Multi-channel fluorescence microscopy imaging control system based on automatic E-FRET imaging of fluorescence probes

Abstract

The present invention relates to a multi-channel fluorescence microscopy imaging control system for automatic E-FRET imaging based on fluorescence probes, which is applied to a multi-channel fluorescence microscopy imaging system. By inputting the relevant information of the imaging system and fluorescence probes, it performs automatic imaging matching according to the optical characteristics of the fluorescence probes and the imaging system, and combines the fluorescence probes that can be imaged in pairs into donor-receptor pairs and then conducts preliminary FRET imaging screening. For the donor-receptor pairs preliminarily screened, the user provides samples for imaging experiments, and based on this, FRET effect judgment and E-FRET parameter correction are carried out. For the donor-receptor pairs determined to be able to produce FRET effects, the corresponding E-FRET shooting process is automatically generated and saved; when the user selects the saved donor-receptor pairs and provides experimental samples for experiments, it controls the imaging system to execute the E-FRET shooting process corresponding to the donor-receptor pairs to conduct imaging experiments on the experimental samples, and calculates the E-FRET results using the results of E-FRET parameter correction.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence microscopy imaging technology, and particularly to a multi-channel fluorescence microscopy imaging control system for automatic E-FRET imaging according to fluorescence probes. Background Art

[0002] A multi-channel fluorescence microscopy imaging system is a highly specialized microscopy technology that allows researchers to simultaneously perform accurate, clear, and interference-free imaging of multiple fluorescence labels in biological samples. The core of this technology lies in its ability to distinguish and record multiple independent fluorescence signals, thereby revealing complex cellular structures, molecular interactions, and dynamic biological processes. A multi-channel fluorescence microscopy imaging system typically includes a light source (including a light source and an excitation filter), an optical device group (including an emission filter, a dichroic mirror, etc.), a detector (CCD, photomultiplier tube, CMOS, etc.), a motorized stage, a control board, etc.

[0003] Current multi-channel fluorescence microscopy imaging systems support imaging by manually selecting a light source and a filter block / group according to recommendations based on fluorescence probes. For such a microscopy imaging system, scientific researchers can only select the fluorescence dyes, i.e., fluorescence probes, preset in the system for imaging. If the system does not have a preset, scientific researchers need to perform a spectral comparison between the new dye and the existing dyes. Imaging can be completed only if the new dye has a similar spectrum to the existing dyes; otherwise, imaging cannot be completed. Moreover, the algorithm for matching imaging according to fluorescence probes in the existing imaging system is unknown. In recent years, with the rapid development of the fluorescence probe field, the types of fluorescence probes have increased rapidly, and the above-mentioned fluorescence microscopy imaging system appears to be very limited.

[0004] Fluorescence resonance energy transfer (Förster resonance energy transfer, abbreviated as FRET)

[0005] As a spectral nanometer molecular ruler, it can achieve nanoscale spatial resolution, and under the irradiation of specific light, the characteristics of energy transfer between two fluorescence dyes are used to monitor the sample in real time. This feature makes it an important research tool in the biological and chemical fields.

[0006] The E-FRET method (i.e., the channel-based sensitized emission method) is a non-destructive three-channel FRET apparent efficiency measurement method. Due to its characteristics such as fast speed and non-invasive nature, the E-FRET method has become the most widely used method for dynamic monitoring of living cells, and can better correct spectral crosstalk and system parameters. The E-FRET method involves two different fluorescent probes. For these two probes, first, each is required to be able to be imaged in the imaging system, and second, these two fluorescent probes are required to exhibit the FRET effect. And before conducting the E-FRET experiment, it is necessary to pre-correct (i.e., parameter correction) the optical detection system and the optical properties of the fluorophores using a standard sample. Moreover, once the correction is completed, no system parameters can be changed in subsequent experiments, which poses a high technical threshold. Currently, the microscope control systems on the market cannot directly perform E-FRET imaging, greatly limiting the development and popularization application of this technology.

[0007] The overall objective of this project is to improve the multi-channel fluorescence microscopy imaging system so that it can automatically identify pairs of fluorescent probes capable of performing E-FRET imaging and quickly perform automatic E-FRET imaging. Summary of the Invention

[0008] The objective of the present invention is to provide a multi-channel fluorescence microscopy imaging control system for automatic E-FRET imaging according to fluorescent probes.

[0009] The objective of the present invention is achieved through the following technical solutions: A multi-channel fluorescence microscopy imaging control system for automatic E-FRET imaging according to fluorescent probes, which is applied to a multi-channel fluorescence microscopy imaging system, and is characterized in that it includes:

[0010] System parameter entry software module: Determine the relevant information required to establish a connection with each electric control in the imaging system and enter the optical characteristic parameters of each optical element in the imaging system;

[0011] Fluorescent probe information entry software module: Enter the optical characteristic parameters of the fluorescent probe;

[0012] Imaging matching software module: Automatically perform imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and save the imaging settings of the fluorescent probe;

[0013] Preliminary FRET imaging screening software module: For the fluorescent probes that can be imaged, combine them in pairs to form donor-acceptor pairs and then perform preliminary FRET imaging screening;

[0014] FRET Effect Judgment and E-FRET Parameter Calibration Software Module: For the donor-receptor pairs preliminarily screened out, generate standard E-FRET imaging settings (after clarifying the donor, receptor, and their respective imaging settings (the imaging settings of each fluorescence probe are clarified by the imaging matching software module), the standard E-FRET imaging settings for this donor-receptor pair can be determined), and then control the imaging system to perform imaging experiments on the standard samples provided by the user according to the standard E-FRET imaging settings, and judge the FRET effect (judge according to the specific imaging situation) and calibrate the E-FRET parameters. For the donor-receptor pairs that are determined to be able to produce the FRET effect, automatically generate the corresponding E-FRET shooting process and save it; the standard samples include single-donor samples, single-receptor samples, and samples containing both the donor and the receptor;

[0015] E-FRET Imaging Execution Software Module: When the user selects a saved donor-receptor pair and provides an experimental sample for the experiment, control the imaging system to execute the E-FRET shooting process corresponding to the donor-receptor pair to perform an imaging experiment on the experimental sample, and calculate the E-FRET result using the result of the E-FRET parameter calibration.

