A fluorescence microscope measurement calibration method and system

Abstract

The present invention relates to the technical field of equipment calibration, and in particular to a fluorescence microscope metrology calibration method and system. A detection end is connected to a host, and the detection end is provided with a standard light source and a micro-hole array plate matching the magnification of an objective lens. The standard light source is used as the standard light source, and the host controls the adjustable luminous intensity with an electrical signal. The micro-hole array plate is used to make the light source uniformly transparent, with equal scale and gradient dot array arrangement, and the objective lens magnification, imaging uniformity, imaging linearity, and optical linearity calibration are sequentially performed. The system is not only traceable, reusable, and controllable, but also has low use cost and high reliability. At the same time, the accuracy and reliability of the measurement data are ensured by gradually calibrating the magnification of the eyepiece, light uniformity, imaging linearity, and optical linearity of the fluorescence microscope being measured.

Description

Technical Field

[0001] The present invention relates to the technical field of equipment calibration, and in particular to a fluorescence microscope metrology calibration method and system. Background Art

[0002] Fluorescence microscopy is a high-precision imaging tool that is widely used in biomedical research, mainly including cell biology: observing the structure inside cells; molecular biology: studying the distribution and interactions of proteins and nucleic acids; and pathology: analyzing tissue samples to identify and study disease-related changes.

[0003] In order to ensure the accuracy of its imaging quality and measurement results, fluorescence microscopes need to be calibrated regularly. In order to ensure the reliability and accuracy of fluorescence microscope detection and quantification, in addition to the self-calibration of the equipment itself, it is also necessary to periodically calibrate the equipment with standard equipment or standard materials, conduct periodic inspections and quality control to ensure that the equipment is controllable.

[0004] In the prior art, fluorescent standard substances or fluorescent samples are used to calibrate the fluorescence microscope, but there are the following problems: 1. The standard fluorescent sample needs to have highly uniform and stable fluorescence characteristics, and any non-uniformity may lead to calibration errors; 2. The fluorescence characteristics may change after long-term exposure; 3. Changes in laboratory environmental conditions (such as temperature and humidity) may affect the fluorescence characteristics of the fluorescent sample, thereby affecting the calibration results; 4. High-quality standard fluorescent samples are expensive; 5. The preparation of standard fluorescent samples suitable for calibration requires complex chemical synthesis and purification steps, and the filters and optical elements used must have appropriate spectral characteristics and high transmittance; 6. The spectral characteristics of the light source used in the fluorescence microscope must match the excitation and emission spectra of the standard sample, otherwise the calibration results may be affected; 7. The difference in the results of each preparation will affect the reproducibility of the calibration results; 8. When performing linear calibration, gradient samples need to be prepared multiple times, which is difficult to prepare and takes a long time to calibrate; 9. Consumables, disposable.

[0005] NIST in the United States has developed calibration slides for calibrating fluorescence microscopes, but they are difficult to prepare and costly, and it is difficult to ensure the uniformity of the coating. They are only suitable for specific excitation wavelengths and do not have the function of resolution calibration. There is a problem of photobleaching after long-term use. The materials and coatings gradually lose their luminescence ability under continuous illumination and have poor stability. After coating, they have defects such as specific intensity and non-adjustability. Argo Light in France has developed multi-dimensional calibration slides. The technology combines photochemistry, optics and material science. Different calibration slides correspond to different functions. Target-shaped calibration slides are used for position calibration. However, they do not have the function of resolution calibration, are difficult to prepare and costly, and do not have traceability. Calibration using slides requires professional operating skills. The coverage spectrum is narrow, and after laser lithography, the intensity is specific and non-adjustable. Summary of the invention

[0006] The invention discloses a fluorescence microscope measurement calibration method and system, aiming to solve the technical problems existing in the prior art.

[0007] The present invention adopts the following technical solutions:

[0008] A fluorescence microscope measurement and calibration method comprises the following steps:

[0009] S1: Obtain the magnification value of the objective lens of the fluorescence microscope to be tested, and horizontally place the detection end with a matching microwell array plate on the stage of the fluorescence microscope so that the microwell array plate is located in the center of the imaging and is clear;

[0010] S2: Magnification error calibration:

[0011] Control the standard light source to emit light to a preset value; adjust the eyepiece diopter of the fluorescence microscope to 0 and then replace the eyepiece with a micrometer eyepiece, respectively obtain the indications of the horizontal and vertical scale spacing images of the detection end, and respectively calculate the horizontal and vertical coordinate objective lens magnifications, and calculate the horizontal and vertical coordinate objective lens magnification error values;

[0012] S3: Collect calibration data:

[0013] Control the standard light source to emit light to a preset percentage luminescence value, wherein the preset percentage luminescence value is set to a plurality of values; under dark field conditions, sequentially use the Rhod, GFP, and DAPI channels of the fluorescence microscope to photograph and image the optical array on the microwell array plate; under bright field conditions, photograph and image the optical array on the microwell array plate, and set the same number of shots and interval exposure time under dark field and bright field conditions;

[0014] S4: Imaging uniformity calibration:

[0015] Acquire an image, select a preset array area, identify the number of measurement points and fluorescence intensity values ​​in the array area, and calculate the fluorescence microscope imaging uniformity R of different channels in the dark field and the bright field, and R should satisfy: R≤1.0cd / m 2 ;

[0016] S5: Imaging linear calibration:

[0017] Acquire the image, select each array area according to the optical array of the imaging, identify the total fluorescence intensity value of each area, and calculate the linearity of the fluorescence microscope imaging in different channels of the dark field and the bright field. ,and Should meet: >0.997;

[0018] S6: Optical linear calibration:

[0019] Acquire the image, select one of the nine microwell areas, analyze its fluorescence intensity value when the standard light source emits different percentages, and calculate the optical linearity ,and Should meet: >0.997;

[0020] S7: Automatically generate a report of fluorescence microscope calibration results.

[0021] In some embodiments, in S1, obtaining the magnification value of the objective lens of the fluorescence microscope under test includes 10x, 20x, and 40x respectively;

[0022] Calculate the horizontal and vertical coordinate objective lens magnification , as shown in formula (1):

[0023] (1)

[0024] Where, Y' is the measured value of the micrometer eyepiece, in mm;

[0025] Y is the scale division value of the horizontal / vertical coordinate of the detection end, the unit is mm, and the horizontal and vertical scale division values ​​are the same;

[0026] Calculate the horizontal and vertical coordinate objective magnification errors respectively , as shown in formula (2):

[0027] (2)

[0028] in, is the nominal value of objective lens magnification;

[0029] And obtain the horizontal and vertical coordinate objective lens magnification error The larger one is the calibration result;

[0030] In some embodiments, it also includes: in S2, controlling the standard light source to emit white light and emitting 20% ​​of the light, adjusting the eyepiece diopter of the fluorescence microscope to 0, removing the eyepiece, installing a micrometer eyepiece, and calculating the horizontal coordinate objective lens magnification and the vertical coordinate objective lens magnification respectively.

[0031] In some embodiments, the method further includes: in S3, controlling the standard light source to emit 20% light, and sequentially using the Rhod, GFP, and DAPI channels of the fluorescence microscope to photograph the optical array on the microwell array plate under dark field conditions, taking pictures 3 times at intervals of 10 seconds, and using the same exposure time for each channel; photographing the optical array on the microwell array plate under bright field conditions, taking pictures 3 times at intervals of 10 seconds, and using the same exposure time as under dark field conditions;

[0032] Some of the preset percentage luminescence values ​​also include luminescence of 40%, luminescence of 80%, and luminescence of 100%, for taking photos under dark field conditions and bright field conditions respectively.

[0033] In some embodiments, the method further includes: in S4, selecting a preset array area, including calculating imaging uniformity by selecting one of nine array areas;

[0034] The imaging uniformity R is calculated at luminescence intensities of 20%, 40%, 80%, and 100%, respectively. The imaging uniformity R includes the fluorescence microscope imaging uniformity of the DAPI channel, GFP channel, and RHOD channel in the bright field and the dark field, as shown in formula (3):

[0035] R (3)

[0036] Where R is the imaging uniformity, in cd / m 2 ;

[0037] is the fluorescence intensity value of the ith measurement point, in cd / m 2 ;

[0038] It is the average illuminance value of all measurement points, in cd / m 2 ;

[0039] is the total number of measurement points.

[0040] In some embodiments, the method further includes: in S4, a middle area, namely, the fifth array area, is selected from the nine array areas to calculate imaging uniformity.

[0041] In some embodiments, the method further includes: in S5, respectively calculating the linearity of fluorescence microscope imaging in different channels of dark field and bright field under the fluorescence intensity conditions of the standard light source with the luminous intensity of 20%, 40%, 80% and 100% of the luminous intensity of the standard light source, , as shown in formula (4):

[0042] (4)

[0043] Where:

[0044] is the total fluorescence intensity value of the jth area in the optical array, in cd / m 2 ;

[0045] is the average of the total fluorescence intensity values ​​of the 9 regions in the optical array, in cd / m 2 ;

[0046] is the logarithmic value of the simulated fluorescence intensity corresponding to the jth region in the optical array;

[0047] is the average value of the logarithmic value of the simulated fluorescence intensity corresponding to each area in the optical array.

