CN114910455A - Visualization method for rare single cell identification contour - Google Patents

Abstract

The invention belongs to the field of liquid biopsy, and particularly relates to a method for visualizing a rare single cell identification profile, which comprises the following steps: specific cell membrane staining is carried out on the circulating tumor cells; adding a fluorescence enhancement reagent, wherein the fluorescence intensity reagent is an organic phase solution of a surfactant or organic alcohol; microscopic imaging; and converting the photo of the microscopic imaging into a digital photo, and amplifying the digital signal in the digital photo to obtain a visualized cell contour map. According to the invention, the specific fluorescence of the cell surface is enhanced by adopting the surfactant or the organic alcohol, so that the signal-to-noise ratio of a surface fluorescence signal is improved; by utilizing a digital signal amplification technology, the visualization of the specific identification contour of the single cell is realized, and the signal loss of the imaging cell is avoided; accuracy in capturing CTC counts and scale estimates can be achieved based on fluorescence visualized cell profiles.

Description

Visualization method for rare single cell identification contour

Technical Field

The invention belongs to the field of liquid biopsy, and particularly relates to a method for visualizing a rare single cell identification profile, which is a method for realizing the visualization of the rare single cell identification profile based on the combination of cell membrane surface marker identification fluorescence enhancement and digital signal amplification.

Background

When the tissue cells of a solid tumor are exfoliated by epithelial-mesenchymal transition, the tumor cells enter venous blood in a single cell state to undergo in vivo circulation, i.e., Circulating Tumor Cells (CTCs), which can attach to or be intercepted within distal organs, i.e., tumor metastasis is achieved. Since CTCs are associated with tumorigenesis and progression and are dose-response to progression, they are considered markers for early diagnosis, in contrast to the clinically diagnosed gold standard "tissue biopsy," also known as fluid biopsy. Fluid biopsies typically have 3 basic steps, (1) obtaining CTCs from venous blood, including extracorporeal blood isolation or capture of CTCs, and in vivo capture of CTCs in venous blood vessels; (2) cell pretreatment before detection, such as cell fixation, blocking, membrane perforation, cell membrane marker recognition by a fluorescent-labeled recognition ligand (such as an antibody and an aptamer), nuclear staining, centrifugation, washing and the like, wherein the specific staining of a captured CTC membrane, namely 'specific membrane staining', is realized by the fluorescent-labeled recognition ligand recognizing the cell membrane marker, and is a key factor for judging the captured cells to be CTC; (3) microscopic imaging and microscopic examination. In order to ensure the accuracy of microscopic examination, when the pretreated cells are imaged, i.e. fluorescent dots in the photograph, and judged as CTCs, laboratory and clinical requirements generally simultaneously satisfy 3 conditions, namely, there are nuclei stained by DNA dye, specific membrane staining can be observed outside the nuclei, and the cell size (usually 10-60 μm) is obviously larger than that of blood cells (usually 3-5 μm). Both of which depend on the fluorescence intensity of the cell membrane-specific staining. If the fluorescence is weak, the specific membrane fluorescence of the imaged CTCs cannot be effectively observed and the CTC counts based on the imaged cells will be low. Meanwhile, the cell size cannot be estimated accurately, because the cell imaging is usually a fluorescent dot or a circle during microscopic examination, and the size of the fluorescent dot is estimated according to an imaging scale to obtain the cell size, which requires that the fluorescent intensity of the cell imaging dot is sufficiently larger than the minimum detection limit of an eye, and meanwhile, a large amount of markers distributed on the whole cell membrane are specifically identified, so that the fluorescence of the imaging dot is uniformly distributed, the defects and irregular shapes of the fluorescent dot are avoided, the imaging outline is ensured to be consistent with the cell outline of the CTC, and the cell size can be estimated accurately based on the complete outline. Therefore, the accuracy of both CTC counts and scale estimation during microscopy requires that cellular imaging have a "complete cell profile that can be visualized" due to the fact that the fluorescence intensity of cell membrane-specific staining is sufficiently strong to be significantly greater than the minimum detection limit of the eye.

The accuracy of CTC enumeration is a prerequisite for CTC detection as a basis for early diagnosis of cancer. Since CTCs are extremely rare in number and are typically rare cells, the mean of detection in patients with intermediate and advanced stages is usually below 10, such as lung cancer 5(Emilsson, v., et al. clin. cancer res.,2016,22:2197), breast cancer 5.5(Nadia s.z., et al. int. j. oncol.,2012,41:1241), and the positive threshold is only 1 CTC. Such low thresholds and extremely low cell counts are likely to result in false negative diagnoses due to poor microscopic imaging of CTCs, which results in poor CTCs. Therefore, enhancing the fluorescence intensity of cell membrane-specific staining to obtain a visualized complete cell profile is key to accurate enumeration and scale estimation of CTCs.

