Fast multicolor imaging method based on dual-channel brightness encoding of fluorescent probes

Abstract

This invention discloses a rapid multicolor imaging method based on dual-channel brightness encoding of fluorescent probes. The method is based on a dual-channel multicolor fluorescence microscopy imaging system and includes the following steps: S1, determining the characteristics of the dual-channel multicolor fluorescence microscopy imaging system and the spectral characteristics of each optical element; S2, analyzing the system components, dividing the system into excitation and emission optical paths, and encoding the components in each optical path; S3, encoding the actual brightness of different fluorescent probes in the system at each excitation wavelength; S4, screening fluorescent probes supported by the system; S5, determining whether the fluorescent probes satisfy simultaneous two-color imaging and time-division imaging; and S6, matching fluorescent probe combinations based on the brightness encoding to achieve multicolor imaging. This invention theoretically calculates whether the brightness of fluorescent probes at each excitation wavelength can be imaged and encoded in the dual channels, evaluates the feasibility of simultaneous detection in dual channels with single-wavelength excitation, and rapidly matches fluorescent probe combinations to achieve fluorescence multicolor imaging.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence microscopy imaging technology, specifically relating to a rapid multicolor imaging method based on dual-channel brightness encoding of a fluorescence probe. Background Technology

[0002] Multicolor fluorescence microscopy refers to an optical microscopy technique that uses multiple fluorescent probes to label samples of interest and then images the labeled samples. Traditional multicolor fluorescence microscopy systems typically use appropriate single-bandpass excitation and emission filters to excite and image different fluorescent probes separately. Because switching filter groups is required for different fluorescent probes, this time-sharing imaging method has a long switching time, low temporal resolution, and the unavoidable mechanical vibrations during switching can affect optical path registration and system stability. Therefore, a dual-channel fluorescence microscopy system using multi-bandpass filters has been proposed. Under certain conditions, this system can excite different probes with a single wavelength and simultaneously image different labels in two channels, i.e., simultaneous imaging.

[0003] Although single-wavelength excitation and multi-passband filter sets are employed, current dual-channel fluorescence microscopy filter sets are still selected based on a few specified probes, such as filter sets chosen for probe groups CFP / YFP / Hoechst / MitotrackerDeepRed. Generally, this fixed filter set is only used for multicolor microscopy imaging of the specified probes. In reality, due to the rapid development of the fluorescence probe field in recent years, the number of available probes is now enormous, and theoretically, this filter set could also be used for multicolor imaging of other probe combinations. However, there is currently a lack of effective methods to quickly screen out suitable probe combinations for multicolor imaging of this system from the vast number of probes, achieve multicolor imaging, and evaluate its imaging quality. Summary of the Invention

[0004] The main objective of this invention is to overcome the shortcomings and deficiencies of the prior art and propose a rapid multicolor imaging method based on dual-channel brightness encoding of fluorescent probes. For dual-channel fluorescence microscopy imaging systems, the method encodes whether the brightness values ​​of the fluorescent probes in the two channels can be imaged under different excitation light sources. Then, based on the encoding, different combinations of fluorescent probes are matched to achieve rapid multicolor fluorescence microscopy imaging.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A rapid multicolor imaging method based on dual-channel brightness encoding of fluorescent probes, the method is based on a dual-channel multicolor fluorescence microscopy imaging system, and includes the following steps:

[0007] S1. Determine the characteristics of the dual-channel multicolor fluorescence microscopy system and the spectral characteristics of each optical element;

[0008] S2. Analyze the system components, divide the system into excitation optical path and emission optical path respectively, and encode the components in the excitation optical path and emission optical path;

[0009] S3. Encode the actual brightness of different fluorescent probes at each excitation wavelength in the dual-channel multicolor fluorescence microscopy system.

[0010] S4. Screen fluorescent probes that support dual-channel multicolor fluorescence microscopy imaging systems.