[0048] In some embodiments, the method further includes: in S6, acquiring an image, selecting one of the nine micropore regions, analyzing the fluorescence intensity values ​​thereof when the luminous intensity of the standard light source is 20%, 40%, 80%, and 100%, respectively, and calculating the optical linearity respectively. , as shown in formula (5):

[0049] (5)

[0050] n is the number of data points;

[0051] The measured value of fluorescence intensity at different percentages of luminescence, in cd / m 2 ;

[0052] is the predicted value, in cd / m 2 ;

[0053] It is the average value of the measured value, in cd / m 2 .

[0054] The present invention also includes a fluorescence microscope measurement and calibration system for implementing the fluorescence microscope measurement and calibration method, including a detection end horizontally arranged on the stage of the fluorescence microscope to be measured and a host controlling the detection end to perform detection and calibration; wherein,

[0055] The detection end includes a bottom PCB circuit board, a top cover plate and a standard light source located at the center between the two plates, which are fixedly connected as one; the PCB circuit board is connected to the host through a flat cable, and a micro-hole array plate provided at the center of the top cover plate corresponds to the center of the standard light source, so that the light of the standard light source is uniformly emitted through the micro-hole array plate;

[0056] The host includes a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the program, it controls the detection end to implement the steps of any of the above-mentioned fluorescence microscope metrology calibration methods.

[0057] In some embodiments, the detection end is provided with different detection ends according to the different magnifications of the objective lens to be measured and the size of the imaging image. The array sizes of the micropore array plate are different, and all are provided with micropores engraved through by laser. The micropore array plate is divided into 9 areas, each area contains 10 / 20 / 30 / 40...90 micropores, which are distributed in a gradient, so that the light uniformly transmitted by the standard light source is distributed in a gradient in each area.

[0058] Beneficial effects:

[0059] The present invention discloses a fluorescence microscope measurement calibration method and system. Compared with the prior art, the present invention has the following advantages:

[0060] The present invention is connected with a host through a detection end which is arranged. The detection end is provided with a standard light source and a micro-hole array plate which matches the magnification of an objective lens. A physical method is adopted, and a standard light source is used as a standard light source. The luminous intensity thereof is adjustable by controlling an electrical signal. The micro-hole array plate is adopted to make the light source uniformly transparent, with equal scale and gradient dot array arrangement. A specific standard signal is input for a fluorescence microscope. The standard light source adopts white light, covering the full spectrum range of the fluorescence microscope. The system is not only traceable, reusable, and controllable, but also has low use cost and high reliability. At the same time, the accuracy and reliability of the measurement data are ensured by gradually calibrating the magnification of the eyepiece, light uniformity, imaging linearity, and optical linearity of the fluorescence microscope to be measured. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, which constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions explain the present invention and do not constitute improper limitations on the present invention. In the drawings:

[0062] Figure 1 A flow chart of a fluorescence microscope measurement and calibration method provided by an embodiment of the present invention;

[0063] Figure 2 A schematic diagram of the structure of a fluorescence microscope measurement and calibration system provided by an embodiment of the present invention;

[0064] Figure 3 for Figure 2 A cross-sectional view of

[0065] Figure 4 for Figure 2 Cross-sectional view of (including filter);

[0066] Figure 5 for Figure 2 A in the middle shows the magnified image (microwell array plate matching the objective lens 40x);

[0067] Figure 6 for Figure 2 A in the middle shows the magnified image (microwell array plate matching the objective lens 20x);

[0068] Figure 7 for Figure 2 A in the middle shows the magnified image (microwell array plate matching the 10x objective lens);

[0069] Figure 8 This is the structural block diagram of the host;

[0070] Fig. 9 It is the linearity of fluorescence microscope imaging under the condition of bright field luminescence 20%;

[0071] Fig.10 It is the linearity of fluorescence microscope imaging under dark field Rhod luminescence 20% condition;

[0072] Fig.11 It is the linearity of fluorescence microscope imaging under dark field GFP luminescence 20% condition;

[0073] Fig.12 Linear fluorescence microscopy imaging under dark field DAPI luminescence 20%.

[0074] In the figure:

[0075] Detection end 1; standard light source 11; PCB circuit board 12; single chip computer 121; upper cover plate 13; filter 14; micropore array plate 131; micropore 1311; host 2; processor 21; memory 22; calculation module 23; acquisition module 24; identification module 25; judgment module 26; cable 3; fastener 4. DETAILED DESCRIPTION

[0076] The following is a description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, including various details of the embodiments of the present invention to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present invention. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0077] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limitations on the present invention.