The specific expression protein distributed in large quantity on the surface of the CTC membrane is used as a recognition receptor, and a specific recognition ligand (such as an antibody and an aptamer) marked by fluorescence is reacted with the CTC to form a receptor-ligand-phosphor compound on the surface of the cell membrane so as to dye the cell membrane specifically. Theoretically, specific stained fluorescent dots can be observed under a microscope, the outline of the dots is consistent with the actual outline of the CTC, the CTC can be counted based on the specific fluorescent dots, and the CTC scale can be estimated according to the complete outline of the dots. It is based on this that the current laboratory and clinic have achieved microscopic CTC counts and scale estimates (Emilsson, v., et al. clin. cancer res.,2016,22: 2197; Li R, et al. acs Applied Materials & Interfaces,2018,10: 16327).

However, the specific fluorescence of CTCs under the mirror is weak, not sufficiently different from background fluorescence (including fluorescence of the substrate, the spots, the slide, etc. of the cell itself), i.e., the signal-to-noise ratio is low, and even some CTCs cannot observe fluorescence; fluorescent dots presented by more cells are incomplete and irregular in shape. The reasons for this are 2, firstly, there are a lot of protein molecules on the surface of cell membrane, they have obvious matrix fluorescence, must deduct while examining microscopically, have led to the intensity of the specificity to discern the fluorescence to weaken apparently, and the cell sap is heterogeneous suspension, the membrane surface specificity discerns the fluorescence intensity too unevenly, the strong fluorescence is observed effectively, the weak fluorescence can not be observed effectively, this has appeared the incomplete of the fluorescent round dot of cell imaging; secondly, the fluorescence excitation light source of the inverted fluorescence microscope widely used in clinic and laboratory is usually a high-pressure mercury lamp with energy significantly lower than that of a laser, which can effectively reduce fluorescence quenching and also reduce the intensity of the excited fluorescence. This must result in poor CTC counts based on the fluorescent dots of the imaged cells and significant deviations in cell scale estimates. In order to solve the above problems, the fluorescence intensity of the cell membrane specific staining of CTCs must be enhanced to increase the difference between the specific fluorescence and the background fluorescence, i.e. to increase the signal-to-noise ratio, so as to make the difference between the fluorescence brightness of the cell image and the background obvious, and when the image is observed, the background is subtracted (i.e. there is no background fluorescence in the photograph, only there is the specific fluorescence of the cell), and the specific fluorescence of the remaining cells has sufficient intensity, which is a precondition for realizing the visualization of the complete outline of the cell. When the residual intensity is obviously greater than the lowest detection limit (namely the lowest observable intensity) of the eyes, visualization is realized, and microscopic observation can be directly carried out; otherwise, visualization still cannot be realized, and microscopic examination is still inaccurate. In fact, more CTCs belong to the latter. Although the fluorescence intensity at this time does not reach the lowest detection limit of the eye, the fluorescence is significantly higher than the background, i.e., the signal-to-noise ratio is significantly improved. In conclusion, 2 problems need to be solved to realize complete visualization of the captured extremely small amount of CTCs, and firstly, the specific recognition fluorescence of the cell membrane is enhanced, and the signal-to-noise ratio of the specific fluorescence is improved; secondly, the intensity of the signal with high signal-to-noise ratio is obviously higher than the lowest detection limit of the eye, so that the cell outline is completely presented, and the cell visualization is realized. Many methods for enhancing fluorescence are available, wherein micelle-enhanced fluorescence is a common strategy, a hydrophobic phosphor is wrapped in an inner layer of a micelle to form a hydrophobic spherical core, a hydrophobic microenvironment is constructed for the phosphor, so that the fluorescence is enhanced, and a hydrophilic spherical shell is formed on an outer layer of the micelle. Firstly, an amphiphilic block copolymer has a non-fluorescent hydrophilic end and a fluorescent lipophilic end, and hydrophilic spherical micelle nanoparticles with the outward hydrophilic end and the inward fluorescent lipophilic end are formed in an aqueous solvent by utilizing hydrophobic interaction; secondly, the non-fluorescent surfactant also has a hydrophilic end and a lipophilic end, and can also form hydrophilic spherical micelles in the water solvent, and hydrophobic fluorescent molecules are fixed in hydrophobic spherical cores of the micelles by a wrapping method in the process of forming the micelles. They have 3 common features. First, enhancing the fluorescence of fluorescent molecules in a free state in a solution, including encapsulated fluorescent small molecules in a free state and fluorescent polymer molecules in a free state accumulated based on hydrophobic interactions; secondly, the fluorescent body is fixed in the hydrophobic spherical inner core, and the whole micelle is hydrophilic spherical nano-particles; third, a large number of fluorophores or fluorescent molecules accumulate in the core of the spherical micelle. The prepared micelle nanoparticles with enhanced fluorescence are used as a fluorescence signal body to mark specific recognition ligands (such as antibodies and aptamers) and then used for recognizing CTC membrane surface markers so as to realize enhancement of CTC membrane recognition fluorescence. The strategy of preparing micelle nanoparticles with enhanced fluorescence, labeling recognition ligands and finally recognizing receptors on a CTC membrane realizes the enhancement of CTC specific recognition fluorescence, wherein a hydrophobic microenvironment required for enhancing the fluorescence of a phosphor is provided by the micelle nanoparticles formed by the accumulation of hydrophobic ends of a surfactant, and the defect of 4 aspects caused by the micelle nanoparticles is inevitably existed. (1) After the recognition ligand is marked by the fluorescent micelle nanoparticle, because the recognition ligand is a molecule, the volume of the recognition ligand is far smaller than that of the micelle nanoparticle connected with the recognition ligand, when the recognition ligand identifies a receptor on the surface of a cell membrane, the micelle connected with the recognition ligand generates great steric hindrance, the recognition capability is obviously weaker than that of the recognition ligand marked by the fluorescent molecule with small volume, the recognized specific fluorescence is weakened, and the cell membrane fluorescence enhanced by the micelle nanoparticle is partially offset. (2) The process for preparing the fluorescence enhanced nanoparticles is complex, and comprises the steps of connecting fluorescent micromolecules with long-chain macromolecules to prepare fluorescent amphiphilic long-chain macromolecules, connecting active groups to the fluorescent amphiphilic long-chain macromolecules to realize the marking of an identification ligand, the structural stabilization of micelle nanoparticles, the membrane dialysis separation of surplus fluorescent micromolecules and the like; (3) the encapsulation rate of the fluorescent micromolecules in the micelle nanoparticles is uneven, even some nanoparticles do not encapsulate the fluorescent micromolecules, so that the fluorescence intensity of the marked recognition ligand is uneven, even no fluorescence exists; (4) the fluorescence intensity of the micelle nanoparticles has a great batch-to-batch difference, which results in poor reproducibility of the recognized fluorescence of the cells. For the above reasons, a great deal of reports have been made on fluorescence-enhanced nanoparticles, but it is difficult to find practical applications in clinical detection.

