A Fluorescent Probe for Combined Expansion-Super-Resolution Microscopy Imaging and a Method for Processing Expanded Samples

Abstract

The present invention discloses a fluorescent probe for expansion-super-resolution microscopy imaging and an expansion sample processing method. The fluorescent probe has a recognition group, an anchoring group, a fluorescent group, and a linking group; the recognition group is used to specifically bind to a specific target protein or structure in a covalent or non-covalent binding manner; the anchoring group is used to anchor the entire fluorescent probe in the expansion hydrogel; the fluorescent group is used to provide a fluorescent signal to label the target structure; the linking group is used to connect the recognition group, the anchoring group, and the fluorescent group in a covalent bond manner to form a complete fluorescent probe. The present invention provides a modular expansion microscopy labeling method based on small molecule fluorescent probes, thereby solving the problem of resolution decline caused by low labeling density and large size of existing fluorescent protein and immunofluorescence labeling methods.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescence imaging, and more specifically, relates to a fluorescence probe for combined expansion-super-resolution microscopy and a method for processing expanded samples. Background Art

[0002] The resolution of an optical microscope refers to the minimum distance between two equally bright point light sources that can be resolved by the microscope. Due to the diffraction effect of light, traditional far-field optical microscopes cannot resolve two points within a distance of about 200 nm in the xy plane - this law was revealed by the German E. Abbe in 1873 and is known as the Abbe diffraction limit in later generations. In practical applications, due to problems such as the accuracy of the optical system and the background noise of the detection element, the resolution of the optical microscope has not reached its theoretical value.

[0003] Eric Betzig, Stefan W. Hell, and William. E. Moerner developed super-resolution fluorescence microscopy, breaking through the 200 nm resolution limit of optical microscopes. Nowadays, using super-resolution fluorescence microscopy (hereinafter referred to as super-resolution imaging technology), scientists can use fluorescent molecules to monitor the interactions of single molecules and subcellular structures inside cells at the nanoscale.

[0004] Shortly afterwards, in 2015, the laboratory of Edward Boyden published a method called Expansion Microscopy (ExM), which can achieve a spatial resolution of about 65 nm using a traditional confocal microscope. This method physically magnifies a biological sample isotropically by about 4 times, enabling samples smaller than the resolution of traditional microscopes to be resolved after expansion. In addition, the expanded sample is almost transparent, with a refractive index basically the same as that of water. By combining with a water lens, spherical aberration in imaging can be easily avoided. Most importantly, since this method only requires a common confocal or wide-field microscope for imaging, super-resolution fluorescence microscopy can be widely used by more researchers.

[0005] Super-resolution imaging techniques are mainly based on two major strategies, which are: (i) super-resolution microscopy techniques based on light modulation, such as Stimulated Emission Depletion Microscopy (STED), (Saturated) Structured Illumination Microscopy ((S)SIM); and (ii) super-resolution microscopy techniques based on single molecule localization (Single Molecule Localization Microscopy, SMLM), such as Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM). In addition, there are many other super-resolution imaging strategies, such as Super-resolution Optical Fluctuation Imaging (SOFI), which are constantly emerging and playing an increasingly important role.

[0006] In practical applications, it is very difficult for super-resolution microscopy to achieve a resolution below 30 nm. However, the combination of expansion microscopy and super-resolution microscopy, namely expansion-super-resolution combined microscopy, provides the possibility to break through this limitation. Scientists have thus attempted expansion-super-resolution combined microscopy. For example, by combining expansion microscopy with SIM (ExM-SIM), researchers obtained 3D assembly structure data of protein complexes with a lateral resolution of approximately 30 nm. By combining expansion microscopy with STED (ExSTED), researchers achieved a resolution 30 times higher than that of traditional microscopes (lateral approximately 10 nm, axial approximately 50 nm).

