An automatic excitation light intensity calibration device and method for quantitative FRET imaging

Abstract

The present invention discloses an automatic calibration device and method for the excitation light intensity in quantitative FRET imaging. The device includes a FRET multi-wavelength combined light source, a beam splitting device, a light intensity detector, a lower computer and an upper computer. The FRET multi-wavelength combined light source is used to generate the excitation light for the donor channel and the acceptor channel during the FRET microscopic imaging process. The beam splitting device is used to split the excitation light. The light intensity detector is used to detect the partial light intensity of the excitation light for the donor and acceptor in the split light path. The lower computer completes channel selection and light intensity control by receiving instructions from the upper computer. The upper computer is used to collect and process the excitation light intensity information of the channels sent by the light intensity detector, and send a compensation instruction to the lower computer. The present invention is designed with independent modules, which can be conveniently placed in the output light path of any multi-wavelength combined light source and is flexibly applicable to different experimental environments. It can control the output intensity of the excitation light for the donor and acceptor and the intensity ratio of the donor and acceptor according to the requirements of the FRET experiment, and is suitable for quantitative FRET detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence resonance energy transfer detection, and particularly relates to an automatic excitation light intensity calibration device and method for quantitative FRET imaging. Background Art

[0002] Fluorescence resonance energy transfer (FRET) microscopy imaging technology can detect the interactions of proteins in living cells in real time and in situ, and has become an important tool for studying the dynamic processes of biochemical molecules in living cells. The FRET method based on acceptor sensitized emission (E-FRET) is considered to be the most suitable FRET quantitative detection technology for dynamic monitoring of living cells due to its high sensitivity, low damage and fast characteristics. In the experimental system of E-FRET, the intensity fluctuations of the multi-color excitation light source will cause changes in the spectral crosstalk coefficient, and the deviation of the spectral crosstalk coefficient will correspondingly lead to D measurement errors of E. Therefore, maintaining the stability of the relative intensity relationship between the donor excitation light and the acceptor excitation light during FRET microscopy imaging is crucial for quantitative FRET. Multi-wavelength combined light sources based on LEDs or lasers are replacing traditional broadband light sources such as halogen lamps and mercury lamps as the mainstream fluorescence microscope light sources due to their high brightness, long lifespan, flexible control, etc. During the use of multi-wavelength combined light sources, the light source and the beam combining device are easily affected by environmental temperature and vibration, etc., resulting in fluctuations in the excitation light intensity entering the imaging system. When a multi-wavelength combined light source is used as a FRET light source, its independent light intensity modulation cannot meet the automatic correction and compensation of the relative intensity relationship between the donor excitation light and the acceptor excitation light. The above problems make it necessary to repeatedly correct the spectral crosstalk coefficient and system parameters when a multi-wavelength combined light source is used as the excitation light source of a FRET experimental system, which brings inconvenience to quantitative FRET detection. Summary of the Invention

[0003] The main object of the present invention is to provide an automatic excitation light intensity calibration device and method for quantitative FRET imaging to overcome or at least mitigate the above-mentioned defects of the prior art.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In one aspect of the present invention, there is provided an automatic excitation light intensity calibration device for quantitative FRET imaging, including a FRET multi-wavelength combined light source, a beam splitting device, a light intensity detector, a lower computer, and an upper computer;

[0006] The FRET multi-wavelength combined light source is used to generate the excitation light for the donor channel and the acceptor channel during FRET microscopy imaging;

[0007] The beam splitting device is used to receive the excitation light generated by the FRET multi-wavelength combined light source and split the light according to a preset ratio;

[0008] The light intensity detector is used to detect the partial light intensity of the donor excitation light and the acceptor excitation light in the light splitting optical path;

[0009] The host computer is respectively connected to the slave computer and the light intensity detector, and is used to receive the excitation light intensity information of the channels sent by the light intensity detector, calculate the deviation of the light intensity ratio of the D / A channels, and send the light intensity compensation instruction to the slave computer;

[0010] The slave computer is connected to the FRET multi-wavelength combined light source, and is used to perform light intensity control according to the instructions of the host computer.