From the analysis of application, the micelle nanoparticle enhanced fluorescence changes the microenvironment of the fluorescence signal molecules, so that the physical enhancement of signals is realized, and the capability (namely the enhancement multiple) of signal enhancement is limited. For example, the bigger the nanoparticle is, the more the encapsulated fluorescent molecules are, the larger the increase of fluorescence is, but when the recognition ligand marked by the fluorescent particle is used for recognizing the receptor on the cell membrane, the larger steric hindrance is generated by the large-volume fluorescent particle, the recognition capability is reduced, and the fluorescence intensity on the cell membrane is reduced on the contrary, thereby forming a mutual restriction relationship. Therefore, the specific recognition fluorescence of the cell membrane is still weak, and after the cell matrix fluorescence is subtracted, the brightness of the imaged cell is low, the complete outline of all cells cannot be clearly observed, and even some cells cannot observe the fluorescence.

Disclosure of Invention

The invention aims to solve the problems and provides a visualization method of a rare single cell identification profile, which can realize the accuracy of CTC (cell-based CTC) capture counting and scale estimation based on the combination of fluorescence enhancement and digital signal amplification of cell membrane surface marker identification and the basis of fluorescence visualization cell profile.

According to the technical scheme of the invention, the method for visualizing the rare single-cell identification contour comprises the following steps,

s1: performing cell membrane fluorescent staining for specific recognition on a rare single cell membrane marker to obtain a cell sap I with a solvent of water or a water solution;

s2: adding a fluorescence enhancement reagent into the cell sap I to obtain cell sap II, wherein the fluorescence enhancement reagent is an organic phase solution of a surfactant or liquid organic alcohol;

s3: performing microscopic imaging on the cell sap II;

s4: and converting the photo of the microscopic imaging into a digital photo, and amplifying the digital signal in the digital photo to obtain a visualized cell contour map.

The invention is before finishing the routine cell preconditioning before microscopic examination, including cell fixation, covering, membrane perforation, specific membrane staining, nuclear staining, centrifugating, washing, etc., later, increase one step of simple operation can realize the enhancement of the specificity and discern fluorescence, namely add organic phase solution of surfactant active, such as N of surfactant active Tween-80, N-dimethyl formamide (DMF) solution (can't use aqueous solution of Tween-80, such as PBS solution of Tween-80, distilled aqueous solution of Tween-80) to get to the cellular fluid after preconditioning directly, and then image according to the routine method microscopic examination, including adding the cellular fluid to the slide glass, standing, microscopic examination; or directly dripping liquid organic alcohol with excellent water solubility, such as ethanol, onto the cell sap of the glass slide, standing, and directly performing microscopic examination. Compared with the conventional method, the method only adds one step of operation of dropping organic phase solution of surfactant or liquid organic alcohol with excellent water solubility in cell fluid after specific recognition, and can perform microscopic examination according to the conventional method without adding other complicated operations such as centrifugation, dialysis and the like, so that the method is simple and consistent with the conventional microscopic examination method, and is easy to popularize and apply in laboratories and clinics. Secondly, the fluorescent micelle nanoparticles do not need to be prepared by a complex process, and the structural defects of the fluorescent micelle nanoparticles, such as uneven encapsulation efficiency and poor process reproducibility, do not exist.