[0007] Due to the high requirements of expansion-super-resolution combined microscopy for labeling density and brightness, simply combining them cannot efficiently improve the resolution - because the digestion proteins in expansion quench the fluorescence labeling, resulting in a decrease in brightness, and the increase in volume leads to a several-fold decrease in labeling density.

[0008] In the attempt to further improve the resolution to sub-10 nm, limited by the fluorescence labeling of the sample, it is very difficult for expansion super-resolution microscopy to achieve the best imaging effect:

[0009] ① The brightness of dye molecules decreases. STED and SMLM are highly selective for dyes. Generally speaking, Alexa Fluor 594 has the best effect in STED, while Alexa Fluor 647 with excellent blinking performance and brightness properties has the best effect in SMLM. However, more than 50% of the brightness of these dye molecules is lost in swollen samples.

[0010] ② The fluorescence labeling density decreases. Since the sample size has increased by four times, it means that the labeling density per unit imaging area has also decreased by about 16 times, and the number of photons emitted per unit area is less than 1% of the sample before swelling. Therefore, methods such as multi-epitope labeling need to be used to increase the labeling density to meet the requirements of super-resolution.

[0011] ③ The sizes of fluorescent proteins (about 5 nm) and antibodies (about 15 nm) are relatively large. After the resolution is improved to below 10 nm, the volume of the label itself will multiply and affect the resolution.

[0012] ④ The color selection of fluorescence labeling is very limited. On the premise of barely meeting the requirements such as labeling density and labeling brightness, it is very difficult to achieve dual-color / multi-color expansion super-resolution microscopy with qualified labeling effects for several colors.

[0013] To address the above problems, compared with antibodies and fluorescent proteins, small molecule fluorescent probes have inherent advantages. ① The size of small molecule probes is small (less than 1 nm), and it has a relatively small overall impact on the resolution when the resolution is below 10 nm; ② The steric hindrance of small molecule probes is small and the labeling density is high. After the sample swells, the number of molecules per unit area / volume is more; ③ The structural design of small molecule probes is flexible, and the brightness of a single probe molecule can be improved; ④ After meeting the above requirements, the color selection range of relatively excellent small molecule probes can be further broadened, and there is potential to achieve better dual-color and multi-color expansion super-resolution microscopy.

[0014] However, up to now, the expansion-super-resolution microscopy combined technology based on small molecule fluorescent probe labeling has not been widely studied, and the main limitations are: ① After the sample is subjected to one-time swelling treatment, its volume has increased by about 64 times. Even though the labeling density of small molecule fluorescent probes is higher than that of antibodies or fluorescent proteins, the swollen sample is still difficult to reach the labeling density required for sub-10 nm resolution; ② The swelling treatment of the sample significantly reduces the brightness of dye molecules, and several dye molecules most suitable for super-resolution imaging, such as Alexa Fluor 647, etc., are not spared; ③ The current swelling sample treatment process is mainly for antibody and fluorescent protein labeling, and there is no treatment scheme for swollen samples labeled with small molecule fluorescent probes. Summary of the Invention

[0015] In view of the above deficiencies or improvement requirements of the prior art, the present invention provides a fluorescent probe and an expanded sample processing method for expansion-super-resolution combined microscopy imaging, aiming to provide a modular expansion microscopy labeling method based on small molecule fluorescent probes, thereby solving the problem of resolution decline caused by low labeling density and large size of existing fluorescent protein and immunofluorescence labeling methods.

[0016] To achieve the above object, according to one aspect of the present invention, there is provided a fluorescent probe for expansion-super-resolution combined microscopy imaging technology, which fluorescent probe has a recognition group, an anchoring group, a fluorescent group and a linking group;

[0017] The recognition group is used to specifically bind to a specific target protein or structure in a covalent or non-covalent binding manner;

[0018] The anchoring group is used to anchor the entire fluorescent probe in the expanded hydrogel;

[0019] The fluorescent group is used to provide a fluorescent signal to label the target structure;

[0020] The linking group is used to connect the recognition group, the anchoring group and the fluorescent group by covalent bonds to form a complete fluorescent probe.

