Multi-target fluorescence imaging method

Abstract

The invention discloses a multi-target fluorescence imaging method which comprises the following steps: S1, exciting each sample by using single-wavelength exciting light with the same power, and continuously collecting fluorescence images to obtain a fluorescence image sequence; s2, extracting a time-varying curve of the total fluorescence signal intensity of the fluorescent protein from each fluorescence image sequence, and performing exponential decay fitting on each fluorescence-time variation curve; and S3, obtaining photobleaching characteristic parameters of the four fluorescent proteins according to the fitting equation, and generating a single fluorescent image of each target from multi-target sample imaging marked by the four fluorescent proteins. The method comprises the following steps: marking targets by adopting fluorescent proteins with the same or similar emission wavelengths, carrying out fluorescence imaging acquisition under the same exciting light condition to obtain a photobleaching curve and bleaching characteristic parameters of each fluorescent protein, and accurately identifying a plurality of targets by taking photobleaching kinetic parameters as new judgment dimensions. And the chromatic aberration problem caused by wavelength difference is fundamentally eliminated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopic imaging technology, in particular to a multi-target fluorescence imaging method. BACKGROUND

[0002] Fluorescence microscopic imaging is an important tool for modern biological research, and multiple molecular targets are usually labeled by different fluorescent probes to achieve simultaneous observation of multiple targets. Existing multi-target fluorescence imaging mainly relies on fluorescent probes with different emission wavelengths (such as green fluorescent protein GFP, red fluorescent protein RFP, cyan fluorescent protein CFP, yellow fluorescent protein YFP, etc.), and signals are distinguished by spectral separation to achieve the differentiation and positioning of different targets.

[0003] However, fluorescent photons with different emission wavelengths are affected by different refractive indices and dispersions in the microscopic imaging system, and will produce wavelength-dependent aberrations (chromatic aberrations). This chromatic aberration causes the images of different fluorescence channels to be offset or distorted in space, thereby reducing the positioning accuracy, especially in high-resolution imaging. At present, although there are optical correction or image registration post-processing methods to reduce the influence of chromatic aberration, these methods are complex, dependent on calibration samples, and difficult to completely eliminate errors. Therefore, there is an urgent need for a new method of multi-target fluorescence imaging without relying on spectral differentiation and without chromatic aberration. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide a multi-target fluorescence imaging method.

[0005] The purpose of the present application is achieved by the following technical solution: a multi-target fluorescence imaging method, comprising the following steps:

[0006] S1: using single-wavelength excitation light with the same power to excite each sample, and continuously acquiring fluorescence images to obtain a fluorescence image sequence;

[0007] S2: extracting the total fluorescence signal intensity curve of the fluorescent protein over time from each fluorescence image sequence, and performing exponential decay fitting on each fluorescence-time curve,

[0008] ;

[0009] wherein, is the total fluorescence signal intensity at time t, is the fluorescence signal intensity at the initial time, is the natural base, is the image acquisition time, is a characteristic constant representing the fluorescence bleaching of a protein; and ​

[0010] S3: obtaining the photobleaching characteristic parameters of the four fluorescent proteins according to the fitting equation 、 、 and , and generating single fluorescent images of each target from the imaging of the multi-target sample labeled by the four fluorescent proteins.

[0011] Preferably, in step S1, the different cell samples are labeled by the four fluorescent proteins of tdTomato, mScarlet-I3, mRuby2 and mRuby3.

[0012] Preferably, in step S1, the fluorescent images are continuously collected for 5 seconds, and the camera has a frame rate of 10 frames per second, so as to obtain a sequence of 50 fluorescent images.

[0013] Preferably, in step S3, the following steps are further included:

[0014] S31: obtaining a mixed imaging sample by simultaneously labeling the same cell sample by the four fluorescent proteins of tdTomato, mScarlet-I3, mRuby2 and mRuby3;

[0015] S32: repeating the steps S1 and S2 for the mixed imaging sample to obtain the fitting results of each pixel;

[0016] S33: comparing the fitting results of each pixel with the photobleaching characteristic parameters 、 、 and , obtaining the photobleaching parameters and the corresponding fluorescent protein closest to the fitting results, and attributing the signal of the pixel to the corresponding fluorescent protein;

[0017] S34: after attributing all the pixels to the targets labeled by the four fluorescent proteins, generating single fluorescent images of each target.

[0018] The present application has the following advantages: the present application labels the targets by using fluorescent proteins with the same or close emission wavelengths, and collects fluorescent images under the same excitation light condition, so as to obtain the photobleaching curve and the bleaching characteristic parameters of each fluorescent protein, uses the photobleaching kinetic parameters as a new dimension for discrimination, realizes accurate recognition of multiple targets, and fundamentally eliminates the color difference problem caused by wavelength difference. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a schematic diagram of the photobleaching curve and the fluorescent spectrum of different fluorescent proteins;

[0020] Figure 2An illustration of the experimental results by multi-target fluorescence imaging. DETAILED DESCRIPTION

[0021] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. The components of the embodiments of the present application described and shown in the drawings can be arranged and designed in various different configurations.

[0022] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative labor are within the scope of protection of the present application.