[0011] S5. Determine whether the fluorescent probe satisfies simultaneous two-color imaging and time-division imaging.

[0012] S6. Based on the brightness encoding, match the fluorescent probe combination to achieve multicolor imaging.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] 1. The method of the present invention achieves rapid multicolor imaging by encoding the brightness of fluorescent probes. In the process of matching brightness encoding, the probe group with higher time resolution that can be excited simultaneously is matched first, and then probes that meet the conditions for time-division excitation are matched as needed. The entire matching process only requires relevant logical operations on the brightness encoding, which is more efficient and faster than conventional multicolor imaging through probe spectral analysis.

[0015] 2. The method of the present invention encodes the excitation light source, optical path devices, brightness and actual brightness value, and includes the imaging information and device information of the entire multicolor imaging process in the encoding; through encoding, information such as the excitation light source, optical path devices, probe and actual brightness value of the probe required for a multicolor imaging can be directly extracted. Attached Figure Description

[0016] Figure 1 This is a flowchart of the method of the present invention;

[0017] Figure 2 This is a schematic diagram of the multicolor imaging system in the embodiment;

[0018] Figure 3 The images shown are fluorescence images of Hoechst-stained cell nuclei in the reflective and transmissive channels under 385nm excitation light in the examples.

[0019] Figure 4 These are fluorescence images of ECFP-transfected cells in the reflected and transmitted channels under 385nm excitation light, as shown in the examples.

[0020] Figure 5 These are fluorescence images of ECFP-transfected cells in the reflected and transmitted channels under 435nm excitation light, as shown in the examples.

[0021] Figure 6 These are fluorescence images of cells transfected with EYFP under 435nm excitation light in the reflective and transmissive channels, respectively, in the example.

[0022] Figure 7 These are fluorescence images of cells transfected with EYFP under 515nm excitation light in the reflective and transmissive channels, respectively, in the example.

[0023] Explanation of reference numerals: 301-Six-wavelength light source FRET-LED fluorescent light source; 302-Excitation filter; 303-Dialect mirror D1; 304-40x oil immersion lens; 305-Experimental sample; 306-Dialect mirror D2; 307-Emission filter for reflection channel; 308-Emission filter for transmission channel; 309-Camera CAM_T; 310-Camera CAM_R. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0025] like Figure 1 As shown, this invention provides a rapid multicolor imaging method based on dual-channel brightness encoding of fluorescent probes. The method is based on a dual-channel multicolor fluorescence microscopy imaging system and includes the following steps:

[0026] S1. Determine the characteristics of the dual-channel multicolor fluorescence microscopy system and the spectral characteristics of each optical element;

[0027] The specific characteristics of the dual-channel multicolor fluorescence microscopy imaging system are as follows:

[0028] It is ensured that the spectral characteristics of the excitation light on the actual irradiated sample remain unchanged, that is, the spectral characteristics of the excitation wavelength of the excitation light path remain unchanged; wherein, the optical components of the excitation light path include, but are not limited to, the light source, the excitation filter, and the excitation dichroic mirror, etc.

[0029] It is ensured that the spectral characteristics of the emitted light actually detected by the detector remain unchanged, that is, the spectral characteristics of the fluorescence emission wavelength of the emission light path remain unchanged; wherein, the optical components of the emission light path include, but are not limited to, emission filters, emission dichroic mirrors, and detectors.