The invention constructs a hydrophobic microenvironment different from micelle-enhanced fluorescence in principle. After the cells are stained with the specific membrane, a "receptor-ligand-fluorophore" complex is formed on the surface of the cell membrane, and the fluorophore extends away from the cell membrane, wherein the fluorophore is located at a distal end, like a "nail that is not completely nailed" on the cell membrane, and the fluorescent molecule is like the head of the nail. The phosphor at this time is in a confined state, unlike fluorescent molecules in general micelle-enhanced fluorescence, which is in a free state. For the identified cells, a hydrophobic microenvironment is also constructed for the phosphor on the membrane by dropping an organic phase solution of a surfactant or dropping a liquid organic alcohol with excellent water solubility on the cell surface.

When the organic phase solution of the surfactant is dripped, the surfactant is uniformly distributed in the solution in a molecular state in the solvent of the organic phase, when the solution is dripped into the identified cell suspension or the cell drops of the glass slide, as the solvent of the cells is an aqueous solvent, such as distilled water, PBS and DPBS, a hydrophilic large environment is provided, the strong hydrophobicity of the fluorophore of the fluorescent body at the head of the nail induces the hydrophobic end of the surfactant free in the water to accumulate towards the fluorescent body, and the hydrophilic end extends outwards to form an accumulation body like an umbrella-shaped cap to be buckled on the fluorescent body outside the cell membrane. The aggregate is called as surface umbrella-shaped micelle, and the fluorescent molecules are surrounded by the umbrella-shaped micelle and cell membrane surface protein under the micelle to form a closed space, so that a hydrophobic microenvironment is formed for the fluorescent molecules. The reason why the hydrophobic micro-environment of the normal spherical micelle cannot be formed is that the receptor-ligand and the cell membrane in the receptor-ligand-phosphor complex on the cell membrane form a spatial restriction, so that the umbrella-shaped micelle is opened, and the opening is just blocked by the cell membrane surface protein, thereby forming a hydrophobic micro-environment of the fluorophore of the inner core.

When liquid organic alcohol with excellent water solubility is dripped into the identified cell sap, the liquid organic alcohol is extremely easy to be mixed with a large amount of water in cell membranes due to the excellent water solubility of the liquid organic alcohol, and the organic alcohol with little water in polyol is extremely easy to volatilize, so that the cell membranes are dehydrated, and wrinkles are generated on the membranes, so that membrane protein molecules are close to each other and are fully exposed. On one hand, the hydrophobic property of the membrane is increased due to membrane dehydration, so that a hydrophobic microenvironment is provided for fluorescent molecules on the surface; on the other hand, the phosphors localized to specific recognition of membrane proteins are close to each other due to membrane folds, and the hydrophobic fluorophores of the phosphors themselves aggregate to provide a hydrophobic microenvironment with each other.

For the cell image obtained by microscopic examination, there still remains the problem that "the brightness of the specific fluorescence on the imaged cell membrane is still low, and the brightness is not uniform, and the complete cell contour cannot be clearly observed, and even some cells cannot observe the fluorescence". For general cell imaging, this is sufficient, and more accurate cell imaging detection can be achieved. The reason is that the conventional cell imaging microscopy generally needs to check 2 parameters, whether a cell is a target cell and the number of the target cells, because the conventional cell number is large, dozens of or even hundreds of cells in a field of view, the total number of the cells which can be microscopically examined is large, a small number of cells cannot be counted due to weak fluorescence, the accuracy of a result cannot be obviously influenced, and false negative cannot be caused. For rare cell CTC, when the imaging cell is subjected to microscopic examination, accurate estimation of cell scale can be realized by requiring complete counting of all cells and complete and clear visible cell outline, so that the requirements of CTC counting and scale estimation cannot be met through the signal enhanced by the specific fluorescence. By dropping an organic phase solution of a surfactant or a liquid-phase organic alcohol, the absolute value of the intensity of the specific recognition fluorescence of the cell membrane is increased, while the relative value, i.e., the signal-to-noise ratio, is also increased. After the photograph is digitized, the digital signal is amplified by a certain factor. Because the signal to noise ratio is improved, the intensity difference between specific fluorescence and non-specific matrix fluorescence is increased, the amplification factor determined by taking the matrix fluorescence as a standard cannot be observed, although the amplification factor is not changed, when the amplification factor is the same, the absolute value of the specific fluorescence intensity is increased, the amplified absolute value is also obviously increased, the intensity of the specific fluorescence intensity is far greater than the minimum detection limit of eyes, and the complete outline visualization of all cells can be realized. In particular, the rare single cells may be Circulating Tumor Cells (CTCs) or fetal-derived cells in maternal blood.

In one embodiment, in step S1, the rare single cell is CTC, the specific membrane protein EpCAM of CTC is used as a recognition receptor, the carboxyfluorescein (FAM) -labeled EpCAM aptamer (Apt-FAM) is used as a recognition ligand, the Apt-FAM specifically recognizes the membrane protein EpCAM, the Apt-FAM is localized on the cell membrane, and the labeled fluorescent FAM is far away from the cell surface and distributed in a large amount on the surface.

