An optical system for rapid quantitative measurement of fluorescence resonance energy transfer

Abstract

The present invention discloses an optical path system for quantitative measurement of rapid fluorescence resonance energy transfer, including an illumination optical path system and an imaging optical path system, wherein the illumination optical path system includes a light source, a collimating optical path component, a first dichroic mirror and an objective lens, wherein a light beam emitted by the light source enters the first dichroic mirror after passing through the collimating optical path component, and is divided into a reflected first light beam and a transmitted second light beam, and the first light beam is focused on the sample surface after passing through the objective lens; the imaging optical path system includes an objective lens, a first dichroic mirror, a first emission filter, a second dichroic mirror, a second emission filter, a first tube lens, a first optical path steering element, a reflection channel detector, a third emission filter, a second tube lens, a second optical path steering element and a transmission channel detector. The present invention improves the sensitivity and accuracy of FRET measurement.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic imaging, and in particular to an optical path system for rapid quantitative measurement of fluorescence resonance energy transfer. Background Art

[0002] Fluorescence resonance energy transfer (FRET) FRET (Resonance Energy Transfer), as a spectral nanomolecular ruler, can achieve nanometer-level spatial resolution and monitor samples in real time through fluorescent dyes, etc. This feature makes it an important research tool in the fields of biology and chemistry.

[0003] Early FRET measurement technology mainly focused on qualitative or semi-quantitative analysis, and its results showed great ambiguity and uncertainty, and were easily affected by a variety of external factors such as solution conditions and the microenvironment of fluorescent probes, which limited its ability to accurately measure distances in complex biomolecular systems and the need to accurately quantify the intensity of interactions. Traditional FRET data is difficult to standardize and consistent across different methods and conditions within laboratories. Therefore, data obtained between different laboratories, or even using different measurement techniques and environmental conditions, are often not directly comparable, which restricts the further expansion of FRET technology in precision biological research and wide application. Although qualitative analysis allows inferences about whether there is interaction between proteins, it is impossible to give definite answers to details such as the extent of such interaction, the specific influencing factors, and the degree of influence of changes in cell state. This has also exacerbated the difficulty of mutual recognition of FRET measurement results in different research contexts, and thus hindered the promotion and recognition of this technology in a wider scientific field. Therefore, quantitative measurement is particularly important.

[0004] In biological research, many molecular events occur instantaneously or dynamically, such as protein interactions, activation of intracellular signal transduction pathways, changes in nucleic acid structure, etc. Since the properties of biological samples themselves (such as metabolism, degradation, diffusion, etc.) will produce errors over time, long-term experiments may introduce more uncontrollable variables, such as temperature fluctuations, light stability and other factors. When the traditional single-channel optical path captures FRET efficiency, it is necessary to mechanically switch the dichroic mirror wheel and filter wheel. For living cell imaging, the increase in experimental time is often fatal. Long-term laser or strong light irradiation may cause phototoxicity to cells, resulting in cell function damage or death, making the observed cell morphology inconsistent with the captured cell morphology, resulting in experimental errors, and thus making the results of FRET quantitative measurement inaccurate. Therefore, the sensitivity and accuracy of FRET measurement are particularly important, especially in living cell and in vivo experiments. Summary of the invention

[0005] The purpose of the present invention is to provide an optical path system for rapid fluorescence resonance energy transfer quantitative measurement, so as to improve the sensitivity and accuracy of FRET measurement.

[0006] The technical solution of the present invention is as follows:

[0007] An optical path system for rapid fluorescence resonance energy transfer quantitative measurement, comprising an illumination optical path system and an imaging optical path system, wherein:

[0008] The illumination optical path system comprises a light source, a collimating optical path component, a first dichroic mirror and an objective lens. The light beam emitted by the light source is collimated by the collimating optical path component to be a collimated light beam that enters the first dichroic mirror. The first dichroic mirror divides the incident light beam into a reflected first light beam and a transmitted second light beam. The first light beam is focused on the sample surface after passing through the objective lens to illuminate the sample.