[0030] The specific spectral characteristics of each optical element are determined as follows:

[0031] The spectral characteristics of each optical element are determined based on the actual system settings, including:

[0032] Light source L kNormalized spectral characteristics: L k (λ ex ), where k represents the existence of k excitation sources;

[0033] Excitation filter F ex Normalized spectral characteristics: F ex (λ ex );

[0034] Normalized transmission / reflection spectral characteristics of excited dichroic mirror D1: D 1,T (λ ex D 1,R (λ ex );

[0035] Emission filter F rm Normalized transmission spectral characteristics: F em (λ em );

[0036] Normalized transmission / reflection spectral characteristics of emission dichroism D2: D 2,T (λ em D 2,R (λ em );

[0037] Detection transmission channel emT emission filter F emT Normalized transmission spectral characteristics: F emT (λ em );

[0038] Detection and reflection channel emR emission filter F emR Normalized transmission spectral characteristics: F emR (λ emR );

[0039] Normalized spectral characteristics of other optical elements in the excitation optical path:

[0040] Normalized spectral characteristics of other optical elements located in the transmission and reflection channels in the emission optical path: EM T (λ em ), EM R (λ em ).

[0041] S2. Analyze the system components, dividing the system into excitation optical path and emission optical path, and encode the components in the excitation optical path and emission optical path; Step S2 specifically includes:

[0042] The optical components in the excitation and emission optical paths are mainly determined as follows:

[0043] The determination of the number of device assembly bits M in the specific optical path and the number of selectable devices N for a single assembly bit;

[0044] The specific steps for encoding the devices in the excitation and emission optical paths are as follows:

[0045] The encoding is divided into two fields: excitation optical path and emission optical path; the encoding of the excitation optical path is determined by the device assembly bit M of the excitation optical path. ex and N ex It is confirmed that the number of selectable devices at the same assembly location generally does not exceed 10. Using 4 bits to encode one device bit, 16 different selectable devices can be represented. Therefore, the encoding length of the entire excitation optical path is 4M. ex bit; the encoding of the transmitting optical path is similar to that of the excitation optical path, consisting of M bits of the device assembly in the excitation optical path. em and N em Sure;

[0046] The entire optical path device encoding is complete, with a total length of 4 (M). ex +M em ) bits.

[0047] S3. Encode the actual brightness of different fluorescent probes in the dual-channel multicolor fluorescence microscopy system; Step S3 specifically involves:

[0048] Determine the fluorescent probe P i Normalized absorption spectral characteristics A i,N (λ ex ) and emission spectral characteristics F N,i (λ em ).

[0049] Fluorescent probe P i The brightness is defined as:

[0050] B i =ε i Q i

[0051] Where, ε i Molar extinction coefficient, in units of M -1 cm -1 Q represents the light absorption characteristics of a fluorescent probe with a thickness of 1 cm and a concentration of 1 mol / L; i Quantum yield is the ratio of emitted photons to absorbed photons.

[0052] In a dual-channel multicolor fluorescence microscopy system, considering the transmittance of the excitation pathway, for the fluorescent probe P... i In the light source L k During irradiation, the actual molar extinction coefficient ε ex,, for:

[0053]

[0054] In a dual-channel multicolor fluorescence microscopy system, considering the transmittance of the emission pathway, for the fluorescent probe P... i Detecting the actual quantum yield Q of the transmission channel i,T And the actual quantum yield Q of the detection reflection channel i,R They are respectively:

[0055]

[0056]

[0057] Among them, S i,T and S i,R S represents the normalized emission spectral area of ​​the transmission channel and the reflection channel, respectively. i For fluorescent probe P i The normalized total area of ​​the emitted light spectrum is expressed as follows:

[0058]

[0059]

[0060]

[0061] For fluorescent probe P i When using light source L k During excitation, the actual brightness of the probe transmission channel and the reflection channel are respectively:

[0062] B i,k,T =ε i,k,ex (λ ex )Q i,T

[0063] B i,k,R =ε i,k,ex (λ ex )Q i,R

[0064] A brightness threshold TH is set. When the brightness is greater than TH, fluorescence is considered detectable; when the brightness is less than TH, fluorescence is considered undetectable.

