shown a structural example of the fluorescence polarization measurement controller 20. The fluorescence polarization measurement controller 20 may be configured with a computer. Figure 3 The fluorescence polarization measurement controller 20 in the example includes a processor 201, a memory (main storage device) 202, an auxiliary storage device 203, an output device 204, an input device 205, and a communication interface (I / F) 207. These components are connected to each other through a bus. The memory 202, the auxiliary storage device 203, or a combination thereof is a storage device, and stores programs and data to be used by the processor 201.

[0069] The memory 202 may be a semiconductor memory and is mainly used to store the program being executed and data. The processor 201 performs various processes according to the program stored in the memory 202. In addition to the operating system not shown, Figure 3 The memory 202 in the structural example in FIG. 2 also stores a device control program 221 and a fluorescent image analysis program 222 .

[0070] The processor 201 operates according to the program to realize various functional units. For example, the processor 201 operates according to the device control program 221 to function as a device control unit, and operates according to the fluorescence image analysis program 222 to function as a fluorescence image analysis unit.

[0071] The auxiliary storage device 203 may be a mass storage device such as a hard disk drive or a solid state drive; it is used for long-term storage of programs and data. Figure 3 The auxiliary storage device 203 in the structural example includes a reference fluorescence sample image database (DB) 231 and a subject fluorescence sample image database 232. The subject fluorescence sample image database 232 stores images acquired from the subject fluorescence sample. The reference fluorescence sample image database 231 stores reference fluorescence sample images based on light intensity values ​​used to correct the subject fluorescence sample images.

[0072] The processor 201 may be one or more processing units and include one or more computing units or multiple processing cores. The processor 201 may be implemented as one or more central processing units, microprocessors, microcomputers, microcontrollers, digital signal processors, state machines, logic circuits, graphics processing units, systems on chips, and / or any device that operates on signals according to control instructions.

[0073] The program and data stored in the auxiliary storage device 203 are loaded to the memory 202 at startup or when necessary, and the program is executed by the processor 201 to perform various processes of the fluorescence polarization measurement controller 20. Therefore, the process performed by the program described below is the process performed by the processor 201 or the fluorescence polarization measurement controller 20.

[0074] The input device 205 is a hardware device for a user to input instructions and information to the fluorescence polarization measurement controller 20. The output device 204 is a hardware device for presenting images for input and output, for example, a display device or a printing device. The communication I / F 207 is an interface for connecting to a network. The input device 205 and the output device 204 are optional, and the fluorescence polarization measurement controller 20 can be accessed by a terminal via a network.

[0075] The functions of the fluorescence polarization measurement controller 20 can be implemented in a computer system, which includes one or more computers, which include one or more processors and one or more storage devices, which include non-transitory storage media. Multiple computers communicate with each other through a network. For example, part of the functions of the fluorescence polarization measurement controller 20 can be implemented in one computer, and another part can be implemented in another computer.

[0076] operate

[0077] In the following, the operation of the fluorescence polarization measurement controller 20 is described. Figure 2 As described, the device control program 221 controls the light source 101, the liquid crystal cell (polarization regulator) 121, and the imaging device 125 of the fluorescence polarization measuring device 10. In the example described below, the liquid crystal cell 121 is switched between on and off. In the off state, the liquid crystal cell 121 transmits only the linear polarization component parallel to the linear polarized light incident on the sample. In the on state, the liquid crystal cell 121 transmits only the linear polarization component perpendicular to the linear polarized light incident on the sample. The liquid crystal cell 121 in the on state rotates the linear polarized light by 90°. Compared with this example, the situation of the liquid crystal cell 121 in the on / off state may be opposite.

[0078] Figure 42 is a flowchart of an example of the operation of the fluorescence polarization measurement controller 20. The device control program 221 acquires reference fluorescence sample images from the fluorescence polarization measurement device 10 and stores them in the reference fluorescence sample image database 231 (S101). In the example described below, the device control program 221 acquires a reference fluorescence sample image obtained by capturing a linear polarization component parallel to the linear polarization direction of the excitation light, and another reference fluorescence sample image obtained by capturing a linear polarization component perpendicular to the linear polarization direction of the excitation light.

[0079] Next, the device control program 221 acquires the object fluorescence sample images from the fluorescence polarization measurement device 10 and stores them in the object fluorescence sample image database 232 (S102). In the example described below, the device control program 221 acquires an object fluorescence sample image obtained by capturing a linear polarization component parallel to the linear polarization direction of the excitation light, and another object fluorescence sample image obtained by capturing a linear polarization component perpendicular to the linear polarization direction of the excitation light.

[0080] Next, the fluorescence image analysis program 222 analyzes the object fluorescence sample image based on the reference fluorescence sample image (S103). In the example described below, the fluorescence image analysis program 222 corrects the object fluorescence sample image based on the reference fluorescence sample image before calculating the fluorescence polarization value of the object fluorescence sample.

[0081] Figure 5 1 is a flowchart of an example of details of step S101 of storing a reference fluorescent sample image. The reference fluorescent sample is placed on the stage 117 by the operator. The device control program 221 supplies a driving voltage to the liquid crystal cell 121 to turn on the liquid crystal cell 121 (S201).

[0082] The device control program 221 causes the imaging device 125 to receive light for a predetermined exposure period to capture a first reference fluorescence sample image (S202). The first reference fluorescence sample image is an image of a linear polarization component of fluorescence that is perpendicular to the polarization direction of the excitation light. The device control program 221 acquires the first reference fluorescence sample image from the imaging device 125 and stores it in the reference fluorescence sample image database 231 (S203).

[0083] Next, the device control program 221 supplies a driving voltage to the liquid crystal unit 121 to turn off the liquid crystal unit 121 (S204). The device control program 221 causes the imaging device 125 to receive light for a predetermined exposure period of time to capture a second reference fluorescence sample image (S205). The second reference fluorescence sample image is an image of a linear polarization component of fluorescence parallel to the polarization direction of the excitation light. The device control program 221 acquires the second reference fluorescence sample image from the imaging device 125 and stores it in the reference fluorescence sample image database 231 (S206).

[0084] Figure 6 The relationship between the driving voltage for the liquid crystal cell 121 and the intensity of light transmitted through the liquid crystal cell 121 according to the device control program 221 is schematically shown. The device control program 221 provides a rectangular wave 401 that periodically oscillates between a predetermined positive voltage and a negative voltage to keep the liquid crystal cell 121 turned on. The liquid crystal cell 121 is turned on at a positive voltage or a negative voltage. A measurement period 421 is defined in a period (on period) 411 when the liquid crystal cell 121 is turned on.

[0085] The device control program 221 causes the imaging device 125 to receive fluorescence during the measurement period 421 to capture an image of the fluorescence. When the liquid crystal unit 121 is turned on, the imaging device 125 receives a polarization component perpendicular to the polarization direction of the excitation light in the fluorescence from the sample. The intensity I⊥ of the received light is represented by the total amount of light received during the measurement period 421 or a time average value.