[0078] like Figure 1 - Fig.12 As shown, the technical solution of the present invention:

[0079] A fluorescence microscope measurement and calibration method comprises the following steps:

[0080] S1: Obtain the objective lens magnification value of the fluorescence microscope to be tested, and horizontally place the detection end 1 provided with a matching microwell array plate 131 on the stage of the fluorescence microscope so that the microwell array plate 131 is located in the center of the image and is clear;

[0081] S2: Magnification error calibration:

[0082] The processor 21 of the host 2 controls the single-chip microcomputer 121 on the PCB circuit board 12 to execute instructions according to the program, and the single-chip microcomputer 121 controls the standard light source 11 to emit 20% of light; after manually adjusting the eyepiece diopter of the fluorescence microscope to 0, the eyepiece is replaced with a micrometer eyepiece, and the indications of the horizontal and vertical graduation spacing images of the detection end are respectively obtained, and the horizontal and vertical coordinate objective lens magnifications are respectively calculated, and the horizontal and vertical coordinate objective lens magnification error values ​​are calculated.

[0083] S3: Collect calibration data:

[0084] The processor 21 of the host 2 controls the single-chip microcomputer 121 on the PCB circuit board 12 to execute instructions according to the program, and controls the standard light source 11 to emit light to a preset percentage of light emission value, wherein the preset percentage of light emission value is set to 20%, 40%, 80%, and 100%; under dark field conditions, the Rhod, GFP, and DAPI channels of the fluorescence microscope are used in turn to photograph and image the optical array on the microwell array plate 131; under bright field conditions, the optical array on the microwell array plate 131 is photographed and imaged, and placed in the storage module 122, and it is set to take 3 photos under dark field and bright field conditions, with an interval of 10s and the same exposure time.

[0085] S4: Imaging uniformity calibration:

[0086] The processor 21 acquires the image and selects a preset array area according to the program. The recognition module 25 recognizes the number of measurement points and the fluorescence intensity value in the array area. The calculation module 23 calculates the fluorescence microscope imaging uniformity R of different channels in the dark field and the bright field respectively, and R should satisfy: R≤1.0 cd / m 2 , the judgment module 26 makes a qualified judgment.

[0087] S5: Imaging linear calibration:

[0088] The processor 21 acquires the image, and controls the acquisition module 24 to select each of the nine array regions according to the optical array of the imaging, and identifies the total fluorescence intensity value of each region. The calculation module 23 calculates the linearity of the fluorescence microscope imaging of different channels in the dark field and the bright field respectively. ,and Should meet: >0.997, the judgment module 26 makes a qualified judgment.

[0089] S6: Optical Linear Calibration:

[0090] The processor 21 acquires the image, selects one of the nine micro-hole areas, and analyzes the fluorescence intensity values ​​of the micro-hole area when the standard light source emits light at different percentages. The calculation module 23 calculates the optical linear ,and Should meet: >0.997, the judgment module 26 makes a qualified judgment.

[0091] S7: The processor 21 automatically generates a calibration result report of the fluorescence microscope under test according to the program.

[0092] The first preferred embodiment disclosed by the present invention is as follows Figure 2-Figure 8 As shown:

[0093] The present invention discloses a fluorescence microscope metrology calibration system for implementing the fluorescence microscope metrology calibration method, comprising a detection end 1 horizontally arranged on a stage of the fluorescence microscope to be measured and a host 2 for controlling the detection end 1 to perform detection and calibration.

[0094] The detection end 1 is a flat plate structure, and the entire detection end 1 is consistent with the length and width of a standard glass slide of a fluorescence microscope so that it can be placed on the stage during calibration, and the preferred size is 75.4mm*25.4mm*5mm.

[0095] A structure of the detection end 1 includes a PCB circuit board 12 at the bottom, an upper cover plate 13 at the top, and a standard light source 11 located at the center between the two plates, which are fixedly connected as a whole by a fastener 4; the PCB circuit board 12 is connected to the host 2 through a flat cable 3, and a micro-hole array plate 131 is provided at the center of the upper cover plate 13, and the micro-hole array plate 131 corresponds to the center of the standard light source 11, so that the light of the standard light source 11 is uniformly emitted through a plurality of micro-holes 1311 provided on the micro-hole array plate 131, such as Figure 2 , Figure 3 shown.

[0096] Another structure of the detection end 1 includes a PCB circuit board 12 at the bottom, an upper cover plate 13 at the top, and a standard light source 11 located at the center between the two plates, which are fixed together by a fastener 4. A microporous array plate 131 is provided at the center of the upper cover plate 13. The microporous array plate 131 corresponds to the center of the standard light source 11. A filter 14 is installed between the standard light source 11 and the microporous array plate 131, which only allows light of a specific wavelength to pass through. The light of the standard light source 11 is uniformly emitted through a plurality of micropores 1311 provided on the microporous array plate 131; the standard light source 11 is preferably a white light LED, and the shape of the LED used is rectangular or circular. The overall area of ​​the LED is larger than 5 times the area of ​​the microporous array plate 131. The microporous array plate 131 is located at its center, and can provide a uniform and stable full-spectrum light source in the microporous array plate 131 area. The standard light source uses white light to cover the full spectrum range of the fluorescence microscope. The PCB circuit board 12 is connected to the host 2 through a flat cable 3, such as Figure 4 shown.