[0023] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0024] It should be noted that: similar reference numerals and letters in the following drawings represent similar items, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0025] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly understood by those skilled in the art, and are only for the convenience of describing the present application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second" and the like are only used to distinguish description, and cannot be understood as indicating or implying relative importance.

[0026] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0027] In the present embodiment, as shown in Figure 1 and Figure 2 , a multi-target fluorescent imaging method comprises the following steps:

[0028] S1: excite each sample using single-wavelength excitation light (561 nm) of the same power, and continuously collect fluorescent images to obtain a fluorescent image sequence; preferably, the fluorescent images are continuously collected for 5 seconds, the camera acquisition frame rate is 10 frames / second, and 50 frames of fluorescent image sequence are obtained. Specifically, the image acquisition device is a laser scanning confocal microscope or a wide-field fluorescent microscope.

[0029] S2: extract the total fluorescent signal intensity-time curve of the fluorescent protein from each fluorescent image sequence, and perform exponential decay fitting on each fluorescent-time curve,

[0030] ;

[0031] wherein, is the total fluorescent signal intensity at time , is the fluorescent signal intensity at the initial time, is the natural base, is the image acquisition time, is a characteristic constant representing the fluorescent bleaching of a protein;

[0032] S3: obtain the photobleaching characteristic parameters , , and of the four fluorescent proteins according to the fitting equation, and generate a single fluorescent image of each target from the multi-target sample imaging labeled with the four fluorescent proteins; specifically, the photobleaching characteristic parameters , , and are used to distinguish the four fluorescent protein-labeled targets in the mixed sample subsequently. By using fluorescent proteins with the same or similar emission wavelengths to label the targets, and collecting fluorescent images under the same excitation light conditions, the photobleaching curve and bleaching characteristic parameters of each fluorescent protein are obtained, the photobleaching kinetic parameters are used as new discriminant dimensions to realize accurate identification of multiple targets, fundamentally eliminate the chromatic aberration problem caused by wavelength difference, and realize multi-target imaging without wavelength aberration.

[0033] Further, in step S1, four fluorescent proteins, tdTomato, mScarlet-I3, mRuby2 and mRuby3, are used to label different cell samples. Specifically, the four fluorescent proteins, tdTomato, mScarlet-I3, mRuby2 and mRuby3, are selected because they have similar or identical emission spectra and are used to label different cell samples.

[0034] Further, in step S3, the following steps are further included:

[0035] S31: The same cell sample is labeled by the four fluorescent proteins, tdTomato, mScarlet-I3, mRuby2 and mRuby3, to obtain a mixed imaging sample;

[0036] S32: The mixed imaging sample is subjected to the steps S1 and S2 again to obtain the fitting results of each pixel;

[0037] S33: The fitting results of each pixel are compared with the photobleaching characteristic parameters 、 、 and to obtain the photobleaching parameters and the corresponding fluorescent protein closest to the fitting results, and the fitting results of the pixel are attributed to the corresponding fluorescent protein;

[0038] S34: After all the pixels are attributed to the targets labeled by the four fluorescent proteins, single fluorescent images of each target are generated. Specifically, since the emission wavelengths of the fluorescent signals are approximately consistent, there is no aberration caused by dispersion, thereby realizing achromatic multi-target imaging.

[0039] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A multi-target fluorescence imaging method, characterized in that: Includes the following steps: S1: Excite each sample with a single-wavelength excitation light of the same power and continuously acquire fluorescence images to obtain a fluorescence image sequence; S2: Extract the curve of the total fluorescence signal intensity of the fluorescent protein versus time from each fluorescence image sequence, and perform exponential decay fitting on each fluorescence-time curve. ； in, For a moment The total fluorescence signal intensity, The fluorescence signal intensity at the initial moment. The base is the natural number. Image acquisition time, This represents a characteristic constant for the fluorescence bleaching of a protein; S3: Photobleaching characteristic parameters of four fluorescent proteins were obtained based on the fitting equation. , , and And generate individual fluorescence images of each target from multi-target sample imaging labeled with four fluorescent proteins.

2. The multi-target fluorescence imaging method according to claim 1, characterized in that: In step S1, different cell samples are labeled using four fluorescent proteins: tdTomato, mScarlet-I3, mRuby2, and mRuby3.

3. The multi-target fluorescence imaging method according to claim 2, characterized in that: In step S1, fluorescence images are continuously acquired for 5 seconds, with the camera acquiring frame rate at 10 frames / second, resulting in a 50-frame fluorescence image sequence.

4. The multi-target fluorescence imaging method according to claim 3, characterized in that: Step S3 further includes the following steps: S31: By simultaneously labeling the same cell sample with four fluorescent proteins—tdTomato, mScarlet-I3, mRuby2, and mRuby3—a mixed imaging sample is obtained. S32: Repeat steps S1 and S2 with the mixed imaging samples to obtain the fitting result for each pixel; S33: Compare the fitting result of each pixel with the photobleaching feature parameters , , and By comparing the results, the photobleaching parameters and corresponding fluorescent proteins that are closest to the fitting results are obtained, and the signal of the pixel is assigned to the corresponding fluorescent protein. S34: After assigning all pixels to targets labeled with the four fluorescent proteins, generate a single fluorescent image for each target.

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