[0021] Further, the recognition group is used to recognize a specific cell sample, specifically the recognition sequence of lysosomes, mitochondria, microfilaments or microtubules,

[0022] Further, the anchoring group is 2-methylacrylic acid (MAA).

[0023] Further, the fluorescent group is a fluorescent dye with strong tolerance to the expansion process, and is selected as a rhodamine derivative, etc.

[0024] Further, the fluorescent group is selected as Abberior STAR RED, Alexa Fluor 546 or Alexa Fluor 488.

[0025] Further, the linking group is adipic acid or lysine-glycine repeat sequence (KG) n 。

[0026] On the other hand, the present invention also provides a method for processing an expanded sample of a fluorescent probe, including the following steps:

[0027] (1) Labeling: Fix the cell sample, incubate the cell sample with the fluorescent probe at 37 °C for 30 min, and then rinse 3 times;

[0028] (2) Gel preparation: Prepare the cross-linking reagent at 4°C (U-ExM, a solution prepared by mixing 10% (wt / wt) tetramethylethylenediamine TEMED and 10% (wt / wt) ammonium persulfate APS in a volume ratio of 180:10:10). Among them, U-ExM is prepared by dissolving 19% (wt / wt) sodium acrylate SA, 10% (wt / wt) acrylamide AA, and 0.1% (wt / wt) N,N'-methylenebisacrylamide BIS in PBS. After rapid mixing, add it to the cell sample after incubation and rinsing to prevent coagulation, and transfer it to an incubator at 37°C for 1 h to obtain the gel.

[0029] (3) Swelling: Use a blade to cut the gel into a suitable shape, add the digestion solution (50 mM Tris (pH 8), 1 mM EDTA, 0.5% Triton X-100, 1 M NaCl), and treat it at 70°C in the dark for 1 h; then add ultrapure water for secondary swelling, and replace the ultrapure water every hour until the gel no longer swells.

[0030] Generally speaking, compared with the prior art, the above technical solutions conceived by the present invention can achieve the following excellent effects:

[0031] (1) The size of the small molecule probe is smaller than that of fluorescent proteins and antibodies, and the labeling density is higher.

[0032] (2) The selection of fluorescent dyes and the design of multi-dye composition multiply the fluorescence brightness of a single molecule itself.

[0033] (3) The modular design of the probe increases the flexibility of the recognition group and dye selection, and can achieve precise localization of subcellular structures according to specific needs, or select fluorescent dyes with corresponding wavelengths according to needs.

[0034] (4) By combining with STED super-resolution imaging technology, sub-10 nm resolution of single-color imaging can be achieved.

[0035] (5) A sample swelling treatment method suitable for small molecule fluorescent probes, aiming at the labeling principle of small molecule probes, proposes an optimized process for preparing swollen samples to achieve the best performance of small molecule fluorescent probes in swollen samples. Description of the Drawings

[0036] Figure 1 It is the MS mass spectrometry detection report of the non-dye part of the probe provided in Example 2.

[0037] Figure 2It is the comparison result of the fluorescence intensity of MAA-Actin-TMR labeled imaging after fixation with fixatives of different components. The components of each fixative are 4% (v / v) paraformaldehyde PFA, 3% (v / v) paraformaldehyde PFA + 0.1% (v / v) glutaraldehyde GA, and 4% (v / v) formaldehyde FA. The fixation conditions are 37°C for 13 min;

[0038] Figure 3 It is the comparison result of the fluorescence intensity of labeled imaging with probes of different concentrations of MAA-Actin-TMR. The concentration gradient is: 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM;

[0039] Figure 4 It is the verification of the co-localization of the probe MAA-Actin-TMR and the fluorescent protein Lifeact-EGFP. Among them, a in the figure is the labeling result of the probe MAA-Actin-TMR, b in the figure is the labeling result of the fluorescent protein Lifeact-EGFP, c in the figure is the superposition result of the two channels, and the Pearson co-localization coefficient between the two is 0.94;