[0086] The device control program 221 provides a zero voltage 402 to turn off the liquid crystal cell 121. A measurement period 422 is defined in a period (off period) 412 in which the liquid crystal cell 121 is off. The device control program 221 causes the imaging device 125 to receive fluorescence during the measurement period 422 to capture an image of the fluorescence. When the liquid crystal cell 121 is off, the imaging device 125 receives a polarization component parallel to the polarization direction of the excitation light in the fluorescence from the sample. The intensity I / / of the received light is represented by the total amount or time average of the light received during the measurement period 422.

[0087] Figure 7 2 is a flowchart of an example of details of step S102 of storing a target fluorescence sample image. The target fluorescence sample is placed on the stage 117 by the operator. The device control program 221 supplies a driving voltage to the liquid crystal cell 121 to turn on the liquid crystal cell 121 (S301).

[0088] The device control program 221 causes the imaging device 125 to receive light for a predetermined exposure period of time to capture a first object fluorescence sample image (S302). The first object fluorescence sample image is an image of a linear polarization component of fluorescence that is perpendicular to the polarization direction of the excitation light. The device control program 221 acquires the first object fluorescence sample image from the imaging device 125 and stores it in the object fluorescence sample image database 232 (S303).

[0089] Next, the device control program 221 supplies a driving voltage to the liquid crystal unit 121 to turn off the liquid crystal unit 121 (S304). The device control program 221 causes the imaging device 125 to receive light for a predetermined exposure period of time to capture a second object fluorescence sample image (S305). The second object fluorescence sample image is an image of a linear polarization component of fluorescence parallel to the polarization direction of the excitation light. The device control program 221 acquires the second object fluorescence sample image from the imaging device 125 and stores it in the object fluorescence sample image database 232 (S306).

[0090] Figure 8 2 is a flowchart of an example of the details of step S103 of analyzing the object fluorescence sample image. The fluorescence image analysis program 222 acquires a first reference fluorescence sample image from the reference fluorescence sample image database 231 (S401). The first reference fluorescence sample image is a reference fluorescence sample image obtained by capturing a linear polarization component perpendicular to the polarization direction of the excitation light. The fluorescence image analysis program 222 calculates a correction coefficient (first correction coefficient) for the first object fluorescence sample image (S402). The details of calculating the first correction coefficient will be described later.

[0091] The fluorescence image analysis program 222 acquires a second reference fluorescence sample image from the reference fluorescence sample image database 231 (S403). The second reference fluorescence sample image is a reference fluorescence sample image obtained by capturing a linear polarization component parallel to the polarization direction of the excitation light. The fluorescence image analysis program 222 calculates a correction coefficient (second correction coefficient) for the second object fluorescence sample image (S404). The details of calculating the second correction coefficient will be described later.

[0092] Next, the fluorescence image analysis program 222 acquires the first object fluorescence sample image and the second object fluorescence sample image from the object fluorescence sample image database 232 (S405). The fluorescence image analysis program 222 calculates the polarization value of the object fluorescence sample based on the first correction coefficient and the second correction coefficient and the first object fluorescence sample image and the second object fluorescence sample image (S406). The fluorescence image analysis program 222 stores the calculated polarization value to the auxiliary storage device 203 and outputs it to the output device 204. The fluorescence image analysis program 222 may also output the first object fluorescence sample image and the second object fluorescence sample image to the output device 204.

[0093] Calibration method

[0094] In the following, a method for calculating correction coefficients based on a reference fluorescent sample image is described. The fluorescent image analysis program 222 calculates a first correction coefficient from a first reference fluorescent sample image. In this example, within the field of view, the area occupied by the reference fluorescent sample includes all regions of interest (ROIs) of the object fluorescent sample. The in-plane fluorescence intensity emitted from the reference fluorescent sample is uniform. If the relationship of the in-plane fluorescence intensity is known, the intensity does not have to be uniform.

[0095] The first correction coefficient is a correction coefficient for an object fluorescence sample image (first object fluorescence sample image) obtained by capturing a linear polarization component perpendicular to the polarization direction of the linear polarization excitation light. The correction coefficient is determined for each pixel. The correction coefficient a⊥(i, j) can be calculated by the following formula:

[0096] a⊥(i,j)=(1 / Ir⊥(i,j))*│Ir⊥(i,j)│,

[0097] Here, Ir⊥(i,j) represents the light intensity (intensity of received light) at pixel (i,j) in the first reference fluorescence sample image, and |Ir⊥(i,j)| represents the average light intensity of multiple pixels constituting a predetermined area including pixel (i,j) in the first reference fluorescence sample image.

[0098] The predetermined area may be the entire captured image (field of view), an area consisting of all pixels having an intensity of fluorescence received from a reference fluorescence sample higher than a threshold, or a partial area of ​​an image including a plurality of ROIs of the subject fluorescence sample. When the predetermined area includes all pixels receiving fluorescence from the reference fluorescence sample, a greater number of pixels can be calibrated to achieve more accurate correction.

[0099] The fluorescence image analysis program 222 calculates a second correction coefficient from the second reference fluorescence sample image. The second correction coefficient is a correction coefficient for an object fluorescence sample image (a second object fluorescence sample image) obtained by capturing a linear polarization component parallel to the polarization direction of the linearly polarized excitation light. The correction coefficient a / / (i, j) can be calculated by the following formula:

[0100] a / / (i,j)=(1 / Ir / / (i,j))*│Ir / / (i,j)│,

[0101] Wherein, Ir / / (i, j) represents the light intensity (intensity of received light) at pixel (i, j) in the second reference fluorescence sample image, and |Ir / / (i, j)| represents the average light intensity of a plurality of pixels constituting a predetermined area including pixel (i, j) in the second reference fluorescence sample image. The predetermined area is an area corresponding to the predetermined area of ​​the first reference fluorescence sample image.

[0102] The fluorescence image analysis program 222 may also use correction factors for background correction. For example, where the sample is contained in a container (such as a microchannel within a cell), the wavelength of light from the cell may be included in the wavelength range to be measured. Eliminating this background noise enables more accurate measurements. A background correction factor (offset value) b⊥(i, j) for a first reference fluorescence sample image and a background correction factor (offset value) b / / (i, j) for a second reference fluorescence sample image are predetermined.

[0103] Based on the correction coefficients a⊥(i,j) and a / / (i,j) calculated from the reference fluorescence sample image and the background correction coefficients b⊥(i,j) and b / / (i,j), the light intensity of each pixel of the object fluorescence sample image is corrected. Specifically, the corrected light intensity of the pixel in the first object fluorescence sample image can be expressed as follows:

[0104] Ic⊥(i,j)=a⊥(i,j)*It⊥(i,j)+b⊥(i,j),

[0105] Here, It⊥(i,j) represents the uncorrected light intensity of the pixel (i,j) in the first object fluorescence sample image captured by the imaging device 125, and Ic⊥(i,j) represents the corrected light intensity of the same pixel (i,j).