[0016] The control system of the present invention can automatically identify the fluorescence probe combinations that can be imaged to form donor-receptor pairs that can produce the FRET effect, and generate the corresponding E-FRET shooting process. The user only needs to select the corresponding donor-receptor pair and provide the experimental sample to achieve the automatic E-FRET imaging of the corresponding FRET pair.

[0017] The method for performing preliminary FRET imaging screening is as follows:

[0018] 1) According to the normalized spectral characteristics of the absorption spectrum of the fluorescence probe , calculate the total area of its absorption spectrum ; according to the normalized spectral characteristics of its emission spectrum , and the wavelength passband of the detection channel recorded in the imaging settings , calculate the total area of its emission spectrum and the proportion of the detectable band in the total area of the emission spectrum ;

[0019] 2) If the donor-receptor pair meets the following two conditions, it is considered that this pair of fluorescence probes is a possible FRET pair:

[0020] ① For both the donor and the receptor, it is necessary to meet that the proportion of the detectable band in the total area of the emission spectrum is greater than or equal to the threshold value compared with the total area of the complete emission spectrum , that is

[0021]

[0022] Threshold value Set empirically to ensure that the emitted light energy of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system.

[0023] ② For the acceptor, it is necessary to satisfy that the ratio of the area of the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of the donor emission spectrum, and the ratio of the area of the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of the acceptor absorption spectrum are both greater than or equal to the threshold value , that is

[0024] ,

[0025] ,

[0026] Threshold value Generally take 0.3.

[0027] In the present invention, the E-FRET parameter correction mainly refers to calculating the E-FRET correction coefficients a, b, c, d, G, k, and it includes the following steps:

[0028] Calculate the cross-talk coefficients a, b, c, d of the single-transfer donor and the single-transfer acceptor correction spectra, where a represents the degree of cross-talk generated by directly exciting the acceptor with the donor excitation light, b represents the degree of cross-talk of the emission light of the acceptor directly excited by the acceptor excitation light into the donor channel, c represents the degree of cross-talk generated by directly exciting the donor with the acceptor excitation light, d represents the degree of cross-talk of the emission light of the donor directly excited by the donor excitation light into the acceptor channel, and their calculation formulas are respectively:

[0029]

[0030]

[0031] And use a sample containing both the donor and the acceptor to calculate the theoretical sensitized emission value Fc:

[0032] ,

[0033] represents the gray value of the detected image, and the subscript meanings are as follows: the first letter represents the excitation light, where D represents the donor excitation light, A represents the acceptor excitation light, the second letter represents the detection channel, where D represents the detection channel of the donor, A represents the detection channel of the acceptor, and the letter in the parentheses represents the sample type, where D represents the single-transfer donor, A represents the single-transfer acceptor, DA represents the sample containing both the donor and the acceptor, for example When the sample is a single transfer receptor, the gray value of the image detected by the detector of the receptor is detected using the excitation light of the donor.

[0034] Then, the sensitization quenching conversion factor G and the concentration correction factor k are calculated. The sensitization quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after receptor photobleaching to the corresponding donor recovery amount, and the concentration correction factor k represents the ratio of the fluorescence intensities of the donor / recipient at equimolar concentrations in the absence of FRET. The calculation formulas are as follows:

[0035]

[0036]

[0037] Among them, E is the theoretical FRET efficiency value of the sample containing both the donor and the receptor.

[0038] When performing automatic imaging matching according to the optical characteristics of the fluorescence probe and the imaging system, the imaging channel is selected according to the brightness of the fluorescence probe in the detection channel of the imaging system. The selected imaging channel has a brightness greater than the set threshold of the detection channel, and preferably the detection channel with the maximum brightness is selected as the imaging channel.

[0039] In the present invention, the detection channel is composed of an excitation optical path and a detection optical path. The excitation optical path refers to the optical path experienced during the excitation process from the light source to the sample, and the detection optical path refers to the optical path experienced by the emitted light generated after the sample is excited to the detector.

[0040] The brightness of the fluorescence probe in the detection channel of the imaging system is determined by the following method:

[0041] Calculate the wavelength passband of the excitation optical path of the detection channel ;

[0042] Calculate the wavelength passband of the detection optical path of the detection channel ;

[0043] Calculate the extinction coefficient of the excitation optical path corrected according to the excitation light source band of the detection channel and the photon utilization rate of the detection optical path , which are respectively defined as:

[0044] ,

[0045] ,

[0046] Among them, is the molar extinction coefficient of the fluorescence probe; is the normalized spectral characteristic of the emission spectrum of the excitation light source; Represents the normalized spectral characteristics of the absorption spectrum of the fluorescent probe; Represents the light transmittance of the objective lens of the detection channel; Represents the quantum yield of the fluorescent probe; Represents the normalized spectral characteristics of the emission spectrum of the fluorescent probe; Represents the quantum efficiency of the detector in the detection channel for light;

[0047] Then calculate the brightness of the fluorescent probe in the detection channel , defined as:

[0048] .

[0049] Before calculating the brightness of the detection channel, it is recommended to select the excitation light source band of the detection channel in the following way:

[0050] Calculate the peak value of the absorption spectrum of the fluorescent probe and the peak value of the emission spectrum of the excitation light source of the imaging system The difference , and compare it with the set threshold and select The light emission band of the corresponding light source; preferably the smallest The light emission band of the corresponding light source.