[0065] According to probe P i The brightness of each channel is obtained under excitation light irradiation and encoded. The encoding consists of an excitation source field, an excitation optical path and an emission optical path device field, a brightness encoding field, and an actual brightness value field; among which, the excitation source field L... kOne byte of encoding can represent 256 excitation wavelengths; the brightness field BC consists of a transmission channel marker bit T and a reflection channel marker bit R, each of which is encoded with one bit; the actual brightness field is encoded by the binary values ​​of the actual brightness values ​​of the transmission channel and the reflection channel, with 20 bits reserved for each, and can represent up to 1 million brightness values.

[0066] The luminance encoding field BC is ultimately divided into four types, as shown in Table 1 below.

[0067]

[0068]

[0069] Table 1

[0070] S4. Screen fluorescent probes that support dual-channel multicolor fluorescence microscopy imaging systems; specifically:

[0071] When an arbitrary light source cannot be used to image the probe in a dual-channel multicolor fluorescence microscopy system, it means that the system does not support the probe. Probes with a brightness encoding field BC of 00 cannot be imaged in the system. Probes with brightness encoding fields BC of 01, 10 and 01 are selected and retained.

[0072] S5. Determine whether the fluorescent probe satisfies simultaneous two-color imaging and time-division imaging; in step S5, determining whether the fluorescent probe satisfies simultaneous two-color imaging specifically involves:

[0073] For any two P from n fluorescent probes i and P j To determine whether simultaneous imaging is possible and to obtain the brightness value, the method is as follows:

[0074] For each fluorescent probe, a set of codes i is obtained under k excitation sources and corresponding optical path devices. n and j n n≤k, i n and j n This includes the excitation source field, excitation optical path and emission optical path device fields, brightness encoding field BC, and actual brightness value field. The excitation source field is represented by L. i and L j express;

[0075] When P i ≠P j And L i =L j =L n At that time, calculate XOR stands for bitwise exclusive OR;

[0076] When Y = 00, it indicates that the excitation source L is...n Under stimulation, P i and P j Simultaneous imaging in either the transmission or reflection channel is not possible;

[0077] When Y = 11, it means that in L n Under stimulation, P i and P j Imaging in different channels without crosstalk, simultaneous imaging is possible, with a brightness of B. i,n,T and B j,n,R ;

[0078] When Y = 01, it means that in L n Under stimulation, P i and P j One probe images in two channels, while the other only images in the transmission channel. Crosstalk exists in the reflection channel; after eliminating the crosstalk, simultaneous imaging is possible, with brightness levels of B. i,n,T and B j,n,R -C·B i,n,T Or B i,n,R -C·B j,n,T and B j,n,T ;

[0079] When Y = 01, it means that in L n Under stimulation, P i and P j One probe images in two channels, while the other only images in the reflection channel, resulting in crosstalk in the transmission channel. Eliminating this crosstalk allows for simultaneous imaging. i and P j The brightness is B respectively i,n,R and B j,n,T -B i,n,R / C or B i,n,T -B j,n,R / C and B j,n,R .

[0080] The specific steps to determine whether a fluorescent probe meets the requirements for time-division imaging are as follows:

[0081] For any two P1 and P2 among n fluorescent probes, the method to determine whether they can be imaged in time-separated manner is as follows:

[0082] The excitation light fields L1 and L2 for P1 and P2 are not equal, meaning the excitation light from the two probes is different. The brightness encoding field is... and and That is, the excitation light corresponding to P1 cannot excite P2, and at the same time, the excitation light of P2 cannot excite P1.

[0083] S6. Based on the brightness encoding, match the fluorescent probe combination to achieve multicolor imaging.

[0084] In a dual-channel fluorescence microscopy system, since only a maximum of two-color imaging can be achieved with a single excitation, for m-color imaging (m≥2), d simultaneous excitations and s = m-2d time-division excitations are required, with a total of md excitations. The d simultaneous excitations must be independent of each other, meaning there should be no crosstalk between each group of simultaneous excitations. This indicates rounding down from m / 2.