[0009] The imaging optical path system includes the objective lens, the first dichroic mirror, the first emission filter, the second dichroic mirror, the second emission filter, the first tube lens, the first optical path steering element, the reflection channel detector, the third emission filter, the second tube lens, the second optical path steering element and the transmission channel detector. The illumination optical path system illuminates the sample to excite the sample to generate a third light beam. The third light beam passes through the objective lens and then transmits through the first dichroic mirror to reach the first emission filter. The first emission filter filters out light of non-target light bands so that the third light beam reaches the second dichroic mirror. The second dichroic mirror divides the incident third light beam into a reflected fourth light beam and a transmitted fifth light beam. The fourth light beam is filtered by the second emission filter to obtain a desired light band and then focused by the first tube lens. The focused light beam is reflected to the reflection channel detector through the first optical path steering element. The fifth light beam is filtered by the third emission filter to obtain a desired light band and then focused by the second tube lens. The focused light beam is reflected to the transmission channel detector through the second optical path steering element.

[0010] The present invention has a reflection channel and a transmission channel to realize dual-channel imaging, wherein the second emission filter is located in the reflection channel, and the third emission filter is located in the transmission channel, both of which play a filtering role, but the emission filters used in the two optical paths are not the same physical entity. The first tube lens and the second tube lens both bear the focusing task, and the sleeve lenses used in the two optical paths of the reflection channel and the transmission channel can be the same device.

[0011] The optical path steering element of the present invention can use an adjustable optical path steering element assembly with the ability to independently adjust the pitch angle and rotation angle, so as to achieve high-precision control of the size and coaxiality of the light beam emitted by the light source after reflection, thereby ensuring that the optical path can strictly maintain a coaxial state, thereby improving the performance and stability of the optical system.

[0012] The first dichroic mirror, the first emission filter, the second dichroic mirror, the second emission filter and the third emission filter in the present invention are the core of the multi-channel design, and have the characteristics of multi-channel filtering capabilities, ensuring efficient and parallel realization of fluorescent multi-color imaging, and at the same time ensuring the rapid response capability of the data acquisition system and the high accuracy of the measurement results when performing quantitative FRET analysis.

[0013] The present invention also has the following preferred design:

[0014] The first dichroic mirror of the present invention comprises at least two passbands for collecting short-wavelength fluorescence of a donor fluorescent dye and for collecting long-wavelength fluorescence of an acceptor fluorescent dye.

[0015] The first dichroic mirror of the present invention also includes any one or more passbands for collecting short-wavelength fluorescence of nuclear fluorescent dyes, long-wavelength fluorescence of mitochondrial fluorescent dyes, and fluorescence of wavelengths corresponding to other dyes such as organelle fluorescent dyes.

[0016] The first emission filter of the present invention is matched with the first dichroic mirror, and at least includes two passbands for filtering out the fluorescence of the donor fluorescent dye and filtering out the fluorescence of the acceptor fluorescent dye. In application, the passbands of the first emission filter and the first dichroic mirror are specifically matched according to the fluorescent dye, and the first emission filter can also filter out the wavelengths of other dyes such as organelle fluorescent dyes. The light emitted by the first dichroic mirror is further screened and filtered by the first emission filter to filter out the emission light that meets the target donor and acceptor fluorescent dyes and other fluorescent dyes.

[0017] The second dichroic mirror of the present invention separates the short-wavelength fluorescence of the donor fluorescent dye from the long-wavelength fluorescence of the acceptor fluorescent dye. At the same time, the second dichroic mirror also has the flexibility to direct the emitted fluorescence from different fluorescent dyes to the appropriate channel. For example, the shorter wavelength fluorescence emitted by the nuclear fluorescent dye can be directed to the donor channel, while the longer wavelength fluorescence of the mitochondrial fluorescent dye is directed to the acceptor channel, thereby improving the accuracy and efficiency of the measurement.