[0051] Beneficial effects:

[0052] 1) The control system of the present invention can automatically identify a combination of fluorescent probes capable of imaging to form a donor-acceptor pair that can produce the FRET effect, and generate a corresponding E-FRET imaging process. The user only needs to select the corresponding donor-acceptor pair and provide an experimental sample to achieve automatic E-FRET imaging of the corresponding FRET pair;

[0053] 2) The present invention also provides a scheme for matching an imaging channel according to a fluorescent probe, and gives a preferred method for calculating the channel brightness. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Is the control flow chart of the control system of the present invention;

[0055] Figure 2 Is the optical path schematic diagram of a dual-channel fluorescence microscopy imaging system;

[0056] Figure 3 Is the actual captured image of a single-turn EGFP sample (after enhancing the contrast);

[0057] Figure 4It is a real - shot image of a single - transfected mCherry sample (after enhanced contrast);

[0058] Figure 5 For a sample containing both GFP and mCherry, three images are taken according to the standard E - FRET procedure. Detailed implementation mode

[0059] The present invention aims to provide a multi - channel fluorescence microscopy imaging control system for automatic E - FRET imaging based on fluorescence probes. The preferred solutions of the present invention will be described in detail below in combination with specific embodiments of the present invention.

[0060] Embodiment 1

[0061] A multi - channel fluorescence microscopy imaging control system for automatic E - FRET imaging based on fluorescence probes, which is applied to a multi - channel fluorescence microscopy imaging system, includes a system parameter entry software module, a fluorescence probe information entry software module, an imaging matching software module, a preliminary FRET imaging screening software module, a FRET effect judgment and E - FRET parameter correction software module, and an E - FRET imaging execution software module.

[0062] System parameter entry software module: Determine the relevant information of the imaging system (specifically including the communication protocols of each electric control in the imaging system, etc. for information transfer with the imaging system) and enter the optical characteristic parameters of each optical element in the imaging system, specifically including:

[0063] (1.1) Enter the wavelength range of the emission spectrum of the multi - band light source of the imaging system and the peak value , where i represents the i - th emission spectrum, and at the same time determine the normalized spectral characteristics of each emission spectrum , and at the same time determine the excitation filter corresponding to each emission light passband and stopband ;

[0064] (1.2) Enter the normalized spectral characteristics of the optical elements in the optical device group of the imaging system: Determine the passband and stopband of the emission filter, where j represents the j - th emission filter; Determine the transmission band and reflection band of the dichroic mirror, where k represents the k - th dichroic mirror. ;

[0065] (1.3)Enter the transmittance of the objective lens in the imaging system for light of different wavelengths ;

[0066] (1.4) Enter the numbers of the detection CMOSs in each channel of the imaging system , where m represents the m-th channel, and determine the quantum efficiency of each CMOS for light of different wavelengths .

[0067] In this embodiment, the wavelength range considered by the imaging system is 300 nm - 800 nm. Correct all the entered wavelength range values. If the entered relevant data exceeds this range, discard it. If there is a data missing in a certain section of this range, uniformly consider it as a stopband or the light intensity is zero.

[0068] Fluorescent probe information entry software module: Enter the optical characteristic parameters of the fluorescent probe. For example, enter the relevant information of the fluorescent probe , where n represents the n-th fluorescent probe. Specifically, it includes:

[0069] Fluorescent probe The peak value of the absorption spectrum , and the normalized spectral characteristics of the absorption spectrum ; the peak value of the emission spectrum , and the normalized spectral characteristics of the emission spectrum ; the molar extinction coefficient of the fluorescent probe ; the quantum yield of the fluorescent probe .

[0070] Imaging matching software module: Automatically perform imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and save the imaging settings of the fluorescent probe. Specifically:

[0071] (3.1) Determine the light source band used when each fluorescent probe is imaged.

[0072] For a certain entered fluorescent probe , first calculate the difference between the peak value of the absorption spectrum of the fluorescent probe and the peak values of all the emission spectra of the light source in sequence. Here, i represents the difference from the peak value of the i-th emission spectrum of the light source. At the same time, set a threshold . When , it is considered that the fluorescent probe can be excited in this system. It is recommended to select the smallest from the excitable options, and the light source emission spectrum peak corresponding to it is used as the excitation light of ; when , it is considered that the fluorescent probe cannot be excited in this system.

[0073] (3.2)For the fluorescent probes that can be excited, calculate the brightness of the fluorescent probes in each detection channel, and then select the imaging channel of the probes according to the brightness.

[0074] After the light source band is determined, for a certain determined fluorescent probe, there is an excitation optical path for the fluorescent probe in the multi-channel fluorescence microscopy imaging system and multiple detection optical paths , where m represents the m-th detection optical path (the same as the number of detectors). The optical elements of the excitation optical path usually include a light source (excitation light and excitation filter), an objective lens (not filtered), and some also include a dichroic mirror. The optical elements of each detection optical path usually include an optical device group (emission filter and dichroic mirror), an objective lens (not filtered), a mirror (not filtered), etc. The detection channel is composed of a certain determined laser optical path and a certain determined detection optical path.

[0075] In the multi-channel fluorescence microscopy imaging system, for the excitation optical path and each detection optical path, considering the optical characteristics of multiple optical elements in the detection path, it is necessary to correct the extinction coefficient and quantum yield of the fluorescent probe.

[0076] First, calculate the wavelength passband of each detection path.

[0077] For the excitation optical path , the optical elements involved in the calculation include a light source (excitation light and excitation filter), and may include a dichroic mirror (optical devices that are not filtered do not affect the wavelength passband, so they are not considered in the following calculations). For the spectral band range of the excitation light and the wavelength passband of the excitation filter and the transmission band (or reflection band) of the dichroic mirror, find the intersection (if the dichroic mirror is included) to obtain the wavelength passband of the excitation optical path , and the calculation formula is:

[0078] (without dichroic mirror)

[0079] (dichroic mirrors are all transmissive)

[0080] (dichroic mirrors are all reflective)

[0081] (dichroic mirrors have both transmission and reflection)

[0082] In the above formulas, i is determined by (3.1).