[0011] As a preferred technical solution, the FRET multi-wavelength combined light source includes at least two light sources with different excitation wavelengths: a donor excitation light source and an acceptor excitation light source; the FRET multi-wavelength combined light source also uses the slave computer to perform channel switching.

[0012] As a preferred technical solution, the beam splitting device is arranged in front of the outgoing optical path of the FRET multi-wavelength combined light source, and one path of its light splitting optical path is introduced into the light intensity detector, and the other path is used as the output light of the FRET light source.

[0013] As a preferred technical solution, the slave computer is respectively connected to the light intensity detector and the host computer through serial communication; after receiving the control instruction of the host computer, the slave computer performs channel selection and light intensity control on the FRET multi-wavelength combined light source in the form of analog voltage modulation of the external trigger through digital-to-analog conversion.

[0014] Another aspect of the present invention also provides an automatic calibration method for the excitation light intensity for quantitative FRET imaging, including the following steps:

[0015] Obtain the D / A channel light source baseline value, dye and channel light intensity ratio configuration;

[0016] Fit the D / A channel light source baseline linear interval according to the D / A channel light source baseline value;

[0017] Obtain the mapping relationship of the D / A channel light intensity linear interval according to the light intensity ratio configuration and obtain the FRET excitation light intensity adjustment function under the current configuration;

[0018] Traverse the modulation range according to the FRET excitation light intensity adjustment function to obtain the theoretical values of the D / A channel light intensity at all points, and use the light intensity detector to measure the actual values of the D / A channel light intensity;

[0019] Calculate the deviation of the D / A channel light intensity ratio according to the theoretical value and the actual value of the D / A channel light intensity;

[0020] Determine whether the light intensity ratio deviation of the D / A channel is less than the set threshold:

[0021] When the deviation is greater than the threshold, compensate the function parameters of the FRET excitation light intensity adjustment function in the linear interval of the D / A channel light intensity through closed-loop control and re-measure and calculate the light intensity ratio deviation of the D / A channel;

[0022] When the deviation is less than the threshold, determine whether the crosstalk coefficient is known according to the automatic calibration result of the excitation light intensity of quantitative fluorescence resonance energy transfer imaging in the above steps; when the crosstalk coefficient is unknown, re-measure the crosstalk coefficient corresponding to the dye and light intensity ratio configuration of this channel, and when the crosstalk coefficient is known, save the FRET excitation light intensity adjustment function and the crosstalk coefficient result under the current configuration.

[0023] As a preferred technical solution, the fitting to obtain the linear interval of the D / A channel light source baseline is specifically:

[0024] Use the Sigmoid function to fit the D / A channel light source baseline by the least squares method, and select the inflection point of the second derivative of the obtained Sigmoid function fitting result as the endpoint of the linear range interval to obtain the light source baseline linear interval.

[0025] As a preferred technical solution, the FRET excitation light intensity adjustment function under the current configuration is specifically:

[0026] Taking the intensity modulation instruction of the upper computer as the independent variable and the D / A channel excitation light intensity obtained by the mapping relationship of the FRET excitation light according to the D / A channel light intensity linear interval as the dependent variable to construct a function mapping relationship, which is the FRET excitation light intensity adjustment function under the current configuration.

[0027] As a preferred technical solution, the calculation of the light intensity ratio deviation of the D / A channel is specifically:

[0028] The light intensity ratio deviation of the D / A channel is the difference between the theoretical value of the D / A channel light intensity mapped by the FRET excitation light intensity adjustment function and the actual value of the D / A channel light intensity measured by the light intensity detector.