[0106] Similarly, the corrected light intensity of a pixel in the second object fluorescent sample image can be expressed as follows:

[0107] Ic / / (i,j)=a / / (i,j)*It / / (i,j)+b / / (i,j),

[0108] Here, It / / (i, j) represents the uncorrected light intensity of the pixel (i, j) in the second object fluorescent sample image captured by the imaging device 125, and Ic / / (i, j) represents the corrected light intensity of the same pixel (i, j).

[0109] The polarization value P in the ROI is expressed as follows:

[0110] P=(Ic / / –Ic⊥) / (Ic / / +Ic⊥),

[0111] Here, Ic / / represents the sum or average value of the corrected light intensities Ic / / (i, j) of multiple pixels in the ROI, and Ic⊥ represents the sum or average value of the corrected light intensities Ic⊥(i, j) of multiple pixels in the ROI.

[0112] The fluorescence image analysis program 222 may correct the light intensity of the pixels in the object fluorescence sample image according to another function. For example, the fluorescence image analysis program 222 may first perform background correction on the light intensity of the pixels in the object fluorescence sample image, and then correct the obtained value using the correction coefficient calculated from the reference fluorescence sample image.

[0113] Specifically, the fluorescence image analysis program 222 calculates the average value of the light intensity (background light intensity) in the background area (area excluding the sample). Taking the unit having a microchannel as an example, the fluorescence image analysis program 222 calculates the average value b of the light intensity in the area other than the microchannel. The area for calculating the average value can have a size equal to the ROI of the sample.

[0114] The fluorescence image analysis program 222 corrects the light intensity It(i, j) of each pixel in the object fluorescence sample image obtained by capturing the linear polarization component parallel or perpendicular to the excitation light according to the following formula:

[0115] Ic(i,j)=a(i,j)*(It(i,j)–b),

[0116] Wherein, Ic(i, j) represents the corrected light intensity of pixel (i, j), a(i, j) represents the correction coefficient calculated from the reference fluorescent sample image, It(i, j) represents the uncorrected light intensity of pixel (i, j), and b represents the background correction coefficient.

[0117] The influence of the dichroic mirror 113 and the light source 101 on the intensity of the light received at the pixel may differ depending on the polarization direction component. Therefore, by using the correction coefficient for the linear polarization component parallel to the linear polarization direction of the excitation light and the correction coefficient for the linear polarization component perpendicular to the linear polarization direction of the excitation light, the fluorescence polarization of the object sample can be measured more accurately. According to the system design, the fluorescence image analysis program 222 can use only one reference fluorescence sample image obtained by capturing any one linear polarization component.

[0118] Example of measurement results

[0119] In the following, examples of measurement results obtained from fluorescent samples are described. Fig. 9 The positions of the ROIs are shown. Nine ROIs are defined in the area of ​​the sample within the captured image (field of view). The numbers in the sample area 501 indicate the number of the ROI. The object fluorescent sample is a liquid material (fluorescently labeled solution) contained in a container. The reference fluorescent sample is a YAG ceramic piece.

[0120] Fig.10 1 is a graph of uncorrected light intensity measured from the object fluorescence sample (when the liquid crystal cell is turned on). The horizontal axis represents the ROI number and the vertical axis represents the light intensity in the ROI. The light intensity in the ROI is the sum of the light intensities at the pixels (pixels of the imaging device 125) included in the ROI. Since the liquid crystal cell 121 is turned on, the measured fluorescence intensity is the light intensity of the linear polarization component perpendicular to the linear polarization direction of the excitation light.

[0121] Fig.11 Graph of light intensity measured from a reference fluorescent sample (when the liquid crystal cell is turned on). The horizontal axis represents the ROI number and the vertical axis represents the light intensity in the ROI. Since the liquid crystal cell 121 is turned on, the measured fluorescence intensity is the light intensity of the linear polarization component perpendicular to the linear polarization direction of the excitation light.

[0122] Fig.12 is a graph showing the results after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample. The horizontal axis represents the ROI number, and the vertical axis represents the light intensity in the ROI. Fig.10 Compared to the chart, Fig.12 The graph of shows small differences (variations) in the measured light intensity between multiple ROIs. The light intensity measured from the subject fluorescent sample should be uniform within the plane. Fig.12 It is taught that by making corrections based on light intensity measured from a reference fluorescent sample, more accurate measurements of a subject fluorescent sample are achieved.

[0123] Fig.13Graph of uncorrected light intensity measured from the target fluorescent sample (when the liquid crystal cell is turned off). The horizontal axis represents the ROI number and the vertical axis represents the light intensity in the ROI. Since the liquid crystal cell 121 is turned off, the measured fluorescence intensity is the light intensity of the linear polarization component parallel to the linear polarization direction of the excitation light.

[0124] Fig.14 Graph of light intensity measured from a reference fluorescent sample (when the liquid crystal cell is turned off). The horizontal axis represents the ROI number and the vertical axis represents the light intensity in the ROI. Since the liquid crystal cell 121 is turned off, the measured fluorescence intensity is the light intensity of the linear polarization component parallel to the linear polarization direction of the excitation light.

[0125] Fig.15 is a graph showing the results after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample. The horizontal axis represents the ROI number, and the vertical axis represents the light intensity in the ROI. Fig.13 Compared to the chart, Fig.15 The graph of shows small differences (variations) in the measured light intensity between multiple ROIs. The light intensity measured from the subject fluorescent sample should be uniform within the plane. Fig.15 It is taught that by making corrections based on light intensity measured from a reference fluorescent sample, more accurate measurements of a subject fluorescent sample are achieved.

[0126] The above structural example controls the liquid crystal cell 121 to extract different polarization components from the fluorescence. Another structural example may use a polarization plate instead of the liquid crystal cell 121. The operator or the fluorescence polarization measurement controller 20 changes the direction of the polarization plate to extract a desired polarization component from the fluorescence.

[0127] The above example uses the measured values ​​in the reference fluorescence sample image when determining the correction coefficients. Another structural example can use two-dimensional curve fitting based on interpolation or regression analysis to correct the light intensity of the pixels of the reference fluorescence sample image. This operation reduces the noise in the reference fluorescence sample image to achieve a more appropriate calibration. Any fitting function can be used; for example, a curve that fits the X and Y directions of the imaging plane (pixel array) to the measured values ​​can be defined by a function of a predetermined order (e.g., sixth order).

[0128] The above example calculates the average value of the light intensity in the ROI including the light intensity of the noise without removing the random fixed pattern noise generated by the image sensor. Another structural example determines fixed pattern noise pixels (so-called hot pixels) from pixels with fixed pattern noise based on a predetermined threshold (image sensor noise threshold) and excludes them.

[0129] To determine a threshold value for the light intensity of a pixel, for example, the fluorescence image analysis program 222 calculates the average value of the light intensity of 24 adjacent pixels (5 (along a vertical line) × 5 (along a horizontal line) −1 (pixel of interest) = 24 pixels). If the light intensity of a pixel is higher than 105% of the calculated average value, the fluorescence image analysis program 222 determines that the pixel is a fixed pattern noise pixel. The fluorescence image analysis program 222 performs this calculation on all pixels in the image to generate an xy coordinate map of the fixed pattern noise pixels. When calculating the light intensity of the object fluorescence sample and the reference fluorescence sample, the fluorescence image analysis program 222 excludes the fixed pattern noise pixels in the xy coordinate map and calculates the light intensity using the remaining pixels.