[0085] Step S6 specifically includes:

[0086] S61. Based on the brightness matching conditions for simultaneous two-color imaging, match the fluorescent probe group {m,n}. N N represents the number of fluorescent probe sets that meet the criteria; specifically:

[0087] Since simultaneous excitation of d times requires them to be independent of each other, that is, there should be no excitation crosstalk between the d probe groups. Excitation crosstalk is the excitation light energy that can only excite one probe group. The excitation light of the d probe groups is different from each other. Therefore, the probe group code of d times bicolor simultaneous excitation matched from the probe code should satisfy:

[0088] The excitation source fields of the d probe groups are all different, that is, the result of the XOR operation is 111; for any group {m,n} in the d probe groups, under the encoding of its corresponding excitation source field, the brightness encoding field BC of the remaining d-1 probe groups containing a total of 2d-2 probes are all 00.

[0089] S62. When d simultaneous excitations cannot meet the imaging requirements, s time-division excitation probes are matched again according to the brightness code, specifically as follows:

[0090] Since the s time-division excitations are also independent of each other, and independent of the previous d simultaneous excitations (i.e., there is no excitation crosstalk between the d simultaneously excited probe groups and the s time-division excited probes), the excitation light of the total d+s excitations is different. Therefore, the probe code of the s time-division excitations matched from the probe code should satisfy:

[0091] The excitation source field codes of the s probes are all different, and they are also different from the excitation source field codes of the first d probe groups, i.e., the result of the XOR operation is 111; for any one of the s probes, under its corresponding excitation source field code, the brightness code field BC of the remaining s-1 probes and the 2d probes contained in the first d probe groups are all 00.

[0092] Example

[0093] In this embodiment, a system capable of multicolor imaging is used, such as... Figure 2As shown, the system includes a six-wavelength FRET-LED fluorescent light source 301, a dichroic mirror D1303, a 40x oil immersion mirror 304, experimental samples 305, a dichroic mirror D2306, a reflection channel emission filter 307, a transmission channel emission filter 308, and two cameras (camera CAM_T309 and camera CAM_R310).

[0094] This embodiment employs six excitation wavelengths of 385 nm, 435 nm, 488 nm, 515 nm, 561 nm, and 630 nm. This embodiment uses an independent, self-tunable fluorescent light source, therefore no additional excitation filter is required. For fluorescent light sources that cannot be self-tuned, an additional excitation filter should be added. Figure 2 A corresponding excitation filter 302 is installed at position 302.

[0095] In this embodiment, the specific model of the dichroic mirror D1 is ZT440 / 514 / 561 / 640rpc; the experimental samples in this embodiment are McF7 cells transfected with ECFP, EYFP, and Hoechst, respectively; the specific model of the dichroic mirror D2 is T525lpxr-UF1; the specific model of the reflective channel emission filter is ET480 / 40m; the specific model of the transmissive channel emission filter is ET520lp; both cameras are Xintu Dhyana 400BSI cameras.

[0096] In this embodiment, the devices in the excitation optical path and the emission optical path are specifically coded as shown in Table 2 below; since not all replaceable filters or dichroic mirrors are attached, the device code at all positions is 0001 in the device coding.

[0097]

[0098] Table 2

[0099] In this embodiment, the encoding of the luminance encoding field BC is shown in Tables 3, 4 and 5; Table 1 shows the luminance calculation values ​​of probes ECFP, EYFP and Hoechst in the two channels;

[0100]

[0101] Table 3

[0102] The probes are luminance-encoded based on the luminance calculation values ​​in Table 3, as shown in Table 4 below.

[0103]

[0104] Table 4

[0105] The final complete encoding of Hoechst is shown in Table 5.

[0106]

[0107] Table 5

[0108] The ECFP and EYFP encoding formats are identical to the complete Hoechst encoding.

[0109] Cell imaging experiments were performed on the three probes using two channels respectively to demonstrate the effect of brightness on imaging as shown in Table 3 and to verify the brightness encoding in Table 4:

[0110] Hoechst is a nuclear dye, such as Figure 3 As shown, the experiment was conducted only under 385nm excitation light and imaging was performed in both channels.