[0083] For the detection optical path , the optical elements involved in the calculation include multiple emission filters and multiple dichroic mirrors. After the detection optical path is determined (i.e., after a is determined), all the optical elements in the optical path and the filtering conditions of the elements are immediately determined (i.e., both j and k have multiple values and the values are determined). The intersection of the wavelength passbands of all the emission filters and the transmission bands (or reflection bands) of all the dichroic mirrors is obtained to get the wavelength passband of the detection optical path. , and the calculation formula is:

[0084] (all dichroic mirrors are transmissive)

[0085] (all dichroic mirrors are reflective)

[0086] (some dichroic mirrors are transmissive and some are reflective).

[0087] Determine the wavelength passband of the excitation optical path and the wavelength passbands of all the detection optical paths After that, due to the characteristics of the absorption and emission of light by fluorescent probes in different wavelength bands, the reception characteristics of the SCMOS photosensitive area, and the transmittance differences of each optical element in the system for light of different wavelengths, it is impossible to directly calculate using the original extinction coefficient and quantum yield of the fluorescent probe. It is necessary to correct them and calculate the extinction coefficient of the excitation optical path of the entire imaging system (including the cell sample transfected with the fluorescent probe) during imaging (the absorption ability for the excitation light, defined as , i corresponds to the excitation light band, and n corresponds to the fluorescent probe number) and the photon utilization rate of each detection optical path (the ratio of the number of photons absorbed by the fluorescent protein to the number of photons that the SCMOS can finally receive, defined as , m corresponds to the detection channel number, and n corresponds to the fluorescent probe number).

[0088] (i is unique and determined by (3.1))

[0089]

[0090] Fluorescent probe The brightness in each detection optical path is defined as:

[0091] (i is unique and determined by (3.1))

[0092] Determine the imaging channels in which the fluorescent probe can be imaged, automatically match the corresponding detector numbers and save all the above settings for one-key imaging.

[0093] Set the brightness threshold , and obtain the fluorescent probe The actual brightness in each detection optical path After If it is considered that the fluorescent probe cannot be imaged in this fluorescence microscopy imaging system; if there is only one m value such that it is considered that the fluorescent probe can only be imaged in one imaging channel, and the system automatically saves the number of the imaging channel and the number of the corresponding detector in the record of this fluorescent probe. After the saving is completed, one-key imaging can be performed; if there are multiple m values such that it is considered that the fluorescent probe can be imaged in multiple imaging channels. The system will take the maximum brightness value greater than the brightness threshold and use this imaging channel as the best imaging channel for the fluorescent probe

[0094] Preliminary FRET imaging screening software module: For the fluorescent probes that can be imaged, they are combined in pairs into donor-acceptor pairs and then preliminary FRET imaging screening is performed.

[0095] The method for performing preliminary FRET imaging screening is as follows:

[0096] 1) According to the normalized spectral characteristics of the absorption spectrum of the fluorescent probe calculate the total area of its absorption spectrum ; according to the normalized spectral characteristics of its emission spectrum and the wavelength passband of the detection channel recorded in the imaging settings calculate the total area of its emission spectrum and the proportion of the detectable band in the total area of the emission spectrum ;

[0097] 2) If the donor-acceptor pair meets the following two conditions, it is considered that this pair of fluorescent probes is a possible FRET pair:

[0098] ① For both the donor and the acceptor, it is necessary to meet the condition that the proportion of the detectable band in the total area of the emission spectrum is greater than or equal to the threshold to

[0099]

[0100] The threshold is set according to experience and is used to ensure that the emitted light energy of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system.

[0101] ②For the acceptor, it is necessary to satisfy that the ratio of the area of the overlapping part between its absorption spectrum and the emission spectrum of the donor to the total area of the donor emission spectrum, and the ratio of the area of the overlapping part between its absorption spectrum and the emission spectrum of the donor to the total area of the acceptor absorption spectrum are both greater than or equal to the threshold value , that is

[0102]

[0103]

[0104] According to the regulations in the literature, the threshold value is usually set to 0.3.

[0105] FRET effect judgment and E-FRET parameter correction software module: For the initially screened donor-acceptor pairs, generate standard E-FRET imaging settings, and then control the imaging system to perform imaging experiments on the standard samples provided by the user according to the standard E-FRET imaging settings, and perform FRET effect judgment (judgment according to the specific imaging situation) and E-FRET parameter correction based on this. For the donor-acceptor pairs that are determined to be able to have FRET effect, automatically generate the corresponding E-FRET shooting process (including light source timing control, camera timing control, and imaging channel selection); the standard samples include single transfected donor samples, single transfected acceptor samples, and samples containing both the donor and the acceptor.

[0106] For the potentially screened FRET pairs, the user needs to provide single transfected samples of the two fluorescent probes and samples containing both the donor and the acceptor for imaging experiments, obtain the actual shooting images of several different experimental samples in the imaging system according to the generated standard E-FRET imaging settings, and then perform FRET effect judgment and E-FRET parameter correction based on these images. It should be noted that the samples containing both the donor and the acceptor need to have a determined theoretical FRET efficiency value E, which is provided by the user to the imaging system.

[0107] The standard E-FRET imaging setup is generated as follows: The FRET effect judgment and E-FRET parameter correction software module preset the E-FRET imaging template. The imaging matching software module in the above text has determined the detection channels (including the detection channel numbers and the detector numbers) when the donor and the receptor are imaged separately, as well as the optimal excitation wavelength band (for the light source in the imaging system) when they are imaged separately, and saved the above two pieces of information. Here, the FRET effect judgment and E-FRET parameter correction software module fills in the optimal excitation wavelength band of the donor fluorescent probe and the information of the donor and receptor detection channels into the E-FRET imaging template, and fills in the optimal excitation wavelength band of the receptor fluorescent probe and the information of the receptor detection channel into the E-FRET imaging template. After saving, when the subsequent user selects this donor-receptor pair, the system will automatically perform one-key E-FRET imaging on all samples of this donor-receptor pair (including single-transfected donor samples, single-transfected receptor samples, and samples containing both the donor and the receptor), complete the E-FRET experiment and perform FRET effect judgment and E-FRET parameter correction.