[0097] The PCB circuit board 12 is provided with a single chip microcomputer 121 for executing instructions.

[0098] In this embodiment, the host 2 includes a memory 22, a processor 21, and a computer program stored in the memory 22 and executable on the processor 21. When the processor 21 executes the program, it controls the detection end 1 to implement the steps of the fluorescence microscope measurement and calibration method; the host 2 also includes a calculation module 23, an acquisition module 24, an identification module 25, and a judgment module 26.

[0099] Different detection ends 1 are provided according to the magnification of the objective lens to be tested and the size of the image to be imaged. The array sizes of the micro-hole array plate 131 are different, and all are provided with micro-holes 1311 which are penetrated by laser engraving. The micro-hole array plate 131 is divided into 9 areas in total, and each area contains 10 / 20 / 30 / 40...90 micro-holes 1311, which are distributed in a gradient, so that the light uniformly transmitted by the standard light source 11 is distributed in a gradient in each area;

[0100] In this embodiment, the different objective lenses to be tested, the magnifications of the objective lenses (10X, 20X, 40X) and the sizes of the imaged images correspond to three different specifications of detection ends 1, and the corresponding array sizes of the microwell array plates 131 are different.

[0101] In this embodiment, the 9 array areas of the micropore array plate 131 have different numbers of micropores 1311 and different light transmittances. The greater the number, the greater the light transmittance, which is a multiple relationship and simulates an exponential gradient relationship.

[0102] In this example, Figure 5As shown, when the magnification of the objective lens is 10X, the corresponding optical array distribution area size is 1420um*1070um, and the diameter of each micropore in the micropore array plate 131 is set to 1μm, which is a through hole and penetrated by laser engraving; the micropore array plate 131 is divided into 9 areas in total, and each area contains 10 / 20 / 30 / 40...90 micropores, which are distributed in a gradient to ensure that the light uniformly transmitted through the standard light source 11 is distributed in a gradient in each area.

[0103] like Figure 6 As shown, when the magnification of the objective lens is 20X, the corresponding optical array distribution area size is 713.65μm*534.73μm, and the diameter of each micropore in the micropore array plate 131 is set to 1μm, which is a through hole and is penetrated by laser engraving; the micropore array plate 131 is divided into 9 areas, each area contains 10 / 20 / 30 / 40...90 micropores, which are distributed in a gradient to ensure that the light uniformly transmitted through the standard light source 11 is distributed in a gradient in each area.

[0104] like Figure 7 As shown, when the magnification of the objective lens is 40X, the corresponding optical array distribution area size is 360.75μm*270.31μm, and the diameter of each micropore in the micropore array plate 131 is set to 1μm, which is a through hole and is penetrated by laser engraving; the micropore array plate 131 is divided into 9 areas in total, and each area contains 10 / 20 / 30 / 40...90 micropores, which are distributed in a gradient to ensure that the light uniformly transmitted through the standard light source 11 is distributed in a gradient in each area.

[0105] The standard light source 11 is used as the standard light source, and its luminous intensity is adjustable by controlling the electrical signal. The microporous array plate 131 is used to make the light source uniformly transparent, with equal scale and gradient dot array arrangement. A specific standard signal is input into the fluorescence microscope. The system can be traced back to the optical metrology standard and has the traceability of measurement value. As a physical device, the system has controllable, reproducible and reusable results, has low storage conditions and is simple to use, providing a convenient and reliable calibration system.

[0106] The second preferred embodiment disclosed by the present invention is as follows Figure 1-Figure 12 As shown:

[0107] A fluorescence microscope measurement and calibration method comprises the following steps:

[0108] S1: The acquisition module 24 obtains the objective lens magnification value of the fluorescence microscope being tested, including 10x, 20x, and 40x respectively; the detection end 1 containing the matching microwell array plate 131 is horizontally placed on the stage of the fluorescence microscope, so that the microwell array plate 131 is located in the center of the imaging and is clear.