[0018] The light source of the present invention is a point light source, which is transmitted into the collimated light path component through a coupling component or an optical waveguide, providing different light source input schemes. The point light source of the present invention is an LED multi-channel electrically controlled light source, and the light source switches between different wavelengths very quickly, with a switching time of microseconds.

[0019] The collimating optical path component of the present invention includes a first plano-convex lens, an achromatic doublet lens, a third optical path steering element and a second plano-convex lens. The light source beam is focused through the plane incident surface of the first plano-convex lens, and then reaches the third optical path steering element after the chromatic aberration is corrected by the achromatic doublet lens. The light source beam is guided by the third optical path steering element and projected onto the convex surface of the second plano-convex lens, and then focused to complete the beam collimation process. The achromatic doublet lens can effectively correct the chromatic aberration problem existing in the entire optical system, ensuring that the light can be accurately converged at the same focus under various light wavelength conditions, thereby greatly improving the imaging clarity. The present invention completes the deep collimation of the light beam through the combination of the first plano-convex lens, the achromatic doublet lens, the third optical path steering element and the second plano-convex lens to ensure the stability and accuracy of the optical path. The third optical path steering element is a preferred design and can be eliminated according to the structural characteristics.

[0020] Wherein, the third light path redirecting element is a reflector with adjustable pitch angle and rotation angle, which can flexibly guide the light beam to travel along a preset path.

[0021] The illumination optical path system of the present invention further includes an absorption element for absorbing the second light beam to prevent the second light beam from interfering with the subsequent imaging process.

[0022] The first light path deflection element and the second light path deflection element of the present invention are preferably reflective prisms, and may also be replaced by other adjustable reflectors.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention innovatively adopts a dual-channel parallel structure design, which can accurately optimize the filter combination for the FRET donor-acceptor pair. The first dichroic mirror, the first emission filter, the second dichroic mirror, the second emission filter and the third emission filter in the present invention are the core of the multi-channel design, and have the characteristics of multi-channel filtering capabilities, ensuring efficient and parallel realization of fluorescent multi-color imaging, and the donor fluorescence and the acceptor fluorescence are collected separately in two channels at the same time. The accuracy level in the quantitative measurement of FRET efficiency is improved, thereby greatly improving the validity and reliability of relevant experimental data.

[0025] 2. The high-precision measurement results of the present invention benefit from its fast image capture capability, and this feature also highlights its work efficiency and accuracy. The present invention completely abandons the mechanical switching link of the turntable by optimizing the optical path design. Therefore, without taking into account the camera exposure period, the execution of the FRET experiment is only limited by the microsecond band switching speed of the light source. Compared with the traditional single channel, the specific percentage of the reduction time is related to the camera exposure time. If the exposure time is 50ms per channel, it is improved by at least 400 times for manual switching and at least 10 times for manual switching. The improvement in time efficiency is particularly obvious in the case of short exposure time imaging settings. The microsecond-level fast switching mechanism realized by the present invention is so fast that it hardly interferes with the natural movement of proteins, ensuring the high speed and high accuracy of FRET measurement, thereby improving the reliability and effectiveness of quantitative analysis.

[0026] 3. The present invention respectively sets a first tube lens and a second tube lens in the front optical path of the reflection channel detector and the transmission channel detector. This improvement measure successfully solves the common image ghosting problem of dual-channel wide-field fluorescence microscopes under traditional single tube lens configuration, thereby effectively improving the imaging quality and signal-to-noise ratio.