[0108] The specific requirements for standard E-FRET imaging are to image the sample with donor excitation light in two channels (donor and receptor detection channels), and image the sample with receptor excitation light in the receptor's detection channel.

[0109] Regarding E-FRET parameter correction, the E-FRET method first needs to use single-transfected donor and single-transfected receptor to correct the spectral crosstalk coefficients a, b, c, d; where a represents the degree of crosstalk generated by directly exciting the receptor with donor excitation light, b represents the degree of crosstalk of the emission light of the receptor excited directly by receptor excitation light to the donor channel, c represents the degree of crosstalk generated by directly exciting the donor with receptor excitation light, and d represents the degree of crosstalk of the emission light of the donor excited directly by donor excitation light to the receptor channel. The calculation formula is:

[0110]

[0111]

[0112] represents the gray value of the image detected in the receptor's detection channel when the sample is a single-transfected receptor and is excited with donor excitation light. The first letter in the subscript DA(A) represents the excitation light (D represents donor excitation light, A represents receptor excitation light), the second letter represents the detection channel (D represents the donor's detection channel, A represents the receptor's detection channel), and the letter in the parentheses represents the sample type (D represents single-transfected donor, A represents single-transfected receptor, DA represents the sample contains both the donor and the receptor). The same applies to the rest.

[0113] After obtaining the spectral crosstalk coefficients of E-FRET, use a sample containing both the donor and the acceptor to calculate the theoretical sensitized emission value Fc:

[0114] 。

[0115] The theoretical FRET efficiency of the sample containing both the donor and the acceptor is E. After obtaining the values of the spectral crosstalk coefficients a, b, c, d and the sensitized emission Fc, calculate the sensitized quenching conversion factor G and the concentration correction factor k. The sensitized quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after acceptor photobleaching to the corresponding donor recovery amount, and the concentration correction factor k represents the ratio of the fluorescence intensities of equimolar concentrations of donor / acceptor in the absence of FRET. The calculation formulas are as follows:

[0116]

[0117]

[0118] E-FRET imaging execution software module: When the user selects a saved donor-acceptor pair and provides an experimental sample for the experiment, control the imaging system to execute the E-FRET shooting process corresponding to the donor-acceptor pair to image the experimental sample, and calculate the E-FRET result using the result of E-FRET parameter correction.

[0119] After calculating all the E-FRET correction parameters, save the E-FRET parameter configuration according to the name of the current FRET pair, and automatically generate the corresponding E-FRET shooting process. After the subsequent user selects the FRET pair according to the name, one-key E-FRET imaging can be performed according to these 6 parameters and the previously generated E-FRET imaging settings, etc., and the E-TRET result can be calculated. The control flow chart of the control system is as Figure 1 shown.

[0120] The following takes the Figure 2 dual-channel fluorescence microscopy system in as an example to specifically introduce the implementation process of the control system in the above-mentioned Embodiment 1.

[0121] Figure 2 Introduction to each optical element of the dual-channel fluorescence microscopy system in:

[0122] Light Source is a fully electronically controllable six-band LED light source that can emit six kinds of excitation lights with wavelength peaks of 385nm, 435nm, 488nm, 515nm, 560nm, and 630nm;

[0123] EX is an excitation filter:

[0124] For the excitation light with a peak wavelength of 385 nm, no excitation filter is set;

[0125] For the excitation light with a peak wavelength of 435 nm, an excitation filter is set;

[0126] For the excitation light with a peak wavelength of 488 nm, an excitation filter is set;

[0127] For the excitation light with a peak wavelength of 515 nm, an excitation filter is set;

[0128] For the excitation light with a peak wavelength of 560 nm, an excitation filter is set;

[0129] For the excitation light with a peak wavelength of 630 nm, an excitation filter is set;

[0130] Objective is the objective lens, an Olympus 60x oil immersion lens. In this example, the observed samples are cells transfected with four fluorescent probes, EGFP, mcherry, EYFP, and ECFP respectively;

[0131] D1 is a dichroic mirror for beam splitting;

[0132] EM1 is an emission filter;

[0133] M1 is a mirror for reflecting the light beam, ignoring losses;

[0134] D2 is a dichroic mirror for beam splitting;

[0135] EM2 is an emission filter;

[0136] EM3 is an emission filter;

[0137] Both SCMOS1 and SCMOS2 are Hamamatsu cameras.

[0138] After connecting all the hardware facilities to build the system, operate according to the procedure in Implementation 1:

[0139] 1) Determine the relevant information of the imaging system and input the optical characteristic parameters of each optical element in the imaging system (only considering the wavelength range of 300 nm - 800 nm).

[0140] According to the optical information files of the light source, filter, and dichroic mirror, input the normalized spectral characteristics of the six bands of the multi-band LED light source, the passband and stopband of the excitation filter corresponding to each band, and the normalized spectral characteristics of the optical elements in the optical device group. For the objective lens, with the model of Olympus 60x oil immersion lens, input the transmittance of this type of objective lens for light of different wavelengths.

[0141] For the two SCMOS, input their quantum yields for light of different wavelengths respectively.

[0142] Modify the wavelength bands related to the optical properties of all the above-entered optical elements, and fix the wavelength band range between 300 - 800 nm. Taking the excitation filter as an example, when entering, only the passband of 427 nm - 445 nm was entered, and the system will automatically supplement the stopbands of this excitation filter as 300 nm - 426 nm and 446 nm - 800 nm. The same applies to the other optical elements.

[0143] 2) Enter the optical property parameters of the fluorescent probe.

[0144] There are 4 fluorescent probes used in this imaging system, namely mCherry, EGFP, EYFP, and ECFP.

[0145] For mCherry, enter the peak value of its absorption spectrum as 587 nm, and enter the spectral characteristics of the absorption spectrum; enter the peak value of its emission spectrum as 610 nm, and enter the spectral characteristics of the emission spectrum; enter the molar extinction coefficient as 72000; enter the quantum yield as 0.22.