[0040] Figure 5 It is the confocal imaging result of the probe MAA-Actin-TMR and the fluorescent protein Lifeact-EGFP labeling and swelling cells. Different false colors represent different depth distributions. Among them, a in the figure is the labeling result of the probe MAA-Actin-TMR, and b and c in the figure are the labeling results of the fluorescent protein Lifeact-EGFP;

[0041] Figure 6 It is the comparison result of the fluorescence intensity of MAA-Actin-TMR labeled imaging with / without protease K added to the digestive solution. After two-tailed t-test, there is a significant difference in the fluorescence intensity results of the final imaging after the samples are treated with the two digestive solutions;

[0042] Figure 7 It is the MS mass spectrometry detection report of the non-dye part of the probe provided in Example 3;

[0043] Figure 8 It is the confocal imaging result of microfilament probes with different fluorescent groups. Among them, a in the figure is the imaging result of MAA-Actin-AF488, b in the figure is the imaging result of MAA-Actin-Atto 495, c in the figure is the imaging result of MAA-Actin-Cy3B, d in the figure is the imaging result of MAA-Actin-Atto 565, e in the figure is the imaging result of MAA-Actin-Cy5, and f in the figure is the imaging result of MAA-Actin- Atto 647N;

[0044] Figure 9 are the confocal and super-resolution imaging results of MAA-Actin-SR labeled cells. In the figure, a is the confocal imaging result before cell swelling, b is the confocal imaging result after cell swelling, c, e, and f are the 2D STED super-resolution imaging results after cell swelling, d is the calculation of the imaging resolution in c (image resolution 20.125 nm, swelling coefficient 4.2, final imaging resolution 4.79 nm), g and h are the fluorescence intensity distribution maps within the dashed boxes in e and f, and i is the 3D STED super-resolution imaging result after cell swelling. Different false colors represent different depth information;

[0045] Figure 10 is the MS mass spectrometry detection report of the non-dye part of the probe provided in Example 4;

[0046] Figure 11 are the confocal imaging results of the probe MAA-Morph-Atto 488 labeled cells. Detailed implementation manners

[0047] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0048] The probe provided by the present invention includes an anchoring group, a recognition group, a linking group and a fluorescent group.

[0049] The anchoring group is methacrylic acid MAA, which can covalently bind to the swelling colloid structure and anchor the entire probe on the colloid framework.

[0050] The recognition group is the recognition sequence of microfilaments, lysosomes or microtubules; the recognition sequence of microfilaments is preferably the polypeptide MGVADLIKKFESISKEE, which can recognize filamentous actin with high specificity; the recognition sequence of lysosomes is preferably N-(2-aminoethyl)morpholine or epoxy probe , which can specifically recognize lysosomes and has very good specificity; the recognition sequence of microtubules is preferably docetaxel, which can specifically recognize tubulin and has very good specificity.

[0051] The linking group may include a lysine-glycine repeat sequence (KG) n, adipic acid Acp or other groups such as glycine G, cysteine C, and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate SMCC that have the function of linking or providing steric hindrance.

[0052] Among them: lysine-glycine repeat sequence (KG) n The lysine residues in it are used to provide free amino groups on the side chains to bind fluorescent groups; while the glycine residues are used to provide a certain steric hindrance; such lysine-glycine repeat sequences can achieve the multi-dye composition of probe molecules, that is, one probe molecule carries multiple dye molecules, greatly improving the brightness of the probe, and is suitable for sample preparation of super-resolution imaging, especially expansion-super-resolution combined imaging; the value of n can be between 3 and 5. The larger the value of n, the higher the brightness of the probe, but the larger the size, which may reduce the labeling density; considering comprehensively, it is appropriate for the value of n to be in the above range. Adipic acid Acp or glycine residue G is used as a linking group to provide steric hindrance; cysteine residue C is used as a linking group to provide a side chain thiol group, so as to react with SMCC; one end of SMCC is connected to the cysteine residue, and the other end is connected to the recognition group docetaxel, thus connecting docetaxel to the probe main body.