[0027] The present invention realizes efficient FRET quantitative analysis, which is conducive to high-throughput experiments, and can greatly accelerate the screening of new drugs, gene function research and the identification of disease-related biomarkers, thereby promoting scientific research progress and clinical transformation. By combining advanced optical imaging technology, rapid FRET measurement can reveal the complex spatial structure and molecular motion laws inside cells in a three-dimensional and real-time manner, and deepen the understanding of biological networks and functional mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A working principle diagram of an optical path system for rapid fluorescence resonance energy transfer quantitative measurement provided by the present invention;

[0029] Figure 2 This is a schematic diagram of a first connection between a light source and a collimating light path component in an embodiment;

[0030] Figure 3 A schematic diagram of a second connection between a light source and a collimating optical path component in an embodiment;

[0031] Figure 4 This is a working principle diagram of the collimating optical path component in the embodiment;

[0032] Figure 5 The left, right and bottom are respectively a fluorescence image 51 of the transmission channel and a fluorescence image 52 of the reflection channel of the cells transfected with C4Y in the embodiment under 435nm excitation light, and a fluorescence image 53 of the transmission channel under 514nm excitation light;

[0033] Figure 6 The left, right and bottom are respectively a fluorescence image 61 of the transmission channel and a fluorescence image 62 of the reflection channel of the cells transfected with C10Y in the embodiment under 435nm excitation light, and a fluorescence image 63 of the transmission channel under 514nm excitation light;

[0034] Figure 7 The left, right and bottom are respectively a fluorescence image 71 of the transmission channel and a fluorescence image 72 of the reflection channel of the cells transfected with C40Y in the embodiment under 435nm excitation light, and a fluorescence image 73 of the transmission channel under 514nm excitation light;

[0035] Figure 8 The left, right and bottom are respectively the fluorescence image 81 of the transmission channel and the fluorescence image 82 of the reflection channel of the cells transfected with C80Y in the embodiment under 435nm excitation light, and the fluorescence image 83 of the transmission channel under 514nm excitation light;

[0036] Fig. 9 The left and right are respectively a fluorescence image 91 of the reflection channel and a fluorescence image 92 of the transmission channel of the cells transfected with CFP in the embodiment under 435 nm excitation light;

[0037] Fig.10 This is a fluorescence image of the transmission channel of the cells transfected with YFP in the example under 515 nm excitation light.

[0038] Description of reference numerals:

[0039] 1-light source; 2-connecting part; 21-optical waveguide; 22-coupling component; 3-collimating optical path component; 3a-first plano-convex lens; 3b-achromatic doublet lens; 3c-third optical path steering element; 3d-second plano-convex lens; 4-first dichroic mirror; 5-objective lens; 6-sample; 7-first emission filter; 8-second dichroic mirror; 9-second emission filter; 10-first tube lens; 11-first optical path steering element; 12-reflection channel detector; 13-third emission filter; 14-second tube lens; 15-second optical path steering element; 16-transmission channel detector; 17-absorption element. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention more obvious, the exemplary embodiments according to the present invention will be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments of the present invention, and it should be understood that the present invention is not limited to the exemplary embodiments described herein. Based on the embodiments of the present invention described in the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the protection scope of the present invention.

[0041] In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is apparent to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0042] It should be understood that the present invention can be implemented in different forms and should not be interpreted as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make the disclosure thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0043] The purpose of the terms used herein is only to describe specific embodiments and is not intended to be limiting of the present invention. When used herein, the singular forms "one", "an" and "said / the" are also intended to include plural forms, unless the context clearly indicates otherwise. It should also be understood that the terms "consisting of" and / or "comprising", when used in this specification, determine the presence of the features, integers, steps, operations, elements and / or parts, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts and / or groups. When used herein, the term "and / or" includes any and all combinations of the relevant listed items.

[0044] In order to fully understand the present invention, a detailed structure will be proposed in the following description to illustrate the technical solution proposed by the present invention. The optional embodiments of the present invention are described in detail as follows, but in addition to these detailed descriptions, the present invention may also have other implementations.

[0045] An optical path system for rapid fluorescence resonance energy transfer quantitative measurement, comprising an illumination optical path system and an imaging optical path system, wherein:

[0046] like Figure 1 As shown, the illumination optical path system includes a light source 1, a collimating optical path component 3, a first dichroic mirror 4 and an objective lens 5. The light beam emitted by the light source 1 is collimated by the collimating optical path component 3 to be a collimated light beam entering the first dichroic mirror 4. The first dichroic mirror 4 divides the incident light beam into a reflected first light beam and a transmitted second light beam. The first light beam is focused on the surface of the sample 6 after passing through the objective lens 5 to illuminate the sample 6.