[0130] Implementation Method 2

[0131] Measurement method

[0132] Correction of the light intensity measured from the target fluorescent sample by another method is described. The difference from Embodiment 1 is mainly described. The device control program 221 applies a voltage that changes in a period longer than the inversion period to the liquid crystal cell 121. Fig.16 FIG. 1 shows an example of a time change of a voltage applied to the liquid crystal unit 121. The horizontal axis represents time, and the vertical axis represents the applied voltage. Fig.16 As shown, the applied voltage reverses between positive and negative values ​​at high frequencies. The positive envelope and the negative envelope are line symmetrical about the horizontal axis. The envelope changes with a period longer than the reversal period.

[0133] Fig.17 Schematically shows that Fig.16 The change in light intensity of the fluorescence transmitted through the liquid crystal cell 121 when the voltage shown is applied to the liquid crystal cell 121. The intensity of the fluorescence changes approximately sinusoidally. The intensity decreases as the absolute value of the applied voltage increases, and increases as the absolute value of the applied voltage decreases. The waveform of the voltage applied to the liquid crystal cell 121 is not limited to a specific waveform as long as the light intensity changes approximately sinusoidally.

[0134] The fluorescence image analysis program 222 calculates the light intensity in the ROI in the image of the fluorescent sample in each time period D1 to D4. The light intensity in each time period is the integral value of the light intensity in the ROI in each time period over time, or the total amount of light received at the pixel in the ROI in each time period (exposure time period). The fluorescence image analysis program 222 evaluates the fluorescence polarization of the sample based on the light intensity in the time period D1 to D4. The imaging device 125 receives a specific polarization component of the fluorescence in each time period D1 to D4. The polarization component in each time period has a certain polarization direction width.

[0135] The overall process of measurement Figure 4 The method is the same as shown in the flowchart. Figure 4 The details of each step are described below. Fig.18 2 is a flowchart of an example of the details of step S101 of storing a reference fluorescent sample image. The device control program 221 provides the liquid crystal unit 121 with the following information: Fig.16 The driving voltage (S501) is periodically varied as shown.

[0136] The device control program 221 controls the imaging device 125 to capture a reference fluorescence sample image in each of the four time periods D1 to D4 (S502). The image acquired in each time period represents the amount of fluorescence received at each pixel of the imaging device 125 in the time period. The device control program 221 stores the reference fluorescence sample images in the four time periods D1 to D4 in the reference fluorescence sample image database 231 (S503).

[0137] Fig.19 2 is a flowchart of an example of the details of step S102 of storing the object fluorescence sample image. The device control program 221 provides the liquid crystal unit 121 with the following information: Fig.16 The driving voltage (S601) is periodically varied as shown.

[0138] The device control program 221 controls the imaging device 125 to capture a subject fluorescence sample image in each of four time periods D1 to D4 (S602). The device control program 221 stores the subject fluorescence sample images in the four time periods D1 to D4 into the subject fluorescence sample image database 232 (S603).

[0139] Fig. 20 2 is a flowchart of an example of the details of step S103 of analyzing the object fluorescence sample image. The fluorescence image analysis program 222 performs steps S701 to S703 on the data of each time period. The fluorescence image analysis program 222 acquires the reference fluorescence sample image in the corresponding time period from the reference fluorescence sample image database 231 (S701). The fluorescence image analysis program 222 calculates the correction coefficient for the object fluorescence sample image in the time period from the acquired reference fluorescence sample image (S702). In this example, the fluorescence image analysis program 222 calculates the correction coefficient from the reference fluorescence sample image having the same polarization component as the polarization component of the object fluorescence sample image. These polarization components do not have to be the same polarization components.

[0140] Method and reference for calculating correction coefficient Figure 8 The method for calculating the first coefficient or the second coefficient described is the same. The fluorescence image analysis program 222 determines the correction coefficient a(i, j) for the light intensity of each pixel according to the following formula:

[0141] a(i,j)=(1 / Ir(i,j))*│Ir(i,j)│,

[0142] Here, Ir(i, j) represents the light intensity of the imaging device 125 at the pixel (i, j) in the reference fluorescent sample image, and |Ir(i, j)| represents the average value of the light intensities at a plurality of pixels constituting the predetermined area.

[0143] The fluorescence image analysis program 222 corrects the light intensity of each pixel (i, j) in the target fluorescence sample image based on the correction coefficient a(i, j) (S703). As described in Embodiment 1, a background correction coefficient may be used together.

[0144] After executing steps S701 to S703 for data of all time periods, the fluorescence image analysis program 222 calculates the polarization value based on the corrected light intensity of the object fluorescence sample image in the four time periods (S704). For example, the fluorescence image analysis program 222 calculates the polarization value P using the following formula:

[0145] P=(π / 4√2)*(B / A)

[0146] A=(LD1+LD2+LD3+LD4)

[0147] B=√{(LD1–LD3) 2 +(LD2–LD4) 2},

[0148] Here, LD1 to LD4 are the corrected light intensities (amounts of received light) in the ROI in the object fluorescence sample image in four time periods.

[0149] Example of measurement results

[0150] In the following, it is described according to the reference Figures 16 to 20 An example of a measurement result obtained from a fluorescent sample by the described method. An example in a time period D1 among four time periods is described below. The object fluorescent sample is a liquid material (fluorescently labeled solution) contained in a container. The reference fluorescent sample is a YAG ceramic piece.

[0151] Fig.21 : is a graph of uncorrected light intensity measured from the target fluorescent sample in time period D1. The horizontal axis represents the ROI number, and the vertical axis represents the light intensity in the ROI. Fig. 22 : is a graph of the light intensity measured from the reference fluorescent sample in the time period D1. The horizontal axis represents the ROI number, and the vertical axis represents the light intensity in the ROI. Fig.23 This is a graph showing the result after the light intensity measured from the target fluorescent sample is corrected based on the light intensity measured from the reference fluorescent sample. The horizontal axis represents the ROI number, and the vertical axis represents the light intensity in the ROI.

[0152] Will Fig.23 The chart and Fig.21 The light intensity measured from the target fluorescent sample should be uniform in the plane. Fig.23 It is taught that by making a correction based on the light intensity measured from the reference fluorescent sample, a more accurate measurement of the subject fluorescent sample is achieved. The correction exhibits the same effect in the other time periods D2 to D4.

[0153] Fig.24A and Fig. 24B A structural example of a unit for containing a liquid sample is schematically shown. Fig. 24B Shown along Fig.24A The cross-sectional structure of the unit taken along line BB in FIG. Fig.24A As shown, the unit 600 has a plurality of microchannels 601 and an inlet 602 for injecting liquid into the microchannels 601. Fig.24A In FIG. 6 , as an example, one of the channels is provided with reference numeral 601 and one of the inlets is provided with reference numeral 602. The width of the channel 601 may be approximately 100 μm.