[0146] For EGFP, enter the peak value of its absorption spectrum as 488 nm, and enter the spectral characteristics of the absorption spectrum; enter the peak value of its emission spectrum as 507 nm, and enter the spectral characteristics of the emission spectrum; enter the molar extinction coefficient as 56000; enter the quantum yield as 0.60.

[0147] For EYFP, enter the peak value of its absorption spectrum as 513 nm, and enter the spectral characteristics of the absorption spectrum; enter the peak value of its emission spectrum as 527 nm, and enter the spectral characteristics of the emission spectrum; enter the molar extinction coefficient as 83400; enter the quantum yield as 0.61.

[0148] For ECFP, enter the peak value of its absorption spectrum as 434 nm, and enter the spectral characteristics of the absorption spectrum; enter the peak value of its emission spectrum as 477 nm, and enter the spectral characteristics of the emission spectrum; enter the molar extinction coefficient as 32500; enter the quantum yield as 0.4.

[0149] 3) Automatically perform imaging matching according to the optical properties of the fluorescent probe and the imaging system, and save the imaging settings of the fluorescent probe.

[0150] Judge the light source wavelength band used when each fluorescent probe is imaged.

[0151] Set the difference threshold to 30.

[0152] For mCherry, the peak value of its absorption spectrum is 587 nm, and it has the smallest difference of 27 from the peak value of the excitation light in the 560 nm wavelength band of the light source, and this is less than the threshold of 30. The system determines that mCherry can be excited within the system, and the peak value of the excitation light wavelength band is 560 nm.

[0153] For EGFP, the peak of its absorption spectrum is 488 nm, with the smallest difference of 0 from the peak of the excitation light in the 488 nm band of the light source, and it is less than the threshold of 30. The system determines that EGFP can be excited within the system, and the peak of the excitation light band is 488 nm.

[0154] For EYFP, the peak of its absorption spectrum is 513 nm, with the smallest difference of 2 from the peak of the excitation light in the 515 nm band of the light source, and it is less than the threshold of 30. The system determines that EYFP can be excited within the system, and the peak of the excitation light band is 560 nm.

[0155] For ECFP, the peak of its absorption spectrum is 434 nm, with the smallest difference of 1 from the peak of the excitation light in the 435 nm band of the light source, and it is less than the threshold of 30. The system determines that ECFP can be excited within the system, and the peak of the excitation light band is 560 nm.

[0156] For the fluorescent probes that can be excited, calculate the brightness of the fluorescent probes in each detection channel.

[0157] As can be seen from (3.1), all four fluorescent probes can be excited. Taking mCherry as an example for analysis.

[0158] For mCherry, the optical elements of the excitation optical path are the light source (using excitation light with a peak of 560 nm), the excitation filter, the first dichroic mirror D1, and the objective lens. Take the intersection of the spectral band range, the reflection band of the dichroic mirror, and the pass band of the excitation filter to obtain the pass band of the optical path as 548 nm - 571 nm. After obtaining the pass band of the optical path, calculate the extinction coefficient of the imaging system as 928077.

[0159] There are two detection optical paths.

[0160] The optical elements of the first detection optical path are the objective lens, the dichroic mirror D1, the emission filter EM1, the dichroic mirror D2, the emission filter EM2, and SCMOS1. Obtain the pass band of the first detection optical path as 445 nm - 448 nm, 604 nm - 617 nm. After obtaining the pass band of the optical path, calculate the photon utilization rate of the first detection optical path of the system as 0.047.

[0161] The optical elements of the second detection optical path are the objective lens, the dichroic mirror D1, the emission filter EM1, the dichroic mirror D2, the emission filter EM3, and SCMOS2. Obtain the pass band of the second detection optical path as 503 nm - 522 nm, 674 nm - 749 nm. After obtaining the pass band of the optical path, calculate the photon utilization rate of the second detection optical path of the system as 0.022.

[0162] After obtaining the photon utilization rates of different detection channels and the extinction coefficient of the system, in the first detection channel, that is, the brightness value detected by SCMOS1 is 43617, and in the second detection channel, that is, the brightness value detected by SCMOS2 is 20418. Set the brightness threshold to 20000. The brightness values of both channels are greater than the threshold, that is, mCherry can be imaged in both channels, and SCMOS1 can achieve better imaging effects.

[0163] After the brightness calculation is completed, the system will automatically configure the one-key automatic imaging process for mCherry. When the user selects mCherry as the fluorescent probe and issues an imaging command, the system will automatically set the light source to emit excitation light with a peak of 560 nm, start SCMOS1, and collect the fluorescent signal emitted by mCherry on SCMOS1 according to the exposure time and light intensity configured by the user and save it locally for the user to view.

[0164] For the four fluorescent probes, the information on the evaluation of the imaging quality of the fluorescent probes analyzed by the system is shown in Table 1.

[0165] Table 1

[0166] Fluorescent probe Peak excitation light Brightness of Channel 1 Brightness of Channel 2 Detector selection Ability to image EGFP 488nm 3394 159421 SCMOS2 Yes mcherry 560nm 43617 20418 SCMOS1 Yes ECFP 435nm 0 0 - No EYFP 515nm 0 0 - No

[0167] 4) For the fluorescent probes that can be imaged, pair them up into donor-acceptor pairs and then conduct preliminary FRET imaging screening.

[0168] (1) It can be determined from 3) that the fluorescent probes that can be imaged in the system are EGFP and mcherry.

[0169] For EGFP, the total area of its emission spectrum is calculated to be 65.04, and the total area of its absorption spectrum is 43.67. It can be determined from 3) that the wavelength passbands of its best detection channels are 503 nm - 522 nm and 674 - 749 nm, and the band that can be detected accounts for 17.59 of the total area of the emission spectrum.