[0109] S2: Magnification error calibration:

[0110] The processor 21 of the host 2 controls the single chip microcomputer 121 on the PCB circuit board 12 according to the program to control the standard light source 11, that is, the white light LED, to emit 20% of light, manually adjust the eyepiece diopter of the fluorescence microscope to 0, remove the eyepiece, install the micrometer eyepiece, obtain the indication of the horizontal and vertical scale spacing image of the detection end 1, and calculate the horizontal and vertical coordinate objective lens magnification respectively. , as shown in formula (1):

[0111] (1)

[0112] Where, Y' is the measured value of the micrometer eyepiece, in mm;

[0113] Y is the scale division value of the horizontal axis / vertical axis of the detection end 1, the unit is mm,

[0114] In this embodiment, the horizontal and vertical axis division values ​​are the same, both set to 0.01 mm. The horizontal and vertical axis division values ​​may also be different according to actual needs.

[0115] The calculation module 23 calculates the horizontal and vertical coordinate objective lens magnification errors respectively , as shown in formula (2):

[0116] (2)

[0117] in, is the nominal value of objective lens magnification;

[0118] The calculation results are stored in the acquisition module 24, and the judgment module 26 obtains the horizontal and vertical coordinate objective lens magnification errors. The larger one is the calibration result.

[0119] Taking a 10X objective lens as an example, the objective lens magnification error calibration results are shown in Table 1:

[0120]

[0121] S3: Collect calibration data:

[0122] The processor 21 of the host 2 controls the single chip microcomputer 121 on the PCB circuit board 12 to execute instructions according to the program, and controls the standard light source 11 to emit light to a preset percentage light value, and the preset percentage light value is set to 20%, 40%, 80%, and 100%.

[0123] The standard light source 11 is controlled to emit 20% light. Under dark field conditions, the Rhod, GFP, and DAPI channels of the fluorescence microscope are used in turn to photograph the optical array on the microwell array plate 131 for imaging. The images are taken 3 times at intervals of 10 seconds, and the exposure time for each channel is the same. The photographing results are stored in the memory 22. Under bright field conditions, the optical array on the microwell array plate 131 is photographed 3 times at intervals of 10 seconds, and the exposure time is the same as that under dark field conditions. The photographing results are stored in the memory 22.

[0124] As described above, the processor 21 controls the single chip computer 121 on the PCB circuit board 12 to execute instructions according to the program, and controls the standard light source 11 to emit light to three light states of 40%, 80%, and 100%, respectively. In the three light states, the following are performed respectively: under dark field conditions, the Rhod, GFP, and DAPI channels of the fluorescence microscope are used in turn to photograph the optical array on the microwell array plate 131, with an interval of 10s and 3 shots, and the exposure time for each channel is the same, and the shooting results are stored in the memory 22; under bright field conditions, the optical array on the microwell array plate 131 is photographed and imaged, with an interval of 10s and 3 shots, and the exposure time is the same as that under dark field conditions, and the shooting results are stored in the memory 22.

[0125] S4: Imaging uniformity calibration:

[0126] The imaging uniformity R is calculated respectively when the luminous intensity of the standard light source 11 is 20%, 40%, 80% and 100%.

[0127] For example, when the luminous intensity of the standard light source 11 is 20%, one array area is selected from the nine array areas to calculate the imaging uniformity. In this embodiment, the central area is selected, that is:

[0128] The processor 21 acquires the image, selects the fifth area array area according to the program, the identification module 25 identifies the number of measurement points and the fluorescence intensity value in the selected array area, and the calculation module 23 calculates the fluorescence microscope imaging uniformity R of the dark field Rhod, GFP, and DAPI channels respectively;

[0129] The calculation module 23 calculates the uniformity R of the fluorescence microscope imaging in the bright field. The calculation formula of the fluorescence microscope imaging uniformity R is as shown in formula (3):

[0130] R (3)

[0131] Where R is the imaging uniformity in cd / m 2 ;

[0132] is the fluorescence intensity value of the ith measurement point, in cd / m2 ;

[0133] It is the average illuminance value of all measurement points, in cd / m 2 ;

[0134] is the total number of measurement points;

[0135] R should meet the following requirements: R≤1.0 cd / m 2 , the judgment module 26 makes a qualified judgment.

[0136] The fluorescence microscope imaging uniformity measurement data and calculation results are shown in Table 2:

[0137]

[0138] S5: Imaging linear calibration:

[0139] The processor 21 controls the standard light source 11 to emit light at 20%, 40%, 80% and 100% of the fluorescence intensity, and calculates the linearity of the fluorescence microscope imaging in different channels of the dark field and the bright field respectively. .