[0047] The imaging optical path system includes an objective lens 5, a first dichroic mirror 4, a first emission filter 7, a second dichroic mirror 8, a second emission filter 9, a first tube lens 10, a first optical path steering element 11, a reflection channel detector 12, a third emission filter 13, a second tube lens 14, a second optical path steering element 15 and a transmission channel detector 16. The illumination optical path system illuminates the sample 6 to generate a third light beam. The third light beam passes through the objective lens 5 and then transmits through the first dichroic mirror 4 to reach the first emission filter 7. The first emission filter 7 filters out non-target light waves. The light of the first segment makes the third light beam arrive at the second dichroic mirror 8, and the second dichroic mirror 8 divides the incident third light beam into a reflected fourth light beam and a transmitted fifth light beam. The fourth light beam is filtered by the second emission filter 9 to obtain the required light band and then focused by the first tube lens 10. The focused light beam is reflected to the reflection channel detector 12 through the first optical path steering element 11. The fifth light beam is filtered by the third emission filter 13 to obtain the required light band and then focused by the second tube lens 14. The focused light beam is reflected to the transmission channel detector 16 through the second optical path steering element 15.

[0048] This embodiment has a reflection channel and a transmission channel to realize dual-channel imaging, wherein the second emission filter 9 is located in the reflection channel, and the third emission filter 13 is located in the transmission channel, both of which play a filtering role, but the emission filters used in the two optical paths are not the same physical entity. The first tube lens 10 and the second tube lens 14 both bear the focusing task, and the sleeve lenses used in the two optical paths of the reflection channel and the transmission channel can be the same device.

[0049] In this embodiment, the first light path steering element and the second light path steering element preferentially use an adjustable reflecting prism assembly with the ability to independently adjust the pitch angle and rotation angle, so as to achieve high-precision control of the size and coaxiality of the light beam emitted by the light source after reflection, thereby ensuring that the light path can strictly maintain a coaxial state, thereby improving the performance and stability of the optical system.

[0050] As a preferred embodiment:

[0051] The light source 1 is a point light source, and the connecting portion 2 between the light source 1 and the collimating light path component 3 can be selected from two structures, such as Figure 2 and Figure 3 As shown, the point light source is transmitted into the collimated light path component 3 through the optical waveguide 21 or the coupling component 22, providing different light source input schemes. In this embodiment, the point light source is an LED multi-channel electrically controlled light source, and the light source switches between different wavelengths very quickly, with a switching time of microseconds.

[0052] like Figure 4As shown, the collimating optical path component 3 includes a first plano-convex lens 3a, an achromatic doublet lens 3b, a third optical path steering element 3c and a second plano-convex lens 3d. The light source beam is focused through the plane incident surface of the first plano-convex lens 3a, and then reaches the third optical path steering element 3c after the chromatic aberration is corrected by the achromatic doublet lens 3b. The light source beam is guided by the third optical path steering element 3c and projected to the convex surface of the second plano-convex lens 3d, and then focused to complete the beam collimation process. The achromatic doublet lens 3b can effectively correct the chromatic aberration problem existing in the entire optical system, ensuring that the light can be accurately converged at the same focus under various light wavelength conditions, thereby greatly improving the imaging clarity. The combination of the first plano-convex lens 3a, the achromatic doublet lens 3b, the third optical path steering element 3c and the second plano-convex lens 3d completes the deep collimation process of the light beam to ensure the stability and accuracy of the optical path.

[0053] In this embodiment, the third light path steering element 3c is a reflector with adjustable pitch angle and rotation angle, which can flexibly guide the light beam to travel along a preset path. In some embodiments, when light path steering is not required, the third light path steering element 3c may not be used, and the light beam of the light source is focused by the plane incident surface of the first plano-convex lens, and then corrected for chromatic aberration by the achromatic doublet lens, and then projected to the convex surface of the second plano-convex lens and focused to complete the light beam collimation process.