[0053] The fluorescent group is a fluorescent dye molecule suitable for expansion-super-resolution combined microscopy imaging, preferably Abberior STAR RED, Abberior STAR ORANGE, Alexa Fluor 546, Alexa Fluor 488, Atto488, etc.

[0054] The probe provided by the present invention can flexibly and widely adapt to various recognition groups and commercially available dyes with excellent optical properties, thereby improving the labeling density and brightness in expansion-super-resolution combined microscopy imaging, and ultimately improving the resolution of imaging.

[0055] The fluorescent probe provided by the present invention preferably adopts the expansion sample treatment method provided by the present invention:

[0056] (1) Labeling: Fix the cell sample, incubate the cells with the above probe at 37°C for 30 min, and rinse 3 times;

[0057] (2) Gel preparation: Prepare the cross-linking reagent at 4°C, immediately add the sample to prevent coagulation, and transfer it to a 37°C incubator for 1 h;

[0058] (3) Expansion: Cut the colloid into a suitable shape with a blade, add the digestion solution without proteinase K, incubate at room temperature and in the dark overnight, then add ultrapure water for secondary expansion, soak for about one hour, change the water, and repeat until the colloid no longer expands.

[0059] This method for processing expanded samples is particularly applicable to expanded microscopy samples labeled with small molecule fluorescent probes. The exclusion of Proteinase K reduces the quenching of fluorescent dyes by proteases, greatly improving the brightness of the final sample, thereby effectively enhancing the imaging quality and resolution.

[0060] Example 1

[0061] A fluorescent probe has the structure of Formula I:

[0062] (MAA)-linker-(dye)-B Formula I

[0063] Wherein: MAA is an anchoring group, linker is a linking group, dye is 5-TAMRA or other dyes, and B is a recognition group.

[0064] MAA is 2-methylacrylic acid;

[0065] Linker is a linking group, including lysine-glycine repeat sequence (KG) n , adipic acid Acp or other groups such as glycine G, cysteine C, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate SMCC that have the function of linking or providing steric hindrance;

[0066] n is 3 - 5;

[0067] B is the polypeptide MGVADLIKKFESISKEE, N-(2-aminoethyl)morpholine , epoxy probe or docetaxel.

[0068] Example 2

[0069] A fluorescent probe has the structure of Formula II:

[0070] (MAA)-K(5-TAMRA)-(Acp)-MGVADLIKKFESISKEE Formula II

[0071] Wherein:

[0072] The anchoring group is 2-methylacrylic acid MAA;

[0073] The recognition group is the polypeptide MGVADLIKKFESISKEE;

[0074] The linking group is lysine residue K, adipic acid Acp;

[0075] The fluorescent group is 5-TAMRA. Based on the selected fluorescent group, the probe is named MAA-Actin-TMR;

[0076] The non-dye part is obtained by the method of solid-phase peptide synthesis, and its MS mass spectrometry detection report is as Figure 1 shown.

[0077] The method for processing the swollen sample of the fluorescent probe is as follows:

[0078] (1) Cell preparation: U2OS cells in good growth state (density 70-90%) were inoculated into a sterile confocal dish (glass bottom Φ15 mm, NEST Biotechnology Co., LTD., China). Cultured overnight at 37°C and 5% CO2 in Mcboy’s 5A medium containing 10% fetal bovine serum.

[0079] (2) Cell fixation: Fixed cells with 3% (v / v) paraformaldehyde PFA + 0.1% (v / v) glutaraldehyde GA for 13 min, reduced with sodium borohydride for 7 min, quenched with 100 mM (PBS) glycine for 10 min. Rinsed three times with phosphate buffer PBS and set aside.