[0029] As a preferred technical solution, the crosstalk coefficient is measured by samples of single donors and single acceptors, specifically:

[0030] a = I DA (A) / I AA (A)

[0031] b = I DD (A) / I AA (A)

[0032] c = I A( (D) / I DD(D)

[0033] d = I DA (D) / I DD (D)

[0034] Among them, the crosstalk coefficients a and b are parameters for correcting the receptor fluorescence crosstalk of channels I DA and I DD The crosstalk coefficients c and d are parameters for correcting the receptor fluorescence crosstalk of channels I AA and I DA ; I DD is the fluorescence intensity detected in the donor channel when using the excitation donor, and I DA is the fluorescence intensity detected in the receptor channel when the donor is excited; I AA is the fluorescence intensity detected in the receptor channel when the receptor is excited; The content in the brackets represents the measured biological sample, where D represents the sample of the single transfected donor and A represents the sample of the single transfected receptor.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] (1) The present invention is designed as an independent module and can be conveniently placed in the output optical path of any multi-wavelength combined beam light source, calibrating various types of multi-wavelength combined beam light sources as the excitation light source of the FRET experimental system. The light source types include LEDs or lasers with corresponding spectra and broadband light sources that filter out the desired excitation wavelength from the broadband spectrum through a filter wheel or filter carrier plate.

[0037] (2) The present invention can not only correct and compensate for the excitation light intensity fluctuation of the multi-wavelength combined beam light source, but also flexibly control the output intensity of the donor and acceptor excitation lights and the donor-acceptor intensity ratio according to the requirements of the FRET experiment, and automatically load the correction factor of the FRET system under the current configuration, solving the problem of repeatedly correcting the spectral crosstalk coefficient and system parameters in the experiment, and thus bringing convenience to quantitative FRET detection. Brief Description of the Drawings

[0038] Figure 1 is the schematic diagram of the principle of an excitation light intensity automatic calibration device for quantitative FRET imaging described in an embodiment of the present invention;

[0039] Figure 2 is the flow schematic diagram of an excitation light intensity automatic calibration method for quantitative FRET imaging described in an embodiment of the present invention;

[0040] Figure 3 is the comparison result diagram of the light source stability of the FRET multi-color combined beam light source after being automatically calibrated by the present invention;

[0041] Figure 4It is a schematic diagram of the effect of the light intensity ratio configuration of different channels after the automatic calibration of the FRET multi-color combined beam light source by the present invention.

[0042] Explanation of the reference numerals in the attached drawings:

[0043] 1. FRET multi-wavelength combined beam light source; 1A. Donor excitation light source; 1B. Receptor excitation light source; 2. Beam splitting device; 3. Light intensity detector; 4. Lower computer; 5. Upper computer. Specific implementation mode

[0044] In order to enable those skilled in the art of this technical field to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of this application.

[0045] Embodiment 1

[0046] As Figure 1 shown, this embodiment provides an automatic calibration device for the excitation light intensity for quantitative FRET imaging, including a FRET multi-wavelength combined beam light source 1, a beam splitting device 2, a light intensity detector 3, a lower computer 4, and an upper computer 5, where:

[0047] The FRET multi-wavelength combined beam light source 1 is used to generate the excitation light for the donor channel and the receptor channel in the FRET microscopy imaging process. The FRET multi-wavelength combined beam light source 1 includes at least two light sources with different excitation wavelengths: a donor excitation light source 1A and a receptor excitation light source 1B. This light source can perform fast channel switching and light intensity modulation using the lower computer 4. Advantageously, this light source has LEDs or lasers corresponding to the spectrum. In one embodiment, the donor excitation light source 1A is a solid-state laser that can generate a wavelength of 488 nm, and the receptor excitation light source 1B is a solid-state laser that can generate a wavelength of 561 nm.