Further, the solvent of the cell sap I is distilled water or an aqueous solution such as PBS, DPBS and the like.

Further, the step S1 is preceded by an operation of fixing the rare single cell; through fixation, rare unicellular cell membrane markers (proteins) are fully exposed, so that ligands (such as antibodies and aptamers) can be recognized conveniently, and the cell membrane specific fluorescent staining effect is improved.

Specifically, fixing the rare single cells by using a fixing solution, wherein the fixing solution comprises acetone and a paraformaldehyde solution.

Further, the step S1 is preceded by a step of blocking non-specific sites of the cell membrane of the circulating tumor cell.

Specifically, the blocking of non-specific sites of the cell membrane of the circulating tumor cells is realized by adding a random base library.

Further, in order to locate the stained cell membrane to determine whether the stained cell membrane is outside the cell nucleus, the step S1 further includes a step of performing cell nucleus staining on the circulating tumor cell. Specifically, nuclear staining can be performed by Hoechst 33342.

Further, in step S2, the fluorescence-enhancing reagent is added directly to the cell fluid I (i.e., the fluorescence-enhancing reagent is added to the cell fluid I first and then transferred to a carrier for microscopic imaging), or is added dropwise to the cell fluid I on the carrier (i.e., the cell fluid I is transferred to the carrier first and then the fluorescence-enhancing reagent is added for microscopic imaging), and the carrier includes a slide, a culture dish, or various well plates.

Specifically, when the fluorescence enhancing reagent is an organic phase solution of a surfactant, both addition modes can be adopted, and when the fluorescence enhancing reagent is liquid organic alcohol, in order to improve the fluorescence enhancing effect, the preferable mode is the cell sap I dropwise added on the carrier. When the organic phase solution in which the fluorescence-enhancing reagent is a surfactant is added directly to the cell sap I, it is necessary to ensure that the solvent for the cell sap I is water or an aqueous solution (distilled water, PBS, DPBS, or the like is all acceptable).

Further, the surfactant is selected from a cationic surfactant, an anionic surfactant or a nonionic surfactant. Preferred are non-ionic surfactants of the Tween series, such as Tween-20, Tween-60 or Tween-80, more preferably Tween-80.

Further, the concentration of the surfactant in the cell sap II is 1-16mmol/L, preferably 7-14 mmol/L.

Specifically, the concentration of the surfactant in the organic phase solution of the surfactant is 3-100mmol/L, and the volume ratio of the organic phase solution of the surfactant to the cell sap I is 1: 3-6.

Further, the liquid organic alcohol is selected from methanol, ethanol, propanol or isopropanol.

Further, in step S3, the specific operation of the microscopic imaging may be as follows: taking a bright field picture; taking fluorescence photographs of nuclear staining and membrane staining under dark fields of ultraviolet light and blue light respectively; and overlapping the bright field picture and the fluorescence picture. During actual detection, only the film dyeing fluorescent photos under a bright field photo and a blue light dark field need to be taken and overlapped to judge whether the film dyeing is leakage-free or not and whether the sizes of the film outlines displayed at the same time are consistent or not.

Further, in step S4, the programmed program is used to convert the microscopic image into a digital image, and the digital image is subjected to specific digital signal amplification or non-specific digital signal amplification to obtain a visualized cell contour map.

Further, the non-specific digital signal amplification is to amplify all digital signals in the picture, and the amplification factor is based on the condition that the matrix fluorescence cannot be observed, and meanwhile, the cell outline is clearly visible.

Further, the specific digital signal amplification is to amplify the signal with the signal-to-noise ratio of more than 3, and the amplification factor is based on the clear visibility of the cell outline.

Compared with the prior art, the technical scheme of the invention has the following advantages:

according to the invention, the surfactant or liquid organic alcohol is adopted to enhance the specific fluorescence of the cell surface, so that the signal-to-noise ratio of a surface specific fluorescence signal is improved; by utilizing a digital signal amplification technology, the membrane fluorescence signal of the imaging cell is amplified, and the outline of the imaging cell is completely and clearly presented, so that the visualization of the specific identification outline of the single cell is realized, and the signal loss of the imaging cell is avoided; accuracy in capturing CTC counts and scale estimates can be achieved based on fluorescence visualized cell profiles.

Drawings

FIG. 1 is a schematic diagram of the fluorophore-induced formation of umbrella-shaped micelles on the surface of single cells before (left column) and after (right column) (row 1) in example 2, and SEM (rows 2 and 3) imaging.

FIG. 2 is a microscopic image (left) of single cells specifically recognized in example 2 and a photograph (right) of the cells with their digital signals amplified (scale: 10 μm).

FIG. 3 is a photograph of a single cell microscopic image (left) without fluorescence enhancement (top) and specific recognition of fluorescence enhancement of example 2 (bottom) and its digital signal amplification (right) (scale: 10 μm).