[0170] For mCherry, the total area of its emission spectrum is calculated to be 64.18, and the total area of its absorption spectrum is 65.35. It can be determined from 3) that the wavelength passbands of its best detection channels are 445 nm - 448 nm and 604 nm - 617 nm, and the band that can be detected accounts for 13.61 of the total area of the emission spectrum.

[0171] Match EGFP with mCherry and check whether the following two conditions are met.

[0172] ① Set the threshold to 0.2. For EGFP, the ratio of the band that can be detected in the best detection channel to the total area of the emission spectrum is 0.27, which is greater than 0.2, meeting the condition;

[0173] For mCherry, the ratio of the area of the emission spectrum that can be detected in the best detection channel to the total area of the emission spectrum is 0.21, which is greater than 0.2, meeting the condition.

[0174] ② Set the threshold to 0.3. Let EGFP be the donor and mCherry be the acceptor. The calculated overlapping area between the absorption spectrum of mCherry and the emission spectrum of EGFP is 22.30. The total area of the emission spectrum of EGFP is 65.04, and the total area of the absorption spectrum of mCherry is 65.35. The ratio of the overlapping area between the absorption spectrum of mCherry and the emission spectrum of EGFP to the total area of the emission spectrum of EGFP is 0.34, which is greater than 0.3, meeting the condition; the ratio of the overlapping area between the absorption spectrum of mCherry and the emission spectrum of EGFP to the total area of the absorption spectrum of mCherry is 0.34, which is greater than 0.3, meeting the condition.

[0175] After judgment, it is determined that EGFP and mCherry are a pair of possible FRET pairs, with EGFP as the donor and mCherry as the acceptor. The system saves them in the selection list and saves the relevant imaging settings of the two fluorescent probes (including single imaging settings and E-FRET imaging settings) for the user to select and perform fluorescence imaging when correcting the E-FRET parameters.

[0176] 5) For the donor-acceptor pairs preliminarily screened out, generate standard E-FRET imaging settings, and then control the imaging system to perform imaging experiments on the standard samples provided by the user according to the standard E-FRET imaging settings, and use this to judge the FRET effect and correct the E-FRET parameters. For the donor-acceptor pairs that are determined to be able to have the FRET effect, automatically generate the corresponding E-FRET shooting process; the standard samples include single-transfected donor samples, single-transfected acceptor samples, and samples containing both the donor and the acceptor.

[0177] For the possible FRET pairs screened out in 4), the user needs to provide single-transfected samples of the corresponding fluorescent probes and samples containing both the donor and the acceptor to correct the E-FRET parameters.

[0178] For the EGFP-mCherry pair, with EGFP as the donor and mCherry as the acceptor, the user provides single-transfected EGFP samples, single-transfected mCherry samples, and samples containing both EGFP and mCherry.

[0179] For the single-transfected EGFP sample, the actual captured image (after enhancing the contrast) is as Figure 3 shown. Figure 3The three images from left to right are the images collected in Channel 2 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the acceptor excitation light (561 nm).

[0180] For the single-transfected mCherry sample, the actual captured images (after enhanced contrast) are as Figure 4 shown Figure 4 The three images from left to right are the images collected in Channel 2 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the acceptor excitation light (561 nm).

[0181] For the sample containing both GFP and mCherry, three images are captured according to the E-FRET standard procedure as Figure 5 shown Figure 5 The three images from left to right are the images collected in Channel 2 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the donor excitation light (488 nm); the images collected in Channel 1 excited by the acceptor excitation light (561 nm).

[0182] Using the above nine images, the following are calculated according to the formula:

[0183] d = 0.433955, c = 0.08367, b = 0.002054, a = 0.415537; G = 1.640552, k = 2.984713.

[0184] 6) When the user selects a saved donor-acceptor pair and provides an experimental sample for the experiment, control the imaging system to execute the E-FRET imaging procedure corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and calculate the E-FRET result using the result of the E-FRET parameter correction.

[0185] After the calculation is completed, the configuration of the above FRET pair, the E-FRET correction parameters, and the E-FRET imaging settings, etc. are saved. After the saving is completed, the user can directly place the sample corresponding to the FRET pair for a one-key fast E-FRET experiment.

[0186] On the one hand, the control system of the present invention can automatically match the imaging channels according to the fluorescence probe information input by the user. In this regard, the present invention not only proposes a method for matching imaging channels according to fluorescence probes, but also provides a preferred method for calculating the channel brightness. It should be noted that those skilled in the art can also use the method for matching imaging channels with fluorescence probes proposed by the present invention to obtain the matching information between the fluorescence probes and the imaging channels, and then directly use this matching information in a multi-channel fluorescence microscopy imaging system to achieve the control of automatic imaging according to fluorescence probes.

[0187] The imaging system, which is the loading object of the control system of the present invention, needs to be equipped with a fully electronically controllable multi-band excitation light source, a detector, etc., so as to ensure that the fluorescence probe can be imaged with one key. For users, they only need to further give relevant parameters for imaging, such as excitation light intensity, exposure time, and photographing instructions, and the imaging system can then allocate the imaging channel with the highest imaging quality to output the imaging result. There is no need for users to judge by themselves the detectors and excitation light bands required for the imaging channel, etc., which reduces the threshold of fluorescence imaging technology.

[0188] On the other hand, the control system of the present invention provides a fast and stable E-FRET solution, which can automatically perform FRET pair matching according to the fluorescence probes in the system and provide users with a complete E-FRET method (the whole process from parameter calibration to E-FRET shooting). Users only need to provide the standard sample corresponding to the FRET pair to complete the one-key E-FRET experiment in this system.