[0048] The beam splitting device 2 is placed in front of the outgoing light path of the FRET multi-wavelength combined beam light source 1, and is used to receive the combined beam light generated by the FRET multi-wavelength combined beam light source 1, and split the light according to a certain ratio, and introduce a part of the light into the light intensity detector 3, and the other part of the light is output as the FRET light source output light. In one embodiment, the beam splitting device 2 is a non-polarizing beam splitting cube, and the splitting ratio of the separated transmitted light and reflected light is 90:10. After the light generated by the FRET multi-wavelength combined beam light source 1 is incident on the beam splitting device 2, the generated reflected light enters the light intensity detector 3, and the transmitted light is output as the FRET light source output light.

[0049] The light intensity detector 3 is placed on a splitting optical path of the beam splitting device 2, and is used to detect the partial light intensities of the donor excitation light and the acceptor excitation light on this splitting optical path. The light intensity detector 3 and the host computer 5 are connected by a cable, and the collected excitation light intensity information of the donor / acceptor (D / A) channel is sent to the host computer 5. In one embodiment, the light intensity detector 3 is a photodiode power meter, and the light intensity information is sent to the host computer 5 through serial communication.

[0050] The slave computer 4 is connected to the FRET multi-wavelength combined light source 1 and the host computer 5 by a cable, and is used to receive the control instructions sent by the host computer 5, and perform gating and light intensity control on the D / A channel of the FRET multi-wavelength combined light source 1. In one embodiment, the slave computer uses an arduino control board, communicates with the host computer 5 through serial communication, and after receiving the control instructions from the host computer 5, performs gating and light intensity control on the solid-state lasers with wavelengths of 488 nm and 561 nm in the form of analog voltage modulation of the external trigger through digital-to-analog conversion.

[0051] The host computer 5 serves as a controller, and is used to collect the excitation light intensity information of the D / A channel sent by the light intensity detector 3, calculate the light intensity ratio deviation of the D / A channel, and then send the light intensity compensation instruction to the slave computer 4. In one embodiment, the host computer is a labview control system based on the NI company.

[0052] Embodiment 2

[0053] As Figure 2 shown, in another embodiment of the present application, an automatic calibration method for the excitation light intensity for quantitative FRET imaging is provided, including the following steps:

[0054] (1) S1, obtain the baseline values of the light sources of the donor / acceptor (D / A) channel, the configuration of the dyes and the light intensity ratios of the channels.

[0055] (1.1) The baseline value of the D / A channel light source is the mapping relationship between different intensity modulation instructions of the host computer 5 and the corresponding measurement values of the light intensity detector 3 within the modulation range of the donor excitation light and the acceptor excitation light of the FRET multi-wavelength combined light source. For different FRET multi-wavelength combined light sources, the baseline values of the D / A channel light sources are also different.

[0056] Furthermore, in this embodiment, the solid-state lasers with wavelengths of 488 nm and 561 nm are selected, and the mean value of the mapping relationship between the intensity modulation instructions of the host computer 5 and the light intensity measurement values of the light intensity detector 3 is measured independently and repeatedly no less than m times (m≥3) as the baseline value of the D / A channel light source;

[0057] (1.2) The dye is a fluorescent labeling group that serves as a donor-acceptor pair for FRET microscopy imaging.

[0058] Furthermore, in this embodiment, the fluorescent protein pair GFP-mCherry is selected as the FRET donor-acceptor pair. Among them, for the donor excitation channel, a laser with a wavelength of 488 nm is used as the excitation light for the donor. For the donor emission channel, a channel with a wavelength of 525±15 nm is used as the detection channel for the emitted fluorescence of the donor. For the acceptor excitation channel, a laser with a wavelength of 561 nm is used as the excitation light for the acceptor. For the acceptor emission channel, a channel with a wavelength of 600±15 nm is used as the detection channel for the emitted fluorescence of the acceptor.

[0059] (1.3) The channel light intensity ratio is the ratio of the donor excitation light intensity and the acceptor excitation light intensity of the FRET multi-wavelength combined beam light source, and is used to configure the most suitable donor-acceptor light intensities under different experimental conditions, so as to ensure that when the FRET three-channel images detected by the detector maintain appropriate intensities, the illumination light intensity of a certain channel will not be too high to cause photobleaching.