[0140] The acquisition module 24 of the host 2 acquires the image, and controls the recognition module 25 and the acquisition module 24 to select each of the 9 array areas according to the optical array of the imaging, and the recognition module 25 identifies the total fluorescence intensity value of each area, and the calculation module 23 calculates the linearity of the dark field Rhod, GFP, DAPI channels and the bright field fluorescence microscope imaging respectively. , the calculation method is as follows:

[0141] (4)

[0142] Where:

[0143] is the total fluorescence intensity value of the jth area in the optical array, in cd / m 2 ;

[0144] is the average of the total fluorescence intensity values ​​of the 9 regions in the optical array, in cd / m 2 ;

[0145] is the logarithmic value of the simulated fluorescence intensity corresponding to the jth region in the optical array;

[0146] is the average value of the logarithmic value of the simulated fluorescence intensity corresponding to each area in the optical array.

[0147] and Should meet: >0.997, the judgment module 26 makes a qualified judgment.

[0148] Imaging linearity is determined by the linearity of the positions of areas with different light transmittances under the same fluorescence intensity.

[0149] The linear measurement data and calculation results of fluorescence microscope imaging are shown in Table 3, and the imaging linearity graph is shown in Figure 9-12 shown.

[0150]

[0151] S6: Optical Linear Calibration:

[0152] The acquisition module 24 acquires the image, and the control recognition module 25 selects one of the nine micropore areas, analyzes the fluorescence intensity values ​​of the micropore area under the standard light source 11 with luminous intensity of 20%, 40%, 80%, and 100%, and calculates the optical linearity respectively. .

[0153] The calculation formula is as follows:

[0154] (5)

[0155] n is the number of data points;

[0156] The measured value of fluorescence intensity at different percentages of luminescence, in cd / m 2 ;

[0157] is the predicted value, in cd / m 2 ;

[0158] It is the average value of the measured value, in cd / m 2 ;

[0159] and Should meet: >0.997, the judgment module 26 makes a qualified judgment;

[0160] The optical linear measurement data and calculation results of the fluorescence microscope are shown in Table 4:

[0161]

[0162] S7: The processor 21 automatically generates a calibration result report of the fluorescence microscope under test according to the program.

[0163] It should be noted that the structure of the detection end 1 can be selected as required. Figure 3As shown or Figure 4 As shown, Figure 4 A filter 14 is installed between the standard light source 11 and the microwell array plate 131 to allow only light of a specific wavelength to pass through. For example, when calibrating the Rhod channel, only light within the Rhod emission wavelength range is allowed to pass through.

[0164] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A fluorescence microscope measurement and calibration method, characterized in that: The steps include: S1: Obtain the magnification value of the objective lens of the fluorescence microscope to be tested, and horizontally place the detection end with a matching microwell array plate on the stage of the fluorescence microscope so that the microwell array plate is located in the center of the imaging and is clear; S2: Magnification error calibration: Control the standard light source to emit light to a preset value; adjust the eyepiece diopter of the fluorescence microscope to 0 and then replace the eyepiece with a micrometer eyepiece, respectively obtain the indications of the horizontal and vertical scale spacing images of the detection end, and respectively calculate the horizontal and vertical coordinate objective lens magnifications, and calculate the horizontal and vertical coordinate objective lens magnification error values; S3: Collect calibration data: Control the standard light source to emit light to a preset percentage luminescence value, wherein the preset percentage luminescence value is set to a plurality of values; under dark field conditions, sequentially use the Rhod, GFP, and DAPI channels of the fluorescence microscope to photograph and image the optical array on the microwell array plate; under bright field conditions, photograph and image the optical array on the microwell array plate, and set the same number of shots and interval exposure time under dark field and bright field conditions; S4: Imaging uniformity calibration: Acquire an image, select a preset array area, identify the number of measurement points and fluorescence intensity values ​​in the array area, and calculate the fluorescence microscope imaging uniformity R of different channels in the dark field and bright field, and R should satisfy: R≤1.0 cd / m 2 ; S5: Imaging linear calibration: Acquire the image, select each array area according to the optical array of the imaging, identify the total fluorescence intensity value of each area, and calculate the linearity of the fluorescence microscope imaging in different channels of the dark field and the bright field. ,and Should meet: >0.997; S6: Optical Linear Calibration: Acquire the image, select one of the nine microwell areas, analyze its fluorescence intensity value when the standard light source emits different percentages, and calculate the optical linearity ,and Should meet: >0.997; S7: Automatically generate a report of fluorescence microscope calibration results.

2. The fluorescence microscope measurement and calibration method according to claim 1, characterized in that: Also includes: In S1, the magnification value of the objective lens of the fluorescence microscope under test is obtained, including 10x, 20x, and 40x respectively; Calculate the horizontal and vertical coordinate objective lens magnification , as shown in formula (1): （1） Where, Y' is the measured value of the micrometer eyepiece, in mm; Y is the scale division value of the horizontal / vertical coordinate of the detection end, the unit is mm, and the horizontal and vertical scale division values ​​are the same; Calculate the horizontal and vertical coordinate objective magnification errors respectively , as shown in formula (2): （2） in, is the nominal value of objective lens magnification; And obtain the horizontal and vertical coordinate objective lens magnification error The larger one is the calibration result.