[0111] ECFP green fluorescent protein (enhanced version), such as Figure 4 and Figure 5 As shown, the experiment was conducted under excitation light of 385nm and 435nm and imaging was performed in both channels.

[0112] EYFP yellow fluorescent protein (enhanced version), such as Figure 6 and Figure 7 As shown, the experiment was conducted under excitation light of 435 nm and 515 nm and imaging was performed only in the transmission channel.

[0113] Experiments verified that the brightness data values ​​and the final dual-channel imaging results showed good consistency, and the brightness encoding matched the experimental imaging results, which can effectively characterize the imaging relationship of the fluorescent probe in the dual channels. Based on brightness encoding matching, Hoechst, ECFP, and EYFP can all achieve two-color imaging between each other, and all three can achieve three-color imaging.

[0114] It should also be noted that, in this specification, terms such as "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0115] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for fast multicolor imaging based on dual-channel intensity coding of fluorescent probes, characterized in that, The method is based on a dual-channel multicolor fluorescence microscopic imaging system, and the method comprises the following steps: S1, determine the characteristics of the dual-channel multicolor fluorescence microscopic imaging system and the spectral characteristics of each optical element; S2, analyze the system device, respectively, the system is divided into excitation light path and emission light path, and the devices in the excitation light path and the emission light path are coded; S3, according to the actual brightness of different fluorescent probes under each excitation wavelength in the dual-channel multicolor fluorescence microscopic imaging system to encode; according to the probe P i The brightness of each channel is obtained under the irradiation of excitation light, which is encoded, and the encoding consists of an excitation light source field, an excitation light path and an emission light path device field, a brightness encoding field and an actual brightness value field; wherein the excitation light source field L k is encoded by 1 byte, which can represent 256 excitation wavelengths; the brightness encoding field BC consists of a transmission channel marker bit T and a reflection channel marker bit R, both of which are encoded by 1 bit; the actual brightness field is encoded by the binary values of the transmission channel actual brightness value and the reflection channel actual brightness value, which are respectively reserved 20 bits, and can represent up to 1 million brightness values; The coding of the brightness coding field BC is finally divided into four kinds: 00, that is, both channels cannot detect fluorescence; 01, that is, the transmission channel cannot detect fluorescence, and the reflection channel can detect fluorescence; 10, that is, the transmission channel can detect fluorescence, and the reflection channel cannot detect fluorescence; 11, that is, both channels can detect fluorescence; S4, screening fluorescent probes supporting the dual-channel multicolor fluorescence microscopic imaging system; S5, judging whether the fluorescent probe meets the two-color simultaneous imaging and time-sharing imaging; S6, according to the brightness coding, matching the fluorescent probe combination to realize multicolor imaging; In the dual-channel multi-color fluorescence microscopic imaging system, since the most two-color imaging can be achieved at a single excitation, for m-color imaging, m≥2, d times of simultaneous excitation and s=m-2d times of time-sharing excitation are required, and the total excitation times is m-d, d times of simultaneous excitation requires mutual independence, that is, no crosstalk occurs between each set of simultaneous excitation; wherein, represents the floor of m / 2; Step S6 specifically includes: S61、According to the brightness matching condition of dichromatic simultaneous imaging, match out the fluorescent probe group {m, n} N N is the number of fluorescent probe groups that meet the conditions; specifically: Since d times of simultaneous excitation requires mutual independence, that is, there is no excitation crosstalk between the d probe groups, there is no excitation crosstalk, that is, the excitation light can only excite one probe group, and the excitation light of the d probe groups is different, therefore, the probe group code matched by the d times of two-color simultaneous excitation from the probe code should satisfy: The excitation light source field of the d probe groups is different, that is, the result of exclusive or operation is 11111111; for any one group {m, n} in the d probe groups, the brightness coding field BC of the other d-1 probe groups containing a total of 2d-2 probes is 00 under the excitation light source field coding corresponding to it; S62, when d times of simultaneous excitation cannot meet the imaging requirements, match s times of time-sharing excitation probes according to the brightness coding again, specifically: Since s times of time-sharing excitation is also independent of each other, and is also independent of the previous d times of simultaneous excitation, that is, there is no excitation crosstalk between the d times of simultaneous excitation and the s times of time-sharing excitation, and the excitation light of the total d+s times of excitation is different, therefore, the probe code matched by the s times of time-sharing excitation from the probe code should satisfy: The excitation light source field coding of the s probes is different, and is also different from the excitation light source field coding of the previous d probe groups, that is, the result of exclusive or operation is 11111111; for any one probe in the s probes, the brightness coding field BC of the other s-1 probes and the 2d probes contained in the previous d probe groups is 00 under the excitation light source field coding corresponding to it.