[0060] Furthermore, the preferred channel light intensity ratios in this embodiment are:

[0061] a) 1:2;

[0062] b) 1:1;

[0063] c) 2:1;

[0064] d) 3:1;

[0065] e) 4:1.

[0066] (2) S2. Obtain the linear interval of the D / A channel light source baseline by fitting the D / A channel light source baseline value.

[0067] The linear interval of the D / A channel light source baseline is the acceptable linear range interval of the output light intensity of the FRET multi-wavelength combined beam light source 1 after receiving the intensity modulation instruction from the host computer 5. In this embodiment, the Sigmoid function is used to fit the D / A channel light source baseline by the least squares method, and the inflection point of the second derivative of the obtained Sigmoid function fitting result is selected as the endpoint of the linear range interval to obtain the light source baseline linear interval, which is a preferred technical solution.

[0068] (3) S3. Configure the mapping relationship of the D / A channel light intensity linear interval according to the light intensity ratio and obtain the FRET excitation light intensity adjustment function under the current configuration.

[0069] Taking the intensity modulation instruction from the host computer 5 as the independent variable and the donor-acceptor channel excitation light intensities obtained from the FRET excitation light according to the mapping relationship of the D / A channel light intensity linear interval as the dependent variable, a functional mapping relationship is constructed, and thus the FRET excitation light intensity adjustment function is obtained.

[0070] Further, in this embodiment, the intensity modulation instruction of the host computer 5 is converted into a voltage value after digital-to-analog conversion and voltage modulation by the slave computer 4, and the FRET excitation light intensity adjustment function is obtained by calculating the measured v-mw curve. The linear interval of the light source baseline of the D / A channel is linearly mapped according to the known D / A channel light intensity ratio r as the slope.

[0071] (4) S4. Traverse the modulation range according to the FRET excitation light intensity adjustment function to obtain the theoretical D / A channel light intensity values of all points; and measure the actual D / A channel light intensity value using the light intensity detector 3.

[0072] (5) S5. Calculate the deviation of the D / A channel light intensity ratio.

[0073] The deviation of the D / A channel light intensity ratio is the difference between the theoretical D / A channel light intensity value mapped by the FRET excitation light intensity adjustment function and the actual D / A channel light intensity value measured by the light intensity detector 3.

[0074] (6) S6. Determine whether the deviation of the D / A channel light intensity ratio is less than the set threshold. When the deviation is greater than the threshold, compensate the parameters of the FRET excitation light intensity adjustment function in the linear interval of the D / A channel light intensity through closed-loop control and re-execute steps S4 - S5; when the deviation is less than the threshold, execute step S7.

[0075] (7) S7. Determine whether the crosstalk coefficients a, b, c, and d are known according to the automatic calibration result of the excitation light intensity of quantitative fluorescence resonance energy transfer imaging in steps 1 - 6. When the crosstalk coefficients are unknown, re-measure the crosstalk coefficients corresponding to the dye and light intensity ratio configuration of this channel, and when the crosstalk coefficients are known, execute step S8.

[0076] The crosstalk coefficients are measured by samples of single donors and single acceptors, specifically:

[0077] a = I DA (A) / I AA (A)

[0078] b = I DD (A) / I AA (A)

[0079] c = I AA (D) / I DD (S)

[0080] d = I SA (S) / I DD (D)

[0081] Among them, the crosstalk coefficients a and b are for I DA and I DDParameters for correcting the receptor fluorescence crosstalk of the channels, where the crosstalk coefficients c and d are for I AA and I DA channels to correct the receptor fluorescence crosstalk; I DD is the fluorescence intensity detected in the donor channel when using the excitation donor, and I DA is the fluorescence intensity detected in the receptor channel when the donor is excited; I AA is the fluorescence intensity detected in the receptor channel when the receptor is excited; the biological sample being measured is indicated in parentheses, where D represents the sample of the single transfected donor and A represents the sample of the single transfected receptor.