[0154] like Fig. 24B As shown, the unit 600 includes: a colored (e.g., black) silicon substrate 612 having a groove corresponding to the channel 601; and a transparent glass plate 611, which covers the silicon substrate 612 to close the groove. The unit 600 is placed on the workbench 117 so that the glass plate 611 faces the imaging device 125. The excitation light passes through the glass plate 611 and is incident on the sample; the fluorescence from the sample passes through the glass plate 611 and travels toward the objective lens 115.

[0155] Hereinafter, examples of measurement results obtained from a sample contained in a cell having a microchannel are described. Fig.25 The positions of the ROIs are shown. Five ROIs are defined in each channel 601 containing a sample. The numbers within the rectangles representing the ROIs indicate the numbers of the ROIs. The object fluorescent sample is a liquid material (fluorescently labeled solution) contained in the cell. The reference fluorescent sample is a Ce:YAG ceramic piece.

[0156] Fig.26 It is shown according to the reference Figures 4 to 8 Graph of polarization values ​​before and after correction using the described method. The horizontal axis represents the ROI number and the vertical axis represents the polarization value. Fig.26Calculated polarization values ​​of samples in different channels before and after correction are shown. Line 701 represents the polarization value of the first channel before correction, and line 702 represents the polarization value of the first channel after correction. Line 703 represents the polarization value of the second channel before correction, and line 704 represents the polarization value of the second channel after correction. Line 705 represents the polarization value of the third channel before correction, and line 706 represents the polarization value of the third channel after correction.

[0157] Comparing the polarization values ​​of the same channel, the difference (variation) in the polarization values ​​between multiple ROIs is reduced after correction. The light intensity measured from the target fluorescent sample should be uniform regardless of the ROI. Fig.26 It is taught that by making corrections based on light intensity measured from a reference fluorescent sample, more accurate measurements of a subject fluorescent sample are achieved.

[0158] Fig. 27 It is shown according to the reference Figures 16 to 20 Graph of polarization values ​​before and after correction using the described method. The horizontal axis represents the ROI number and the vertical axis represents the polarization value. Fig. 27 Calculated polarization values ​​of a sample contained in one channel before and after correction are shown. Line 751 represents the calculated polarization value before correction, and line 752 represents the calculated polarization value after correction. After correction, the difference (variation) of polarization values ​​between multiple ROIs is reduced. Regardless of the ROI, the light intensity measured from the object fluorescent sample should be uniform. Fig. 27 It is taught that by making corrections based on light intensity measured from a reference fluorescent sample, more accurate measurements of a subject fluorescent sample are achieved.

[0159] As described in Embodiment 1, this Embodiment 2 may also be configured to exclude the xy coordinates of the fixed pattern noise pixels and calculate the light intensities of the object fluorescent sample and the reference fluorescent sample using the remaining pixels.

[0160] Implementation 3

[0161] Device structure

[0162] A measurement method using a fluorescence polarization measurement device having another device structure is described. The differences from Embodiment 1 are mainly described. Fig.28 is configured as shown. Figure 1 The structure of embodiment 1 in the embodiment 3 is different from the following two points: (1) the device in embodiment 3 does not include a polarizing plate or a liquid crystal unit as a polarization regulator on the emission side; (2) the device in embodiment 3 includes a polarization camera 127 instead of the usual imaging device.

[0163] A polarization camera is an imaging device that includes a polarization filter located just above a sensor array (image sensor) to obtain polarization information about an object. The image sensor has a plurality of pixels, and the polarization filter includes a plurality of polarizers that transmit light linearly polarized in different directions in fluorescence. The linearly polarized light transmitted through the polarizer enters one or more pixels.

[0164] The polarization camera can simultaneously capture the same number of images of light that is linearly polarized in different directions and transmitted through the polarization filter within a specified exposure time. In the example described below, the polarization filter of the polarization camera 127 transmits linearly polarized light parallel (at 0°) and perpendicular (at 90°) to the polarization direction of the excitation light.

[0165] operate

[0166] Hereinafter, the operation of the fluorescence polarization measurement controller 20 is described. Fig.29 As shown, the device control program 221 controls the light source 101 and the polarization camera 127 of the fluorescence polarization measuring device 10. The polarization camera 127 in the example described below can take images of light polarized in two directions (0° and 90°). A fluorescence image polarized in the 0° direction is acquired by transmitting a linear polarization component parallel to the linear polarized light incident on the sample. A fluorescence image polarized in the 90° direction is acquired by transmitting a linear polarization component perpendicular to the linear polarized light incident on the sample. Compared with this example, the situation of fluorescence polarized in the 0° and 90° directions can be opposite. The remaining operations are the same as those described in Implementation Example 1. The differences from Implementation Example 1 are mainly described.

[0167] For example, the fluorescence polarization measurement controller 20 keeps the light source 101 turned on to irradiate the sample with excitation light during measurement. The fluorescence polarization measurement controller 20 also controls the exposure time (imaging time) of the polarization camera 127, or the start time of imaging and the duration of imaging, to obtain image data of the polarization component of the desired fluorescence. The fluorescence polarization measurement controller 20 analyzes the acquired image data. The fluorescence polarization measurement controller 20 corrects the value measured from the object sample based on the image of the reference sample. This operation corrects the image changed by the device structure of the fluorescence polarization measurement device 10 to achieve more accurate measurement.

[0168] For example, the fluorescence polarization measurement controller 20 controls the exposure time (imaging time) of the polarization camera 127, or the start time of imaging and the duration of imaging, so as to capture the multiple polarization components in the fluorescence from the object fluorescence sample in the same length of time period as the multiple polarization components in the fluorescence from the reference fluorescence sample. This structure eliminates the difference between images caused by time variation, thereby achieving accurate measurement.

[0169] Measurement method

[0170] Like Embodiment 1, Embodiment 3 is configured to acquire a subject fluorescence sample image and a reference fluorescence sample image by respectively capturing linear polarization components parallel to and perpendicular to the linear polarization direction of the excitation light.

[0171] The overall process of measurement Figure 4 The method of the embodiment 1 shown in the flowchart of FIG. Fig.30 2 is a flowchart of an example of the details of step S101 of storing the reference fluorescence sample image. The device control program 221 sets the polarization camera 127 to a mode of capturing images of fluorescence polarized in two directions (0° and 90°) (S1501), and simultaneously captures images of the reference fluorescence sample in two polarization directions (0° and 90°) (S1502). The image with a polarization direction of 0° is a reference fluorescence sample image obtained by capturing a polarization component parallel to the linear polarization direction of the excitation light, and the image with a polarization direction of 90° is a reference fluorescence sample image obtained by capturing a polarization component perpendicular to the linear polarization direction of the excitation light. The device control program 221 stores the acquired images in the reference fluorescence sample image database 231 (S1503).