[0054] The illumination optical path system further includes an absorption element 17 for absorbing the second light beam to prevent the second light beam from interfering with the subsequent imaging process.

[0055] In this embodiment, the first dichroic mirror 4 includes at least two passbands for collecting short-wavelength fluorescence of the donor fluorescent dye and long-wavelength fluorescence of the acceptor fluorescent dye. The first dichroic mirror 4 may also include any one or more passbands for collecting short-wavelength fluorescence of the cell nucleus fluorescent dye, collecting long-wavelength fluorescence of the mitochondrial fluorescent dye, and collecting fluorescence of wavelengths corresponding to other dyes such as the organelle fluorescent dye.

[0056] The first emission filter 7 is matched with the first dichroic mirror 4, and at least includes two passbands for filtering out the fluorescence of the donor fluorescent dye and filtering out the fluorescence of the acceptor fluorescent dye. In application, the passbands of the first emission filter 7 and the first dichroic mirror 4 are specifically matched according to the fluorescent dye, and the first emission filter 7 can also filter out the fluorescence wavelengths of other dyes such as organelle fluorescent dyes. The light emitted from the first dichroic mirror 4 is further screened and filtered by the first emission filter 7 to filter out the emission light that meets the target donor and acceptor fluorescent dyes and other dyes such as organelle fluorescent dyes.

[0057] The second dichroic mirror 8 separates the short-wavelength fluorescence of the donor fluorescent dye from the long-wavelength fluorescence of the acceptor fluorescent dye. At the same time, the second dichroic mirror 8 also has the flexibility to direct the emitted fluorescence from different fluorescent dyes to the appropriate channel. For example, the shorter-wavelength fluorescence emitted by the nuclear fluorescent dye can be directed to the donor channel, while the longer-wavelength fluorescence of the mitochondrial fluorescent dye can be directed to the acceptor channel, thereby improving the accuracy and efficiency of the measurement.

[0058] In this embodiment, the incident angle of the first dichroic mirror 4 and the incident angle of the second dichroic mirror 8 are both 45°.

[0059] The following is a description of the common fluorescence resonance energy transfer CFP-YFP system as an example. CFP (Cyan Fluorescent Protein) is selected as the donor of fluorescence resonance energy transfer, and YFP (Yellow Fluorescent Protein) is selected as the acceptor of fluorescence resonance energy transfer. If the donor molecule and the acceptor molecule are to undergo FRET, three conditions must be met. The selected CFP-YFP here has met the two conditions that the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor and the directions of the dipoles of the donor molecule and the dipoles of the acceptor molecule are not perpendicular to each other. FRET can occur as long as the distance between CFP and YFP is close enough (usually less than 10nm).

[0060] In the present invention, the cell samples transfected with CFP and YFP genes are selected for measurement and calculation. Figure 1 The optical path configuration shown in the figure obtains the fluorescence intensity I of the fluorescence image of the CFP sample in the reflection channel under the action of 435nm excitation light. DD And the fluorescence intensity I of the fluorescence image in the transmission channel DA , the fluorescence intensity I of the fluorescence image of the YFP sample in the transmission channel under the action of 515nm excitation light AA , respectively presented in Fig. 9 and Fig.10 middle.

[0061] Among them, I DD : fluorescence intensity detected by the donor channel under donor excitation light; I DA : fluorescence intensity detected by the acceptor channel under donor excitation light; I AA : Fluorescence intensity detected by the acceptor channel under acceptor excitation light.

[0062] Calculate the values ​​of a, b, c, and d according to the following formula:

[0063] a=I DA (A) / I AA (A) (1),

[0064] b=I DD (A) / I AA (A) (2),

[0065] c=I AA (D) / I DD (D) (3),

[0066] d=I DA (D) / I DD (D) (4).