[0080] (3) Cell labeling: Take the probe stock solution, dilute it to a final volume of 100 μL with PBS solution, add it to the confocal dish, and incubate at 37°C and 5% CO2 for 30 minutes. Quickly rinse three times with PBS and immediately proceed to the next step.

[0081] (4) Crosslinking: Prepare the crosslinking reagent U-ExM, 10% (wt / wt) tetramethylethylenediamine TEMED, and 10% (wt / wt) ammonium persulfate APS solution and mix them in a volume ratio of 180:10:10. Among them, U-ExM is prepared by dissolving 19% (wt / wt) sodium acrylate SA, 10% (wt / wt) acrylamide AA, and 0.1% (wt / wt) N,N’-methylenebisacrylamide BIS in PBS, pre-cooled in a 4°C refrigerator respectively, quickly mixed and added to the incubated and rinsed cell samples to prevent coagulation, transferred to a 37°C incubator and cultured for 1 h to obtain a colloid.

[0082] (5) Swelling: Take out the confocal dish and cool it to room temperature. Cut the colloid into a suitable shape with a blade, add the digestion solution (50 mM Tris (pH 8), 1 mM EDTA, 0.5% (v / v) Triton X-100, 1 M NaCl), and treat it at 70°C in the dark for 1 h; then add ultrapure water for secondary swelling, and replace the ultrapure water every hour until the colloid no longer swells.

[0083] (6)Preparation before imaging: Prepare a glass-bottom culture dish (large diameter) for imaging in advance. Incubate the glass-bottom dish with 0.1% (w / v) polylysine PLL at 60 rpm for 10 min, aspirate the PLL, add water, incubate for 30 s, dry it, suck the water on the colloid clean, transfer it to the glass culture dish with a glass slide and a brush, and image immediately.

[0084] For the fluorescent probe provided in this example, the imaging experiment is as follows:

[0085] Wide-field fluorescence microscopy imaging experiment: Observe through a wide-field fluorescence microscope Olympus CKX53. Figure 2 It shows that in the cells fixed with 4% (v / v) paraformaldehyde PFA, 3% (v / v) paraformaldehyde PFA + 0.1% (v / v) glutaraldehyde GA, and 4% (v / v) formaldehyde FA fixatives for 13 min respectively, the fluorescence intensities after the probe MAA-Actin-TMR labels U2OS cells are different. Among them, the fluorescence intensity of the combination of PFA+GA is the highest, indicating that this fixative is most suitable for the preparation of this swollen sample. Figure 3 In it, when incubating the probe MAA-Actin-TMR at a concentration of 1-10 μM, the labeled fluorescence intensity increases with the increase of the probe concentration, indicating that increasing the incubation concentration can effectively improve the labeled fluorescence intensity.

[0086] Laser scanning confocal microscopy imaging experiment: Observe through a laser scanning confocal microscope Nikon C2 or A1. Figure 4 In it, the co-localization of the probe MAA-Actin-TMR and the label of the fluorescent protein Lifeact-EGFP was verified, and the two channels were superimposed. Figure 4 In c of it, the two signals can be well co-localized, and the Pearson co-localization coefficient is 0.94, proving that the structure labeled by the probe MAA-Actin-TMR is microfilament. Figure 5 It shows that after the probe MAA-Actin-TMR labels and swells U2OS cells, compared with the labeling method of the fluorescent protein Lifeact-EGFP, the swollen cell sample after the probe MAA-Actin-TMR labeling shows richer microfilament fiber information. Figure 6 It shows that there is a significant difference in the fluorescence intensity of the swollen sample imaging under the conditions of adding / without adding proteinase K to the digestive solution (P < 0.0001, two-tailed t-test), proving that without adding proteinase K can significantly improve the fluorescence intensity of the swollen sample labeling.