3. The fluorescence microscope measurement and calibration method according to claim 1, characterized in that: Also includes: In S2, the standard light source is controlled to emit white light and to emit 20% of the light, the eyepiece of the fluorescence microscope is adjusted to have a diopter of 0, the eyepiece is removed, a micrometer eyepiece is installed, and the magnification of the horizontal coordinate objective lens and the magnification of the vertical coordinate objective lens are calculated respectively.

4. The fluorescence microscope measurement and calibration method according to claim 3, characterized in that: Also includes: In S3, the standard light source is controlled to emit 20% light, and under dark field conditions, the optical array on the microwell array plate is photographed and imaged using the Rhod, GFP, and DAPI channels of the fluorescence microscope in sequence, with an interval of 10 seconds and 3 shots, and the exposure time for each channel is the same; under bright field conditions, the optical array on the microwell array plate is photographed and imaged, with an interval of 10 seconds and 3 shots, and the exposure time is the same as that under dark field conditions; Some of the preset percentage luminescence values ​​also include luminescence of 40%, luminescence of 80%, and luminescence of 100%, for taking photos under dark field conditions and bright field conditions respectively.

5. The fluorescence microscope measurement and calibration method according to claim 4, characterized in that: Also includes: In S4, a preset array area is selected, including calculating imaging uniformity by selecting one of nine array areas; The imaging uniformity R is calculated at luminescence intensities of 20%, 40%, 80%, and 100%, respectively. The imaging uniformity R includes the fluorescence microscope imaging uniformity of the DAPI channel, GFP channel, and RHOD channel in the bright field and the dark field, as shown in formula (3): （3） Where R is the imaging uniformity, in cd / m 2 ; is the fluorescence intensity value of the ith measurement point, in cd / m 2 ; It is the average illuminance value of all measurement points, in cd / m 2 ; is the total number of measurement points.

6. The fluorescence microscope measurement and calibration method according to claim 5, characterized in that: Also includes: In S4, a middle area, namely the fifth array area, is selected from the nine array areas to calculate imaging uniformity.

7. The fluorescence microscope measurement and calibration method according to claim 5, characterized in that: Also includes: In S5, the linearity of fluorescence microscopy imaging in different channels of dark field and bright field is calculated respectively under the fluorescence intensity conditions of the standard light source luminescence intensity of 20%, 40%, 80% and 100%. , as shown in formula (4): （4） Where: is the total fluorescence intensity value of the jth area in the optical array, in cd / m 2 ; is the average of the total fluorescence intensity values ​​of the 9 regions in the optical array, in cd / m 2 ; is the logarithmic value of the simulated fluorescence intensity corresponding to the jth region in the optical array; is the average value of the logarithmic value of the simulated fluorescence intensity corresponding to each area in the optical array.

8. The fluorescence microscope measurement and calibration method according to claim 5, characterized in that: Also includes: In S6, an image is acquired, one of the nine micropore areas is selected, and its fluorescence intensity values ​​when the luminous intensity of the standard light source is 20%, 40%, 80%, and 100% are analyzed, and the optical linearity is calculated respectively. , as shown in formula (5): （5） n is the number of data points; The measured value of fluorescence intensity at different percentages of luminescence, in cd / m 2 ; is the predicted value, in cd / m 2 ; It is the average value of the measured values, in cd / m 2 .

9. A fluorescence microscope measurement and calibration system, characterized in that: The method for measuring and calibrating a fluorescence microscope as described in any one of claims 1 to 8 above comprises a detection end horizontally arranged on the stage of the fluorescence microscope to be measured and a host controlling the detection end to perform detection and calibration; wherein, The detection end includes a bottom PCB circuit board, a top cover plate and a standard light source located at the center between the two plates, which are fixedly connected as one; the PCB circuit board is connected to the host through a flat cable, and a micro-hole array plate provided at the center of the top cover plate corresponds to the center of the standard light source, so that the light of the standard light source is uniformly emitted through the micro-hole array plate; The host includes a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the program, the steps of the fluorescence microscope metrology calibration method described in any one of the above items are implemented.

10. The fluorescence microscope measurement and calibration system according to claim 9, characterized in that: The detection end is provided with different detection ends according to the different magnifications of the objective lens to be measured and the size of the imaging image. The array sizes of the micropore array plate are different, and all are provided with micropores that are penetrated by laser engraving. The micropore array plate is divided into 9 areas, each area contains 10 / 20 / 30 / 40...90 micropores, which are distributed in a gradient, so that the light uniformly transmitted by the standard light source is distributed in a gradient in each area.

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