FIG. 4 is a photograph of single cell microscopic imaging (top) of Tween-20 (left), Tween-60 (center) and example 2 (right) with fluorescence enhancement and its digital signal amplification (bottom) (scale: 10 μm).

FIG. 5 is a photograph of a 7.5mmol/L (top) and 13mmol/L (bottom) Tween-80 fluorescence-enhanced single-cell microscopy (left) and its digital signal-amplified cell (right) (scale: 10 μm).

FIG. 6 is a photograph of a single cell microscopic image (left) without fluorescence enhancement (top) and specific recognition of fluorescence enhancement of example 5 (bottom) and its digital signal amplification (right) (scale: 10 μm).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The reagents and instrumentation in the following examples are as follows:

EpCAM aptamer (Apt-FAM, sequence 5 'FAM-T TTT TTT TTT CAC TACAGA GGT TGC GTC TGT CCC ACG TTG TCATGG GGG GTT GGC CTG-3', Shanghai Production); a random library of 48 bases (Shanghai Producer); pancreatin cell digestive juice, penicillin-streptomycin double antibody, culture medium RPMI-1640, fetal calf serum (Shanghai Biyuntian); MCF-7 human breast cancer cells (Suzhou university college of medicine), and other reagents were analytically pure. Inverted fluorescence microscope (WMF-3650, Shanghai Nemliki optical instruments Co., Ltd.).

Example 1: culture and digestion of cells

For convenience of example, single cells of very low density obtained by digestion and resuspension of cultured cells were used to mimic the very small amount of CTCs captured.

The breast cancer cell MCF-7 is placed in RPMI-1640 complete medium at 37 ℃ and 5% CO ₂ And (4) incubating. When the logarithmic phase is reached, the culture flask is taken out, the culture solution is discarded, 3mL of DPBS solution is added for cleaning, and the operation is repeated twice. Then, 1mL of trypsin was added to digest at 37 ℃ for 3min, the digest was discarded, 3mL of complete medium was added, and the adherent cells were blown with a pipette gun. And (3) sucking the cell suspension, adding the cell suspension into a 15mL centrifuge tube, centrifuging at 1500rpm/min for 3min, discarding the supernatant, and then resuspending with 3mL PBS (isotonic phosphate buffer solution) for later use.

Example 2: visualization of rare single cell identification profiles

1. Cell pretreatment

1mL of suspension (obtained in example 1) was centrifuged at 3000rpm/min for 3min in a brown centrifuge tube, the supernatant was discarded, 1mL of DPBS was used for resuspension, the supernatant was centrifuged again and discarded, 300. mu.L of acetone was added to resuspend the cells, and after 10min, the cells were centrifuged at 3000rpm/min for 3min and the supernatant was discarded, thus obtaining 3 tubes of fixed cells. Of these, 2 tubes were stored at 4 ℃ for later use. To the other fixed cell precipitation tube, 1mL of triple distilled water was added for resuspension, centrifugation, and the supernatant was discarded to wash off the residual acetone. Resuspending with 1mL of 1% Triton X-100 (polyethylene glycol octylphenyl ether) solution, centrifuging after 10min, discarding the supernatant, adding 1mL of triple-distilled water for resuspending, centrifuging, discarding the supernatant, and washing off the residual Triton X-100 to realize cell membrane permeabilization. Taking 20 mu L of triple distilled water to resuspend the cells, adding 10 mu L of 10 mu m/L base random library, adding 500 mu L of triple distilled water after 30min, centrifuging, and discarding the supernatant, so that the non-specific sites of the cell membranes are blocked, and the cells are reserved.

2. Specific cell membrane staining

And taking 20 mu L of triple-distilled water for resuspending the cells, adding 10 mu L of LApt-FAM aptamer solution (10 mu m/L) in the dark condition, uniformly blowing and beating, and dyeing for 30min, wherein the blowing and beating are carried out for 1 time every 5 min. Then, 500. mu.L of triple distilled water is added, the mixture is evenly blown and centrifuged, the supernatant is discarded, and 1mL of triple distilled water is used for resuspending the cells to obtain the cells with membrane specific staining. Add 8. mu.L of Hoechst 33342 in DMSO (2mg/mL), blow and mix well, spin and mix well for 15 min. Centrifuging, discarding the supernatant, resuspending in 1mL of triple-distilled water, repeating the operation for 3 times, washing off redundant Hoechst 33342 fluorescent dye, avoiding obvious basal fluorescence interference, and obtaining cell suspension for membrane staining and nuclear staining.