Claims

1. A multi-channel fluorescence microscopy imaging control system based on automatic E-FRET imaging of fluorescent probes, which is applied to a multi-channel fluorescence microscopy imaging system, characterized in that: include: System parameter input software module: determining the relevant information required for establishing connection with each electric control in the imaging system and inputting the optical characteristic parameters of each optical element in the imaging system; Fluorescent probe information input software module: input the optical characteristic parameters of the fluorescent probe; Imaging matching software module: performing automatic imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and saving the imaging settings of the fluorescent probe; Preliminary FRET imaging screening software module: for the fluorescent probes capable of imaging, two of them are combined into donor-acceptor pairs and then preliminary FRET imaging screening is performed; FRET effect judgment and E-FRET parameter correction software module: for the donor-acceptor pairs preliminarily screened, a standard E-FRET imaging setting is generated, and then the imaging system is controlled according to the standard E-FRET imaging setting to perform imaging experiments on the standard samples provided by the user, and the FRET effect judgment and E-FRET parameter correction are performed accordingly, and for the donor-acceptor pairs determined to be capable of FRET effect, the corresponding E-FRET shooting process is automatically generated and saved; the standard samples include single-transfer donor samples, single-transfer acceptor samples, and samples containing both donors and acceptors; E-FRET imaging execution software module: when the user selects a saved donor-acceptor pair and provides an experimental sample for an experiment, the imaging system is controlled to execute the E-FRET shooting process corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and the E-FRET result is calculated using the result of the E-FRET parameter calibration; Perform a preliminary FRET imaging screen as follows: 1) According to the normalized spectral characteristic AB of the fluorescent probe absorption spectrum n (λ n,ab ), calculate the total area of ​​its absorption spectrum According to the normalized spectral characteristics EM of its emission spectrum n (λ n,em ), and the wavelength passband λ of the detection channel recorded in the imaging setup m,BP,DC , calculate the total area of ​​its emission spectrum and the detectable wavelengths occupy the total area of ​​the emission spectrum 2) If the donor-acceptor pair meets the following two conditions, the pair of fluorescent probes is considered to be a possible FRET pair: ① For both the donor and the acceptor, the ratio of the total area of ​​the detectable band to the total area of ​​the complete emission spectrum must be greater than or equal to the threshold TH3, that is, The threshold TH3 is set based on experience to ensure that the emission light of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system; ② For the acceptor, the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the donor emission spectrum, as well as the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the acceptor absorption spectrum, must be greater than or equal to the threshold TH4, that is, The threshold TH4 is set to 0.

3.

2. The control system according to claim 1, characterized in that: E-FRET parameter calibration mainly refers to calculating the E-FRET correction coefficients a, b, c, d, G, k, which includes the following steps: Calculate the corrected spectral crosstalk coefficients a, b, c, and d of the single-turn donor and single-turn acceptor, where a represents the degree of crosstalk caused by the direct excitation of the acceptor by the donor excitation light, b represents the degree of crosstalk of the emission light of the acceptor directly excites the acceptor by the acceptor excitation light, c represents the degree of crosstalk caused by the direct excitation of the donor by the acceptor excitation light, and d represents the degree of crosstalk of the emission light of the donor directly excites the donor by the donor excitation light. Their calculation formulas are: The theoretical sensitized emission value Fc is calculated using a sample containing both donor and acceptor: Fc=I DA(DA) -a(I AA(DA) -cI DD(DA) )-d(I DD(DA) -bI AA(DA) ) I represents the gray value of the detected image, and its subscripts have the following meanings: the first letter represents the excitation light, where D represents the donor excitation light and A represents the acceptor excitation light; the second letter represents the detection channel, where D represents the donor detection channel and A represents the acceptor detection channel; the letters in brackets represent the sample type, where D represents a single-transfer donor and A represents a single-transfer acceptor; and DA represents that the sample contains both donors and acceptors; Then the sensitization-quenching conversion factor G and the concentration correction factor k are calculated. The sensitization-quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after acceptor photobleaching to the corresponding donor recovery amount. The concentration correction factor k represents the ratio of the donor / acceptor fluorescence intensities of equimolar concentrations in the absence of FRET. The calculation formula is: Where E is the theoretical FRET efficiency of a sample containing both donor and acceptor.

3. The control system according to claim 1, characterized in that: When automatic imaging matching is performed according to the optical characteristics of the fluorescent probe and the imaging system, the imaging channel is selected according to the brightness of the fluorescent probe in the detection channel of the imaging system, and the imaging channel selects the detection channel whose brightness is greater than the set threshold TH2.

4. The control system according to claim 3, characterized in that: Select the detection channel with the highest brightness as the imaging channel.

5. The control system according to claim 3 or 4, characterized in that: The brightness of the fluorescent probe in the detection channel of the imaging system is determined by: Calculate the wavelength passband λ of the excitation light path of the detection channel BP,EC ; Calculate the wavelength passband λ of the detection optical path of the detection channel m,BP,DC ; Calculate the extinction coefficient εS of the excitation light path corrected according to the excitation light source band of the detection channel i,n and the photon utilization rate Q of the detection optical path m,n , which are defined as: Among them, ε n is the molar extinction coefficient of the fluorescent probe; S i (λ ex ) is the normalized spectral characteristic of the emission spectrum of the excitation light source; AB n (λ n,ab ) represents the normalized spectral characteristics of the fluorescent probe absorption spectrum; T(λ ob ) represents the transmittance of the objective lens of the detection channel to light; QY n represents the quantum yield of the fluorescent probe; EM n (λ n,em ) represents the normalized spectral characteristics of the fluorescent probe emission spectrum; QE m (λ C ) represents the quantum efficiency of the detector of the detection channel for light; Then calculate the brightness B of the fluorescent probe in the detection channel m,n , defined as: B m,n =εS i,n Q m,n 。 6. The control system according to claim 5, characterized in that: Before calculating the brightness of the detection channel, the excitation light source band of the detection channel is selected in the following manner: Calculate the absorption spectrum peak λ of the fluorescent probe a,ab The peak λ of the emission spectrum of the excitation light source of the imaging system i The difference Dif i and compare it with the set threshold TH1, select Dif i ≤TH1 corresponds to the light emission band of the light source.

7. The control system according to claim 6, characterized in that: Select the smallest Dif i The corresponding light source emits light band.

Images