2. The method according to claim 1, wherein, In step S1, the characteristics of the dual-channel multicolor fluorescence microscopic imaging system are determined as follows: The spectral characteristics of the excitation light actually irradiating the sample remain unchanged, that is, the excitation wavelength spectral characteristics of the excitation light path remain unchanged; wherein the optical elements of the excitation light path include light source, excitation filter and excitation dichroic mirror; The spectral characteristics of the detected emission light remain unchanged, that is, the spectral characteristics of the fluorescence emission wavelength of the emission light path remain unchanged; wherein the optical elements of the emission light path include emission filter, emission dichroic mirror and detector.

3. The method according to claim 2, wherein, In step S1, the spectral characteristics of each optical element are determined as follows: The spectral characteristics of each optical element are determined according to actual system settings, including: Light source L k Normalized spectral properties of L k (λ ex ), k denotes that there are k excitation light sources; Excitation filter F ex Normalized spectral characteristic: F ex (λ ex ); Normalized transmission and reflection spectral characteristics of the excitation dichroic mirror D1 : D 1,T (λ em ), D 1,R (λ ex ); Emission filter F em Normalized transmission spectral properties: F em (λ em ); Normalized transmission and reflection spectral characteristics of the transmitting dichroic mirror D2: D 2,T (λ em ), D 2,R (λ em ); Probe transmission channel emT emission filter F emT normalized transmission spectral properties: F emT (λ em ); Probe reflection channel emR emission filter F emR Normalized transmission spectral properties: F emR (λ em ); Normalized spectral properties of other optical elements in the excitation light path: EX(λ ex ); Normalized spectral properties of other optical elements in the emission light path located between the transmission and reflection channels: EM T (λ em ), EM R (λ em ).

4. The method according to claim 1, wherein, The step S2 is specifically: The optical devices in the excitation light path and the emission light path are determined, including: The determination of the number M of device assembly positions in the specific optical path and the determination of the number N of optional devices in a single assembly position; The coding of the devices in the excitation light path and the emission light path is specifically: The coding is divided into two fields: excitation light path and emission light path; the coding of excitation light path is determined by the number of device assembly positions M ex and the number of optional devices N of single assembly position ex ; one device position is coded by 4 bits, indicating 16 different optional devices, so the coding length of the whole excitation light path is 4M ex bits; the coding of emission light path is similar to that of excitation light path, determined by the number of device assembly positions M em and the number of optional devices N of single assembly position em ; The whole optical path device encoding is completed, and the total length is 4(M ex +M em ) bits.