[0082] The methods for measuring the crosstalk coefficients are only schematically shown in Figure 2 as they are well known in the prior art.

[0083] (8) S8, save the FRET excitation light intensity adjustment function and the crosstalk coefficient results under the current configuration.

[0084] As Figure 3 shown is the comparison of the light source stability results before and after the automatic calibration of the present invention. Taking the receptor excitation light channel with a wavelength of 561 nm as an example, the independent variable of the obtained light source baseline linear interval is modulated and sent to the lower computer as an instruction of the host computer, measured by the light source detector 3, and the actual measured v-mw curve distribution can be obtained by feedback to the host computer. The results of 60 independent repeated measurements in a stable experimental environment show that the light source stability is significantly improved after automatic calibration, meeting the requirements of quantitative FRET imaging.

[0085] In this embodiment, the preferred channel light intensity ratios are 1:2, 1:1, 2:1, 3:1, 4:1. Taking the channel with a wavelength of 561 nm as an example, after 30 independent repeated measurements in a stable experimental environment, the effects of different channel light intensity ratio configurations after the automatic calibration of the present invention are as Figure 4 shown. After the laser calibration, the actual ratios are very close to the preset ratios such as 1:2, 1:1, etc., and the error is very small.

[0086] It should be understood that each part of the present application can be implemented by hardware, software, firmware or a combination thereof. In the above embodiments, multiple steps or methods can be implemented by software or firmware stored in the memory and executed by a suitable instruction execution system. For example, if implemented by hardware, as in another embodiment, any one or a combination of the following well-known technologies in the art can be used: discrete logic circuits with logic gate circuits for implementing logical functions on data signals, application-specific integrated circuits with appropriate combinational logic gate circuits, programmable gate arrays (PGAs), field-programmable gate arrays (FPGAs), etc.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for automatic calibration of excitation light intensity for quantitative FRET imaging, characterized in that: The following steps are involved: Obtain D / A channel light source baseline value, dye and channel light intensity ratio configuration; The linear interval of the D / A channel light source baseline is obtained by fitting the D / A channel light source baseline value, specifically: The Sigmoid function is used to fit the D / A channel light source baseline through least squares fitting, and the inflection point of the second-order derivative of the Sigmoid function fitting result is selected as the endpoint of the linear range interval to obtain the light source baseline linear interval; According to the light intensity ratio configuration, the mapping relationship of the linear interval of the D / A channel light intensity is obtained and the FRET excitation light intensity adjustment function under the current configuration is obtained; According to the FRET excitation light intensity adjustment function, the modulation range is traversed to obtain the theoretical value of the D / A channel light intensity at all points, and the actual value of the D / A channel light intensity is measured using a light intensity detector; Calculate the D / A channel light intensity ratio deviation based on the D / A channel light intensity theoretical value and actual value; Determine whether the D / A channel light intensity ratio deviation is less than the set threshold: When the deviation is greater than the threshold, the FRET excitation light intensity adjustment function parameters in the linear interval of the D / A channel light intensity are compensated through closed-loop control and the D / A channel light intensity ratio deviation is remeasured and calculated; When the deviation is less than the threshold, determining whether the crosstalk coefficient is known based on the automatic calibration result of the excitation light intensity of the quantitative fluorescence resonance energy transfer imaging in the above step; When the crosstalk coefficient is unknown, re-measure the crosstalk coefficient corresponding to the channel dye and light intensity ratio configuration. When the crosstalk coefficient is known, save the FRET excitation light intensity adjustment function and crosstalk coefficient results under the current configuration.