[0087] Example 3

[0088] A fluorescent probe has the structure of formula Ⅲ:

[0089] Formula Ⅲ

[0090] Wherein:

[0091] The anchoring group is 2-methylacrylic acid MAA;

[0092] The recognition group is the polypeptide MGVADLIKKFESISKEE;

[0093] The linking group is the lysine-glycine repeat sequence (KG)3;

[0094] The fluorescent group dye is Abberior STAR RED, Alexa Fluor 546, Alexa Fluor 488, etc.;

[0095] Based on the selected fluorescent group, the probes are named MAA-Actin-SR, MAA-Actin-AF546, MAA-Actin-AF488, etc. respectively;

[0096] The non-dye part is obtained by the method of solid-phase peptide synthesis, and its MS mass spectrometry detection report is as Figure 7 shown.

[0097] The method for processing the swollen samples of the fluorescent probe is the same as that in Example 3; the cell lines used are BSC-1 cells cultured in MEM and U2OS cells cultured in Mcboy’s 5A.

[0098] For the fluorescent probe provided in this example, the imaging experiment is as follows:

[0099] Laser scanning confocal microscopy imaging experiment: Observed by a laser scanning confocal microscope Nikon C2 or A1. After the probes MAA-Actin-AF488, MAA-Actin-Atto 495, MAA-Actin-Cy3B, MAA-Actin-Atto 565, MAA-Actin-Cy5, MAA-Actin- Atto 647N labeled BSC-1 cells, Figure 8 It shows that different dyes have an impact on the specific labeling of the probe. Among them, MAA-Actin-AF488 and MAA-Actin-Cy5 have higher specificity in microfilament labeling. However, the cyanine dyes have poor tolerance to the swelling process, so MAA-Actin-AF488 is more suitable.

[0100] Stimulated emission depletion super-resolution imaging experiment: Observed by a self-built stimulated emission depletion super-resolution imaging STED system. Figure 9 It shows that MAA-Actin-SR can achieve the labeling of microfilaments ( Figure 9a), and maintain a strong fluorescence signal in expansion-STED imaging, sufficient to present the fiber winding in microfilaments ( Figure 9 c); meanwhile, the resolution of this imaging can reach 20.125÷4.2 = 4.79 nm ( Figure 9 d-h). In the 3D STED imaging mode, more abundant information on fiber tortuous winding can be presented ( Figure 9 i).

[0101] Example 4

[0102] A fluorescent probe has the structure of Formula IV:

[0103] Formula IV

[0104] Wherein:

[0105] The anchoring group is 2-methylacrylic acid MAA;

[0106] The recognition group is N-(2-aminoethyl)morpholine ;

[0107] The linking group is the lysine-glycine repeat sequence (KG)3;

[0108] The fluorescent group dye is Abberior STAR RED, Abberior STAR ORANGE, Alexa Fluor546, Alexa Fluor 488, Atto 488, etc.;

[0109] Based on the selected fluorescent group, the probes are named MAA-Morph-SR, MAA-Morph-SO, MAA-Morph-AF546, MAA-Morph-AF488, MAA-Morph-Atto 488, etc. respectively;

[0110] The non-dye part is obtained by the method of solid-phase peptide synthesis, and its MS mass spectrometry detection report is as Figure 10 shown.

[0111] The method for processing the expanded sample of the fluorescent probe is the same as that in Example 3.

[0112] For the fluorescent probe provided in this example, the imaging experiment is as follows:

[0113] Laser scanning confocal microscopy imaging experiment: Observe through a laser scanning confocal microscope Nikon C\(_2\) or A1. Figure 11 It shows that after the probe MAA-Morph-Atto 488 labels U2OS cells, the morphology and distribution of lysosomes are presented.