[0172] Fig.31 2 is a flowchart of an example of the details of step S102 of storing the object fluorescence sample image. The device control program 221 sets the polarization camera 127 to a mode of capturing images of fluorescence polarized in two directions (0° and 90°) (S1601), and simultaneously captures the object fluorescence sample images in the two directions (0° and 90°) (S1602). The image with a polarization direction of 0° is the object fluorescence sample image obtained by capturing the polarization component parallel to the linear polarization direction of the excitation light, and the image with a polarization direction of 90° is the object fluorescence sample image obtained by capturing the polarization component perpendicular to the linear polarization direction of the excitation light. The device control program 221 stores the acquired images in the object fluorescence sample image database 232 (S1603).

[0173] As in the first embodiment, the fluorescence image analysis program 222 follows Figure 8 The flowchart calculates correction coefficients (first correction coefficient and second correction coefficient) for a first subject fluorescence sample image and a second subject fluorescence sample image.

[0174] The fluorescence image analysis program 222 uses the obtained correction coefficient to correct the light intensity of each pixel (i, j) in each object fluorescence sample image. As described in Embodiment 1, the background correction coefficient can be used together.

[0175] As described in Embodiment 1, this Embodiment 3 may also be configured to exclude the xy coordinates of the fixed pattern noise pixels and calculate the light intensities of the object fluorescent sample and the reference fluorescent sample using the remaining pixels.

[0176] Example of measurement results

[0177] Hereinafter, an example of a measurement result obtained from a fluorescent sample using the polarization camera described in Embodiment 3 is described. Hereinafter, an example of an image of a linear polarization component parallel to the linear polarization direction of the excitation light acquired when the polarization direction of the polarization camera 127 is 0° is described. The target fluorescent sample is a liquid material (fluorescently labeled solution) contained in a container. The reference fluorescent sample is a YAG ceramic piece.

[0178] Fig.32 The average value of the uncorrected light intensity and the average value of the corrected light intensity measured by the polarization camera 127 from the object fluorescence sample filled in the five channels of the microchannel unit are provided. The object fluorescence sample filled in the five channels is at the same concentration level, and the polarization direction of the polarization camera 127 is 0° to capture an image of light polarized in a direction parallel to the linear polarization direction of the excitation light. The measured light intensity is corrected based on the light intensity measured from the reference fluorescence sample. The vertical axis represents the average light intensity among the five channels. Fig.33 The standard deviation (σ) of uncorrected light intensity and the standard deviation (σ) of corrected light intensity measured from the object fluorescence sample filled in the five channels of the microchannel unit by the polarization camera 127 are provided. The vertical axis represents the standard deviation among the five channels.

[0179] Fig.34 The dispersion (σ / average) of the uncorrected light intensity and the dispersion (σ / average) of the corrected light intensity measured by the polarization camera 127 from the object fluorescence sample filled in the five channels of the microchannel unit are provided. The vertical axis represents the dispersion (σ / average) of the light intensity between the five channels. Fig.34 The difference (variation) in the corrected light intensity between channels is small compared to the deviation before and after correction in the . The light intensity measured from the target fluorescent sample should be uniform in the plane. Figure 32 to Figure 34 It is taught that by making corrections based on light intensity measured from a reference fluorescent sample, more accurate measurements of a subject fluorescent sample are achieved.

[0180] Implementation 4

[0181] Device structure

[0182] A measurement method using a fluorescence polarization measuring device having another structure is described. Differences from Embodiment 1 are mainly described. The fluorescence polarization measuring device is configured as follows. Fig.35 As shown. Figure 1 The differences in structure from embodiment 1 are the following two points: (1) the polarization adjuster on the emission side in embodiment 4 is not a polarization plate or a liquid crystal unit, but a polarization beam splitter 110; (2) the device includes two imaging devices 125.

[0183] The polarization beam splitter 110 transmits a specific linear polarization component (p) of the fluorescence, and reflects a component (direction of the polarization beam splitter 110: s) orthogonal to the aforementioned polarization component (direction of the polarization beam splitter 110: p). Specifically, the polarization beam splitter 110 transmits p linear polarization light polarized parallel to the polarization direction of the excitation light, and reflects s ​​linear polarization light polarized perpendicular to the polarization direction of the excitation light.

[0184] operate

[0185] Hereinafter, the operation of the fluorescence polarization measurement controller 20 is described. Fig.36 As shown, the fluorescence polarization measurement system 1 includes a fluorescence polarization measurement device 10, a fluorescence polarization measurement controller 20, and a D / A converter (DAC) 30. The fluorescence polarization measurement device is as shown in FIG. Fig.36 The fluorescence polarization measurement controller 20 controls the fluorescence polarization measurement device 10 and analyzes a fluorescence image captured by the fluorescence polarization measurement device 10 .

[0186] Specifically, the fluorescence polarization measurement controller 20 controls the light source 101 and two imaging devices 125 of the fluorescence polarization measurement device 10. In the example described below, the polarization adjuster on the emission side is a polarization beam splitter 110 so that the imaging device 125 can capture images of light polarized in directions p and s. The image of light polarized in direction p is a fluorescence polarization image of a linear polarization component parallel to the linear polarized light incident on the sample. The image of light polarized in direction s is a fluorescence polarization image of a linear polarization component perpendicular to the linear polarized light incident on the sample. Compared with this example, the polarization conditions in directions p and s may be opposite. The remaining operations are the same as those described in Embodiment 1. The differences from Embodiment 1 are mainly described.

[0187] For example, the fluorescence polarization measurement controller 20 keeps the light source 101 turned on to irradiate the sample with excitation light during measurement. The fluorescence polarization measurement controller 20 also controls the exposure time (imaging time) of the two imaging devices 125, or the start time of imaging and the duration of imaging through the D / A converter 30 to obtain image data of the desired polarization component of fluorescence. The fluorescence polarization measurement controller 20 analyzes the acquired image data. The fluorescence polarization measurement controller 20 corrects the value measured from the object sample based on the image of the reference sample. This operation corrects the image changed by the device structure of the fluorescence polarization measurement device 10 to achieve more accurate measurement.

[0188] For example, the fluorescence polarization measurement controller 20 controls the exposure time (imaging time) of the imaging device 125, or the start time of imaging and the duration of imaging using the processor, so as to measure the polarization components of the two directions in the fluorescence from the object fluorescence sample in the same measurement (exposure) time period. Further, the fluorescence polarization measurement controller 20 controls the imaging device 125 to measure the polarization components of the two directions in the fluorescence from the reference fluorescence sample in the same measurement (exposure) time period as the measurement time period of the object fluorescence sample. This structure eliminates the difference between images caused by time changes, thereby achieving accurate measurement.

[0189] Measurement method

[0190] Like Embodiment 1, Embodiment 4 is configured to acquire a subject fluorescence sample image and a reference fluorescence sample image by respectively capturing linear polarization components parallel to and perpendicular to the linear polarization direction of the excitation light.

[0191] The overall process of measurement Figure 4 The method of the embodiment 1 shown in the flowchart of FIG. Fig.37 2 is a flowchart of an example of the details of step S101 of storing the reference fluorescence sample image. The device control program 221 identically configures the exposure conditions of the two imaging devices 125 (S1701). The device control program 221 synchronously drives the two imaging devices 125 to simultaneously capture the reference fluorescence sample images of the fluorescence signals in the two polarization directions p and s from the polarization beam splitter (S1702). The image in the polarization direction p is a reference fluorescence sample image obtained by capturing the polarization component parallel to the linear polarization direction of the excitation light, and the image in the polarization direction s is a second reference fluorescence sample image obtained by capturing the polarization component perpendicular to the linear polarization direction of the excitation light. The device control program 221 stores the acquired images in the reference fluorescence sample image database 231 (S1703).