[0067] a, b, c, and d are all crosstalk factors, a represents the excitation crosstalk degree of the acceptor, b represents the emission crosstalk degree of the acceptor, c represents the excitation crosstalk degree of the donor, and d represents the emission crosstalk degree of the donor. (A) means that the sample of the measured object is a single-turnover acceptor. In this embodiment, YFP is a sample of a single-turnover acceptor. (D) means that the sample of the measured object is a single-turnover donor. In this embodiment, CFP is a sample of a single-turnover donor.

[0068] The present invention selectively studies cell samples transfected with C4Y, C10Y, C40Y, and C80Y genes. C4Y means that there are four base pairs between the two fluorescent proteins CFP and YFP, and the meanings of C10Y, C40Y, and C80Y are similar. Figure 1 The optical path configuration shown in the figure accurately obtains the fluorescence images of these samples in the transmission and reflection channels and obtains the fluorescence intensity. The sensitized emission intensity F is calculated according to the following formula (5): C After the results are obtained, the reference FRET efficiency values ​​are substituted into formulas (6) and (7) to calculate the G factor and K factor in turn. G represents the conversion factor of the energy of the donor molecule into the sensitization quenching of the acceptor due to FRET, and K is the fluorescence intensity ratio of the donor and acceptor at equal concentrations. The E in formula (6) is the ED value obtained by performing FRET experiments on cell samples transfected with C4Y, C10Y, C40Y, and C80Y genes under the condition of a donor-acceptor concentration ratio of 1:1. For the specific experimental principle, please refer to Butz ES, Ben-Johny M, Shen M, et al. Quantifying macromolecular interactions inliving cells using FRET two-hybrid assays[J]. Nature protocols, 2016, 11(12): 2470-2498.

[0069]

[0070]

[0071] Finally, E is calculated according to formula (8) and formula (9):D FRET efficiency relative to donor concentration and donor-acceptor concentration ratio R C The quantitative FRET measurement is performed based on the value of Figures 5 to 8 middle.

[0072]

[0073] Figures 5 to 8 The fluorescence characteristics of C4Y, C10Y, C40Y, and C80Y cells under two different excitation light conditions are shown in turn, mainly used to extract the fluorescence intensity of their fluorescence images First, under the action of 435nm excitation light, the fluorescence intensity of each type of cell in the transmission channel fluorescence image was recorded. Fluorescence intensity of the fluorescence image in the reflectance channel Secondly, for the same group of cell samples, we further collected the fluorescence intensity of the transmission channel fluorescence image under 514nm excitation light.

[0074] The high-precision measurement results of the present invention benefit from its fast image capture capability, which also demonstrates its work efficiency and accuracy. Specifically, in the traditional single-channel system, the time for a FRET experiment includes the filter disk switching time, the filter wheel switching time and the camera exposure time. It usually takes at least 10 seconds to rotate various wheels by manual single operation; and the fine alignment process can even be extended to more than ten seconds or even longer. In contrast, the traditional single-channel system using electric filter disks and electric filter wheels, although there is a certain improvement in speed, is still not comparable to the efficiency of the present invention. For example, when using the Zaber electric filter wheel, the filter replacement time is 57ms; when using the electric filter disk, the switching time is 500ms. In summary, the traditional single-channel system needs to switch the filter disk twice and the filter wheel twice for a FRET experiment. It takes at least 40s to manually switch various disks and at least 1.114s to electrically switch various disks.

[0075] The present invention completely abandons the mechanical switching link of the turntable by optimizing the optical path design. Therefore, the execution of the FRET experiment is only limited by the microsecond band switching speed of the light source without taking into account the camera exposure period. Compared with the traditional single channel, the specific percentage of the reduction time is related to the camera exposure time. If the exposure time is 50ms per channel, it is improved by at least 400 times for manual switching and at least 10 times for manual switching. The improvement in time efficiency is particularly obvious in the case of short exposure time imaging settings.