2. The method for automatic calibration of excitation light intensity for quantitative FRET imaging according to claim 1, wherein: The FRET excitation light intensity adjustment function under the current configuration is specifically: The intensity modulation instruction of the host computer is used as the independent variable, and the D / A channel excitation light intensity obtained by mapping the FRET excitation light according to the linear interval of the D / A channel light intensity is used as the dependent variable to construct a function mapping relationship, which is the FRET excitation light intensity adjustment function under the current configuration.

3. The method for automatic calibration of excitation light intensity for quantitative FRET imaging according to claim 1, wherein: The calculation of the D / A channel light intensity ratio deviation is specifically as follows: The D / A channel light intensity ratio deviation is the difference between the theoretical value of the D / A channel light intensity mapped by the FRET excitation light intensity adjustment function and the actual value of the D / A channel light intensity measured by the light intensity detector.

4. The method for automatic calibration of excitation light intensity for quantitative FRET imaging according to claim 1, wherein: The crosstalk coefficient is measured by a single transfection donor sample and a single transfection acceptor sample, specifically: a =( I DA ( A ))⁄( I AA ( A ))； b =( I DD ( A ))⁄( I AA ( A ))； c =( I AA ( D ))⁄( I DD ( D ))； d =( I DA ( D ))⁄( I DD ( D ))； Among them, the crosstalk coefficient a and b Yes I DA and I DD The parameter for correcting the acceptor fluorescence crosstalk of the channel, the crosstalk coefficient c and d Yes I AA and I DA Parameters for correcting the acceptor fluorescence crosstalk of the channel; I DD is the fluorescence intensity detected in the donor channel when using the excited donor, I DA is the fluorescence intensity detected in the acceptor channel upon donor excitation; I AA is the fluorescence intensity detected in the receptor channel when the receptor is excited; the brackets indicate the biological sample being measured, where D Representative samples of single-transfection donors, A Representative samples of single transfected receptors.

5. An automatic calibration device for excitation light intensity for quantitative FRET imaging, characterized in that: A method for automatic calibration of excitation light intensity for quantitative FRET imaging according to any one of claims 1 to 4, comprising a FRET multi-wavelength beam combining light source, a beam splitting device, a light intensity detector, a slave computer, and a master computer; The FRET multi-wavelength beam combining light source is used to generate excitation light for the donor channel and the acceptor channel during FRET microscopic imaging; The beam splitting device is used to receive the excitation light generated by the FRET multi-wavelength beam combining light source and split the light according to a preset ratio; The light intensity detector is used to detect the partial light intensity of the donor excitation light and the acceptor excitation light in the split light path; The host computer is connected to the slave computer and the light intensity detector respectively, and is used to receive the excitation light intensity information of the channel sent by the light intensity detector and calculate the D / A channel light intensity ratio deviation, and send the light intensity compensation instruction to the slave computer; The lower computer is connected to the FRET multi-wavelength beam combining light source and is used to control the light intensity according to the instructions of the upper computer.

6. The automatic calibration device for excitation light intensity for quantitative FRET imaging according to claim 5, characterized in that: The FRET multi-wavelength beam combining light source includes at least two light sources with different excitation wavelengths: a donor excitation light source and an acceptor excitation light source; the FRET multi-wavelength beam combining light source also uses a lower computer to perform channel switching.

7. The automatic calibration device for excitation light intensity for quantitative FRET imaging according to claim 5, characterized in that: The beam splitting device is arranged before the outgoing light path of the FRET multi-wavelength combined light source, one of the split light paths is introduced into the light intensity detector, and the other is used as the FRET light source to output light.

8. The automatic calibration device for excitation light intensity for quantitative FRET imaging according to claim 5, characterized in that: The lower computer is connected to the light intensity detector and the upper computer respectively through serial communication; after receiving the control instruction of the upper computer, the lower computer selects the channel and controls the light intensity of the FRET multi-wavelength beam combining light source by analog voltage modulation external triggering through digital-to-analog conversion.

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