[0114] Example 5

[0115] A fluorescent probe has the structure of Formula V:

[0116] Formula V

[0117] Wherein:

[0118] The anchoring group is 2-methylacrylic acid MAA;

[0119] The recognition group is an epoxy probe ;

[0120] The linking group is a lysine-glycine repeat sequence (KG)3;

[0121] The fluorescent group dye is Abberior STAR RED, Abberior STAR ORANGE, Alexa Fluor546, Alexa Fluor 488, Atto 488, etc.;

[0122] Based on the selected fluorescent group, the probes are named MAA-Epoxy-SR, MAA-Epoxy-SO, MAA-Epoxy-AF546, MAA-Epoxy-AF488, MAA-Epoxy-Atto 488, etc.;

[0123] Example 6

[0124] A fluorescent probe has the structure of Formula VI:

[0125] Formula VI

[0126] Wherein:

[0127] The anchoring group is 2-methylacrylic acid MAA;

[0128] The recognition group is docetaxel;

[0129] The linking group is a lysine-glycine repeat sequence (KG)3, cysteine C, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate SMCC;

[0130] The fluorescent group dye is Abberior STAR RED, Abberior STAR ORANGE, Alexa Fluor546, Alexa Fluor 488, Atto 488, etc.;

[0131] Based on the selected fluorescent groups, the probes are named MAA-Tubulin-SR, MAA-Tubulin-SO, MAA-Tubulin-AF546, MAA-Tubulin-AF488, MAA-Tubulin-Atto 488, etc.;

[0132] It is easy for those skilled in the art to understand that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for expansion-super-resolution combined microscopy imaging technology, characterized in that, This fluorescent probe is suitable for the combined imaging of expansion microscopy and super-resolution microscopy; it has a recognition group, an anchoring group, a fluorescent group, and a linking group; The recognition group is used to specifically bind to a target protein or structure; the recognition group is a recognition sequence of microfilaments, lysosomes or microtubules; the recognition sequence polypeptide of microfilaments is MGVADLIKKFESISKEE, and the recognition sequence of lysosomes is N-(2-aminoethyl)morpholine or epoxy probe , and the recognition sequence of microtubules is docetaxel; The anchoring group is used to anchor the entire fluorescent probe in the swollen hydrogel; the anchoring group is 2-methylacrylic acid MAA; The fluorescent group is used to provide a fluorescent signal to label the target structure; the fluorescent group is a fluorescent dye molecule suitable for expansion-super-resolution combined microscopy, such as Abberior STAR RED, Abberior STAR ORANGE, AlexaFluor 546, Alexa Fluor 488, or Atto 488; The linking group is used to covalently link the recognition group, the anchoring group, and the fluorescent group to form a complete fluorescent probe; the linking group includes a lysine-glycine repeat sequence (KG) with n ranging from 3 to 5 n .

2. A method for processing an expanded sample based on the fluorescent probe described in claim 1, characterized in that, It includes the following steps: Labeling: Fix the cell sample, incubate the cell sample with the fluorescent probe at 37°C for 30 min, and then rinse it 3 times; Gel preparation: Prepare a cross-linking reagent by mixing U-ExM, 10% (mass fraction) of N,N,N',N'-tetramethylethylenediamine TEMED, and 10% (mass fraction) of ammonium persulfate APS solution in a volume ratio of 180:10:10 at 4°C. Among them, U-ExM is prepared by dissolving 19% (mass fraction) of sodium acrylate SA, 10% (mass fraction) of acrylamide AA, and 0.1% (mass fraction) of N,N'-methylenebisacrylamide BIS in PBS. After rapid mixing, add the incubated and rinsed cell sample to prevent coagulation, transfer it to a 37°C incubator and culture for 1 h to obtain a gel; Expansion: Cut the gel into a suitable shape with a blade, add a digestion solution prepared with 50 mM Tris, pH 8, 1 mM EDTA, 0.5% (volume fraction) Triton X-100, and 1 M NaCl. No protease is added to this digestion solution. Treat it at 70°C in the dark for 1 h; then add ultrapure water for secondary expansion, and replace the ultrapure water every hour until the gel no longer expands.

Images