[0192] Fig.38 1801. The device control program 221 identically configures the exposure conditions of the two imaging devices 125 (S1801). The device control program 221 synchronously drives the two imaging devices 125 to simultaneously capture the object fluorescence sample images of the fluorescence signals in the two polarization directions p and s from the polarization beam splitter (S1802). The image in the polarization direction p is the object fluorescence sample image obtained by capturing the polarization component parallel to the linear polarization direction of the excitation light, and the image in the polarization direction s is the object fluorescence sample image obtained by capturing the polarization component perpendicular to the linear polarization direction of the excitation light. The device control program 221 stores the acquired images in the object fluorescence sample image database 232 (S1803).

[0193] As in the first embodiment, the fluorescence image analysis program 222 follows Figure 8 The flowchart calculates correction coefficients (first correction coefficient and second correction coefficient) for a first subject fluorescence sample image and a second subject fluorescence sample image.

[0194] The fluorescence image analysis program 222 uses the obtained correction coefficient to correct the light intensity of each pixel (i, j) in each object fluorescence sample image. As described in Embodiment 1, the background correction coefficient can be used together.

[0195] As described in Embodiment 1, this Embodiment 4 may also be configured to exclude the xy coordinates of the fixed pattern noise pixels and calculate the light intensities of the object fluorescent sample and the reference fluorescent sample using the remaining pixels.

[0196] Example of measurement results

[0197] Hereinafter, an example of a measurement result obtained from a fluorescent sample using the imaging device described in Embodiment 4 is described. Described below is an image of a linear polarization component parallel to the linear polarization direction of the excitation light acquired when the polarization direction of the polarization beam splitter 110 is p. The object fluorescent sample is a liquid material (fluorescently labeled solution) contained in a container. The reference fluorescent sample is a YAG ceramic piece.

[0198] Fig.39 The average value of the uncorrected light intensity and the average value of the corrected light intensity measured from the object fluorescence samples filled in the nine channels of the microchannel unit are provided. The object fluorescence samples filled in the nine channels are at the same concentration level, and the polarization direction of the polarization beam splitter is p to take an image of light polarized in a direction parallel to the linear polarization direction of the excitation light. The measured light intensity is corrected based on the light intensity measured from the reference fluorescence sample. The vertical axis represents the average light intensity among the nine channels. Fig.40 The standard deviation (σ) of the uncorrected light intensity and the standard deviation (σ) of the corrected light intensity measured from the object fluorescence sample filled in the nine channels of the microchannel unit are provided. The vertical axis represents the standard deviation among the nine channels.

[0199] Fig.41 The dispersion of uncorrected light intensity (σ / mean value) and the dispersion of corrected light intensity (σ / mean value) measured from the object fluorescent sample filled in the nine channels of the microchannel unit are provided. The vertical axis represents the dispersion of light intensity (σ / mean value) between the nine channels. Fig.41 The dispersion before and after correction is small, and the difference (variation) of the corrected light intensity between channels is small. The light intensity measured from the target fluorescent sample should be uniform in the plane. Figure 39 to Figure 41It is taught that by making a correction based on the light intensity measured from a reference fluorescent sample, a more accurate measurement of the subject fluorescent sample is achieved. With respect to the other polarization direction s of the polarization beam splitter, the correction exhibits the same effect.

[0200] As mentioned above, multiple embodiments of the present invention have been described, however, the present invention is not limited to the aforementioned embodiments. Within the scope of the present invention, those skilled in the art can easily modify, add or convert each element in the aforementioned embodiments. A part of the structure of an embodiment can be replaced by the structure of another embodiment, or the structure of an embodiment can be incorporated into the structure of another embodiment.

Claims

1. A fluorescence polarization measurement system, comprising: Fluorescence polarization measurement controller; light source; Dichroic mirror; a first polarization modifier between the light source and the dichroic mirror; Image sensor; as well as a second polarization adjuster, the second polarization adjuster being located between the image sensor and the dichroic mirror and being configured to switch between on and off to transmit a linear polarization component that is parallel or perpendicular to the linear polarization light transmitted through the first polarization adjuster, wherein the fluorescence polarization measurement controller is configured to obtain a first reference fluorescence sample image, a second reference fluorescence sample image, a first object fluorescence sample image, and a second object fluorescence sample image captured by the image sensor using the dichroic mirror, wherein the first reference fluorescent sample image is an image obtained by irradiating a reference fluorescent sample whose in-plane fluorescence intensity relationship is known with linearly polarized light transmitted from the light source through the first polarization adjuster and capturing a polarization component perpendicular to the linearly polarized light of the fluorescence from the reference fluorescent sample, wherein the second reference fluorescent sample image is an image obtained by illuminating the reference fluorescent sample with linearly polarized light transmitted from the light source through the first polarization adjuster and capturing a polarization component parallel to the linearly polarized light of the fluorescent light from the reference fluorescent sample, wherein the first object fluorescence sample image is an image obtained by irradiating the object fluorescence sample with linearly polarized light and capturing a polarization component perpendicular to the linearly polarized light of the fluorescence from the object fluorescence sample, wherein the second object fluorescence sample image is an image obtained by illuminating the object fluorescence sample with linearly polarized light and capturing a polarization component parallel to the linearly polarized light of fluorescence from the object fluorescence sample, and Wherein, the fluorescence polarization measurement controller is constructed as follows: determining, using the first reference fluorescent sample image, a first correction coefficient for correcting for non-uniformity in measured light intensity between pixels of a captured image; determining a second correction coefficient for correcting non-uniformity in measured light intensity between pixels of a captured image using the second reference fluorescent sample image; correcting light intensities of pixels of the first object fluorescent sample image using the first correction coefficient; and The light intensity of the pixels of the second object fluorescent sample image is corrected using the second correction coefficient.

2. The fluorescence polarization measurement system according to claim 1, wherein: The in-plane intensity of the fluorescence from the reference fluorescence sample is uniform.

3. The fluorescence polarization measurement system according to claim 1, wherein: The fluorescence polarization measurement controller is configured to correct light intensities of pixels of the first object fluorescence sample image and the second object fluorescence sample image based on a predetermined offset value.

4. The fluorescence polarization measurement system according to claim 1, wherein: The fluorescence polarization measurement controller is configured to: determining background light intensity in areas other than an area of ​​the object fluorescence sample in the first object fluorescence sample image and the second object fluorescence sample image; as well as Light intensities of pixels of the first and second subject fluorescence sample images are corrected based on the background light intensity.

5. The fluorescence polarization measurement system according to claim 1, wherein: The fluorescence polarization measurement controller is configured to determine light intensities of pixels in the first reference fluorescence sample image and the second reference fluorescence sample image based on a predetermined fitting function.