[0076] For proteins and organelles, they are in continuous motion inside the cell. For example, the microtubule motor proteins (such as kinesin and dynein) that move along actin fibers travel along the microtubules at a speed of approximately 0.5 to 2 micrometers per second. When using traditional single-channel and various turntable methods for fluorescence resonance energy transfer (FRET) experiments, these fast-moving microtubule motor proteins will cause slight changes in the sample position due to the long mechanical switching time, thereby introducing artifacts in the process of capturing fluorescent images, and ultimately affecting the accuracy of the experimental results. In contrast, the microsecond-level fast switching mechanism implemented by the present invention is so fast that it hardly interferes with the natural movement of proteins, ensuring the high speed and high accuracy of FRET measurements, thereby improving the reliability and effectiveness of quantitative analysis.

[0077] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An optical system for rapid fluorescence resonance energy transfer quantitative measurement, comprising an illumination optical system and an imaging optical system, characterized in that: The illumination optical path system comprises a light source, a collimating optical path component, a first dichroic mirror and an objective lens. The light beam emitted by the light source is collimated by the collimating optical path component to be a collimated light beam that enters the first dichroic mirror. The first dichroic mirror divides the incident light beam into a reflected first light beam and a transmitted second light beam. The first light beam is focused on the sample surface after passing through the objective lens to illuminate the sample. The imaging optical path system comprises the objective lens, the first dichroic mirror, the first emission filter, the second dichroic mirror, the second emission filter, the first tube lens, the first optical path steering element, the reflection channel detector, the third emission filter, the second tube lens, the second optical path steering element and the transmission channel detector. The illumination optical path system illuminates the sample to excite the sample to generate a third light beam. The third light beam passes through the objective lens and then transmits through the first dichroic mirror to reach the first emission filter. The first emission filter filters out light of non-target light bands so that the third light beam reaches the second dichroic mirror. The second dichroic mirror divides the incident third light beam into a reflected fourth light beam and a transmitted fifth light beam. The fourth light beam is filtered by the second emission filter to obtain a desired light band and then focused by the first tube lens. The focused light beam is reflected to the reflection channel detector through the first optical path steering element. The fifth light beam is filtered by the third emission filter to obtain a desired light band and then focused by the second tube lens. The focused light beam is reflected to the transmission channel detector through the second optical path steering element. The first dichroic mirror includes at least two passbands for collecting short-wavelength fluorescence of the donor fluorescent dye and long-wavelength fluorescence of the acceptor fluorescent dye. The first emission filter matches the first dichroic mirror and includes at least two passbands for filtering out the fluorescence of the donor fluorescent dye and filtering out the fluorescence of the acceptor fluorescent dye. The second dichroic mirror separates the short-wavelength fluorescence of the donor fluorescent dye from the long-wavelength fluorescence of the acceptor fluorescent dye.

2. The optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to claim 1, characterized in that: The light source is a point light source, and the point light source is transmitted into the collimated light path component through a coupling component or an optical waveguide.

3. The optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to claim 2, characterized in that: The collimating optical path component includes a first plano-convex lens, an achromatic doublet lens, a third optical path steering element and a second plano-convex lens. The light source beam is focused through the plane incident surface of the first plano-convex lens, and then corrected for chromatic aberration through the achromatic doublet lens and projected onto the convex surface of the second plano-convex lens for focusing to complete the beam collimation process.

4. The optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to claim 3, characterized in that: The third light path deflection element is a reflector with adjustable pitch angle and rotation angle.

5. An optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to any one of claims 1 to 4, characterized in that: The illumination optical path system further comprises an absorption element for absorbing the second light beam.

6. The optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to claim 5, characterized in that: The first light path deflection element and the second light path deflection element are reflectors.

7. The optical path system for rapid fluorescence resonance energy transfer quantitative measurement according to claim 5, characterized in that: The first dichroic mirror also includes any one or more passbands for collecting short-wavelength fluorescence of nuclear fluorescent dyes, collecting long-wavelength fluorescence of mitochondrial fluorescent dyes, and collecting fluorescence of wavelengths corresponding to organelle fluorescent dyes.

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