5. The method according to claim 3, wherein, The step S3 is specifically: determining the normalized absorption spectral properties A i (λ i,N ) and emission spectral properties F i,N (λ em ) of the fluorescent probe P ex ​ The fluorescence probe P i The luminance of the light source is defined as: B i = ε i Q i wherein ε i Molar extinction coefficient, unit: M -1 cm -1 , represents the absorption characteristics of the fluorescent probe with a thickness of 1 cm and a concentration of 1 mol / L; Q i is the quantum yield, which represents the ratio of the number of emitted photons to the number of absorbed photons; In the dual-channel multicolor fluorescence microscopic imaging system, the transmittance of the excitation path is considered, and for the fluorescent probe P i , the light source L k is arranged on the side of the objective lens. ex,i,k The actual molar extinction coefficient ε ex,i,k is: In a dual-channel multicolor fluorescence microscopy system, considering the transmittance of the emission pathway, for the fluorescent probe P... i Detecting the actual quantum yield Q of the transmission channel i,T And the actual quantum yield Q of the detection reflection channel i,R They are respectively: where S i,T and S i,R are the normalized emission spectrum areas of the detection transmission channel and reflection channel, respectively, S i is the total normalized emission spectrum area of the fluorescent probe P i , and are expressed as: For the fluorescent probe P i In use of the light source L k The actual brightness of the detection transmission channel and the reflection channel when excited are respectively: B i,k,T = ε ex,i,k Q i,T B i,k,R = ε ex,i,k Q i,R The brightness threshold TH is set, and when the brightness is greater than TH, it is considered that the fluorescence can be detected, and when the brightness is less than TH, it is considered that the fluorescence cannot be detected.

6. The method according to claim 5, wherein, The step S4 is specifically: When any light source in the dual-channel multi-color fluorescence microscopic imaging system cannot be imaged in the detection channel after excitation, that is, the system does not support the probe, the probe with the brightness coding field BC of 00 cannot be imaged in the system, and the probes with the brightness coding field BC of 01, 10 and 11 are screened and retained.

7. The method according to claim 5, wherein, In step S5, the judgment of whether the fluorescent probe meets the two-color simultaneous imaging is specifically: For any two P i and P j in the n fluorescent probes, it is determined whether they can be simultaneously imaged, and the brightness values are obtained by the method comprising the following steps: For each fluorescent probe, a set of codes i is obtained under k excitation light sources and corresponding optical path devices respectively n and j n , n≤k, i n and j n includes the excitation light source field, the excitation light path and the emission light path device field, the brightness code field BC and the actual brightness value field, and the excitation light source field is represented by L i and L j ; When P i ≠ P j and L i = L j = L n then compute XOR denotes bitwise XOR; According to the calculation result, when Y = 00, it is indicated that the excitation light source L n P i and P j At the same time, the transmission channel or the reflection channel cannot be imaged simultaneously; When Y = 11, it means that in L n P i and P j are imaged in different channels and without crosstalk, and can be imaged simultaneously, with brightness B i,n,T and B j,n,R ; When Y = 10, it means that in L n P i and P j One of the two channels is imaged, and the other probe is only imaged in the transmission channel, there is reflection channel crosstalk, and after eliminating the crosstalk, it can be imaged simultaneously, and the brightness is B i,n,T and B j,n,R -C·B i,n,T or B i,n,R -C·B j,n,T and B j,n,T ; When Y = 01, it means that in L n P i and P j one of the two channels imaging, the other probe only in the reflection channel imaging, there is a transmission channel crosstalk, eliminate crosstalk can be imaged simultaneously, P i and P j brightness is B i,n,R and B j,n,T -B i,n,R / C or B i,n,T -B j,n,R / C and B j,n,R .

8. The method of claim 5, wherein the method is a fluorescence probe-based dual-channel intensity encoding fast multi-color imaging method. In step S5, the judgment of whether the fluorescent probe meets the time-sharing imaging is specifically: For any two P1 and P2 of the n fluorescent probes, the method for judging whether they can be time-sharing imaged is: P1 and P2 excitation light source field L1≠L2, that is, the excitation light of the two probes is different, and the brightness field is and and That is, the excitation light corresponding to P1 cannot excite P2, and at the same time the excitation light of P2 cannot excite P1.

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