6. The fluorescence polarization measurement system according to claim 1, wherein: The fluorescence polarization measurement controller is configured to: detecting fixed pattern noise of the image sensor from pixels of the first reference fluorescence sample image and the second reference fluorescence sample image and pixels of the first object fluorescence sample image and the second object fluorescence sample image based on a predetermined threshold of image sensor noise; When determining the first correction coefficient and the second correction coefficient, excluding pixels having noise from the first reference fluorescence sample image and the second reference fluorescence sample image; as well as When determining light intensities of pixels of the first and second subject fluorescence sample images, pixels having noise are excluded from the first and second subject fluorescence sample images.

7. The fluorescence polarization measurement system according to claim 1, wherein: The first correction coefficient and the second correction coefficient are determined for each pixel respectively.

8. A fluorescence polarization measurement system, comprising: Fluorescence polarization measurement controller; light source; Dichroic mirror; a polarization modifier between the light source and the dichroic mirror; as well as a polarization camera comprising an image sensor and a polarization filter configured to transmit polarization components parallel and perpendicular to the linearly polarized light transmitted through the polarization adjuster, wherein the fluorescence polarization measurement controller is configured to obtain a first reference fluorescence sample image, a second reference fluorescence sample image, a first object fluorescence sample image, and a second object fluorescence sample image captured by the image sensor using the dichroic mirror, wherein the first reference fluorescent sample image is an image obtained by irradiating a reference fluorescent sample whose in-plane fluorescence intensity relationship is known with linearly polarized light transmitted from the light source through the polarization adjuster and capturing a polarization component perpendicular to the linearly polarized light of the fluorescence from the reference fluorescent sample, wherein the second reference fluorescent sample image is an image obtained by illuminating the reference fluorescent sample with linearly polarized light transmitted from the light source through the polarization adjuster and capturing a polarization component parallel to the linearly polarized light of the fluorescence from the reference fluorescent sample, wherein the first object fluorescence sample image is an image obtained by illuminating the object fluorescence sample with linearly polarized light transmitted from the light source through the polarization adjuster and capturing a polarization component perpendicular to the linearly polarized light of the fluorescence from the object fluorescence sample, wherein the second object fluorescence sample image is an image obtained by illuminating the object fluorescence sample with linearly polarized light transmitted from the light source through the polarization adjuster and capturing a polarization component parallel to the linearly polarized light of fluorescence from the object fluorescence sample, and Wherein, the fluorescence polarization measurement controller is constructed as follows: determining, using the first reference fluorescent sample image, a first correction coefficient for correcting for non-uniformity in measured light intensity between pixels of a captured image; determining a second correction coefficient for correcting non-uniformity in measured light intensity between pixels of a captured image using the second reference fluorescent sample image; correcting light intensities of pixels of the first object fluorescent sample image using the first correction coefficient; and The light intensity of the pixels of the second object fluorescent sample image is corrected using the second correction coefficient.

9. The fluorescence polarization measurement system according to claim 8, wherein: The first correction coefficient a⊥(i, j) of pixel (i, j) is calculated by the following formula: a⊥(i,j)=(1 / Ir⊥(i,j))*│Ir⊥(i,j)│, wherein Ir⊥(i, j) represents the light intensity at the pixel (i, j) in the first reference fluorescent sample image, |Ir⊥(i, j)| represents the average value of the light intensities of a plurality of pixels constituting a predetermined area including the pixel (i, j) in the first reference fluorescent sample image, and The second correction coefficient a⊥(i, j) of pixel (i, j) is calculated by the following formula: a / / (i,j)=(1 / Ir / / (i,j))*│Ir / / (i,j)│, Wherein Ir / / (i, j) represents the light intensity at pixel (i, j) in the second reference fluorescence sample image, and |Ir / / (i, j)| represents the average light intensity of multiple pixels constituting a predetermined area including the pixel (i, j) in the second reference fluorescence sample image.

10. A fluorescence polarization measurement system, comprising: Fluorescence polarization measurement controller; light source; Dichroic mirror; a first polarization modifier between the light source and the dichroic mirror; Image sensor; as well as a second polarization adjuster, the second polarization adjuster being located between the image sensor and the dichroic mirror and periodically changing an intensity of a first predetermined polarization component of light transmitted through the second polarization adjuster, wherein the fluorescence polarization measurement controller is configured to obtain a reference fluorescence sample image and an object fluorescence sample image captured by the image sensor using the dichroic mirror, wherein the reference fluorescent sample image is an image obtained by illuminating a reference fluorescent sample whose in-plane fluorescence intensity relationship is known with linearly polarized light transmitted from the light source through the first polarization adjuster and capturing the first predetermined polarization component of the fluorescence from the reference fluorescent sample, wherein the object fluorescence sample image is an image obtained by irradiating the object fluorescence sample with linearly polarized light and capturing the first predetermined polarization component of fluorescence from the object fluorescence sample, and Wherein, the fluorescence polarization measurement controller is constructed as follows: determining, using the reference fluorescent sample image, a correction factor for correcting for non-uniformities in measured light intensity between pixels of a captured image; correcting light intensities of pixels of the object fluorescent sample image using the correction coefficients; and The polarization value P is calculated using the following formula: A=(LD1+LD2+LD3+LD4) Wherein LD1 to LD4 are the light intensities transmitted through the second polarization adjuster and corrected with the correction coefficients in four consecutive time periods in the region of interest ROI in the object fluorescent sample image.

11. A fluorescence image analysis method, comprising: obtaining, using a dichroic mirror, a first reference fluorescent sample image, a second reference fluorescent sample image, a first subject fluorescent sample image, and a second subject fluorescent sample image captured by an imaging device; using the first reference fluorescent sample image, determining a first correction coefficient for correcting non-uniformity in light intensity measured between pixels of a captured image; using the second reference fluorescent sample image, determining a second correction coefficient for correcting non-uniformity in light intensity measured between pixels of a captured image; correcting light intensity of pixels of the first object fluorescent sample image using the first correction coefficient; as well as correcting the light intensity of the pixels of the second object fluorescent sample image using the second correction coefficient, wherein the first reference fluorescent sample image is an image obtained by irradiating a reference fluorescent sample whose in-plane fluorescence intensity relationship is known with linearly polarized light transmitted from a light source through a first polarization adjuster and capturing a polarization component perpendicular to the linearly polarized light of the fluorescence from the reference fluorescent sample, wherein the second reference fluorescent sample image is an image obtained by illuminating the reference fluorescent sample with linearly polarized light transmitted from the light source through the first polarization adjuster and capturing a polarization component parallel to the linearly polarized light of the fluorescent light from the reference fluorescent sample, wherein the first object fluorescence sample image is an image obtained by irradiating the object fluorescence sample with linearly polarized light and capturing a polarization component perpendicular to the linearly polarized light of fluorescence from the object fluorescence sample, and Wherein, the second object fluorescence sample image is an image obtained by illuminating the object fluorescence sample with linearly polarized light and capturing a polarization component parallel to the linearly polarized light of fluorescence from the object fluorescence sample.

Images