3. Fluorescence enhancement (surfactant) and microscopy (addition to cell suspension) for specific staining

The membrane-stained and nucleus-stained cell suspensions were centrifuged, and the supernatant was discarded. 500. mu.L of triple distilled water was quickly added dropwise, blown, the cells were resuspended, and 100. mu.L of LTween-80 in DMF (80mmol/L) was quickly added dropwise using a pipette. And then, blowing and beating the bottom of the test tube to quickly and uniformly mix the mixture, and standing for 20min to obtain the cell with enhanced membrane fluorescence. 100 mul of cell suspension is taken out and put on a glass slide, and the cell suspension is kept still for about 5min to ensure that the cells are settled on the glass slide. The cells were observed for morphological integrity in the bright field and a photograph taken in the bright field. Thereafter, fluorescence photographs of nuclear staining and membrane staining were taken under dark fields of violet light (exposure time 800ms) and blue light (exposure time 2000ms), respectively. And overlapping the three pictures by using ImageView software to obtain overlapped pictures of the detected cells.

4. Fluorescence signal amplification for cell membrane specific recognition (non-specific signal amplification)

Fluorescent photographs taken with a blue light dark field (fluorescent photographs of membrane specific staining) were programmed with the Matlab software itself to obtain digitized photographs. All digital signals in the digital picture are amplified by a certain factor, wherein the factor is based on the condition that the substrate fluorescence cannot be observed, and meanwhile, the cell outline is clearly visible, namely, a visual cell outline picture is obtained.

Example 3: visualization of rare single cell recognition profiles

Step 3 was replaced on the basis of example 2 by:

3' fluorescence enhancement of specific staining (absolute ethanol) and microscopy (addition to cell suspension)

The membrane-stained and nucleus-stained cell suspensions were centrifuged and the supernatant was discarded. 500. mu.L of absolute ethanol was added dropwise rapidly. And then, blowing and beating the bottom of the test tube to quickly and uniformly mix the mixture, and standing for 20min to obtain the cell with enhanced membrane fluorescence. 100 mul of cell suspension is taken out and put on a glass slide, and the cell suspension is kept still for about 5min to ensure that the cells are settled on the glass slide. The cells were observed for morphological integrity in the bright field and a photograph taken in the bright field. Thereafter, fluorescence photographs of nuclear staining and membrane staining were taken under dark fields of violet light (exposure time 800ms) and blue light (exposure time 2000ms), respectively. And overlapping the three pictures by using ImageView software to obtain overlapped pictures of the detected cells.

Example 4: visualization of rare single cell identification profiles

Step 3 was replaced on the basis of example 2 by:

3' fluorescence enhancement of specific staining (surfactant) and microscopy (addition of drops of cell fluid onto slides)

100 mu L of cell suspension liquid of membrane staining and nuclear staining is taken to be placed on a glass slide and kept still for about 5min, so that the cells are guaranteed to be settled on the glass slide. A solution of Tween-80 in DMF (80mmol/L) was added dropwise to the cell drops on the slides. Standing for 5-8min, observing whether the cell morphology is complete under a bright field, and taking a bright field picture. Thereafter, fluorescence photographs of nuclear staining and membrane staining were taken under dark fields of violet light (exposure time 800ms) and blue light (exposure time 2000ms), respectively. And overlapping the three pictures by ImageView software to obtain overlapped pictures of the detected cells.

Example 5: visualization of rare single cell recognition profiles

Step 3 was replaced on the basis of example 2 by:

3' fluorescence enhancement of specific staining (absolute ethanol) and microscopy (addition of drops of cell fluid onto slides)

100 mu L of cell suspension liquid of membrane staining and nuclear staining is taken to be placed on a glass slide and kept still for about 5min, so that the cells are guaranteed to be settled on the glass slide. Anhydrous ethanol is dripped into the cell drops on the glass slide. Standing for 5-8min, observing whether the cell morphology is complete under a bright field, and taking a bright field picture. Thereafter, fluorescence photographs of nuclear staining and membrane staining were taken under dark fields of violet light (exposure time 800ms) and blue light (exposure time 2000ms), respectively. And overlapping the three pictures by ImageView software to obtain overlapped pictures of the detected cells.

Example 6: visualization of rare single cell identification profiles

Step 4 is replaced by step 2

4. Fluorescent signal amplification for cell membrane specific recognition (specific signal amplification)

Fluorescent photographs taken with a blue light dark field (fluorescent photographs of membrane specific staining) were programmed with the Matlab software itself to obtain digitized photographs. And amplifying the signal with the signal-to-noise ratio of more than 3 in the digital picture, wherein the amplification factor is based on the condition that the cell contour is clearly visible, namely obtaining a visual cell contour map.

Analysis of results

1. As shown in FIG. 1, the fluorescence enhancement principle of single cell surface recognition in example 2 was analyzed, and it can be seen that:

because the specific protein EpCAM is over-expressed on the surface of the tumor cell membrane, such as human breast cancer cell MCF-7, when a fluorescence-labeled EpCAM ligand, such as a FAM-labeled aptamer FAM-Apt, is contacted with the tumor cell, the EpCAM ligand can specifically identify a large number of EpCAM receptors on the cell membrane, and a large number of distributed receptor-ligand complexes EpCAM (Apt-FAM) are formed on the cell membrane, so that a large number of labeled fluorescence FAMs are distributed on the surface of the cell membrane, and the displayed membrane fluorescence profile is the tumor cell profile in a fluorescence field, as shown in the left diagram of the first row in FIG. 1. Because Tween is a nonionic surfactant and has a non-ionized hydrophilic end and a non-ionized hydrophobic end, when the Tween is contacted with a receptor-ligand complex EpCAM (Apt-FAM) on a cell membrane, the Tween cannot be accumulated on the cell membrane due to charge interaction, and the hydrophobic end of the Tween can be subjected to 2 kinds of accumulation on the cell due to hydrophobic interaction. First, hydrophobic chains of Tween lay flat on the membrane due to the hydrophobicity of the cell membrane, which do not interact with the fluorescent FAM; second, when Tween comes into contact with the fluorophore of the FAM molecule in the receptor-ligand complex EpCAM. Apt-FAM, the hydrophobicity of the fluorophore will induce the hydrophobic ends of Tween to accumulate centered on the FAM due to hydrophobic interactions, while FAM labels several tens of bases of aptamers (e.g., 58 bases in this application), which will keep the FAM away from the cell membrane. If the labeled recognition ligand is an antibody, the FAM is also kept away from the cell membrane by the inter-arm action of the antibody macromolecules. This in turn provides enough room for the accumulation of Tween to form an umbrella-shaped micelle around the FAM, as shown in the right-hand first row of FIG. 1. Because the umbrella-shaped micelle is buckled on the cell membrane, the membrane protein below the umbrella-shaped micelle just seals the opening of the umbrella-shaped micelle, a hydrophobic microenvironment is constructed for the fluorescent body FAM in the umbrella-shaped micelle, the spherical micelle also shows the property of the common spherical micelle for enhancing the fluorescence, which is completely composed of the surfactant, and the enhancement of the specific recognition fluorescence of the cell membrane is realized. From SEM characterization, umbrella-shaped micelle nanoparticles can be observed on the cell surface of the MCF-7 with Tween enhanced specific fluorescence (the right row 3 in FIG. 1), and a large number of surface nanoparticles are observed (the right row 2 in FIG. 1); for cells without specific fluorescence enhanced by Tween, the surface contour is clear, and umbrella-shaped micelle nanoparticles are not found (lines 2 and 3 in FIG. 1), thereby indicating that the umbrella-shaped micelle nanoparticles are formed by Tween.

2. As shown in fig. 2, the single cell microscopic imaging (fluorescence) of specific recognition in comparative example 2 and the cell photograph of digital signal amplification thereof revealed that:

since each specific recognition site on the surface of the CTC membrane can only form a surface ligand-acceptor fluorescent complex EpCAM · Apt-FAM with one Apt-FAM, even after the fluorescence is enhanced by the umbrella-shaped micelles formed by Tween-80, it often appears as weak fluorescence under a fluorescence microscope, plus the heterogeneity of the cell suspension, which is generally weakly fluorescent, incompletely outlined cell outlines under a microscope (fig. 2 left). The inevitable resulting number of CTCs in the microscopic examination, which is less, or even significantly less, than the number of CTCs captured, may be a significant factor in the false negatives. For specifically identified single cell microscopic imaging (fig. 2 left), it can be found that the FAM fluorescence (actually green) of the cells is weak, a small number of cells can observe a complete brighter green circle on the scale of about 10 μm, i.e. a visualized complete cell outline, more are circles with weak brightness, even incomplete cell outlines with weak brightness (cell imaging within a rectangular box in the figure), and cells that cannot be effectively observed (cell imaging within a circle in the figure). When the digital signal is amplified (right in fig. 2), the brightness of all cells is significantly increased, and the cell profile of about 10 μm size is completely visualized. In particular, 3 cells that cannot be effectively observed (cells within a circle in the left image of fig. 2) also enable visualization of the complete contour, which provides an idealized cytogram for accurate counting of imaged cells and cell scale estimation based on the complete contour. Of course, in the digital signal-amplified photograph, a large number of high-luminance noise spots appear. Because the digital signal amplification is used for amplifying all signals of the microscopic imaging, including fluorescence signals, background signals and miscellaneous point signals of cells, by the same magnification factor, namely nonspecific signal amplification, the brightness of the background signals and the miscellaneous point signals is obviously increased. However, this does not affect the count and scale estimation of the imaged cells. Because the green circles of cells are relatively uniform in size and much larger than the high brightness noise spot, the background signal is usually very weak, and can not be observed effectively by selecting the signal amplification factor to make it weaker than the detection limit.

3. As shown in fig. 3, comparing example 2 with single cell microscopic imaging (fluorescence) without fluorescence enhanced specific recognition and its digital signal amplified cell photograph, it can be seen that:

the surfactant Tween enhances the fluorescence of the labeled fluorescent molecule FAM on the ligand-receptor complex EpCAM · Apt-FAM on the cell surface, which is a specific fluorescence enhancement that will increase the difference between the cell fluorescence and the background fluorescence, thereby increasing the signal-to-noise ratio of the imaging detection, which is a prerequisite for whether digital signal amplification can visualize the complete contour, see fig. 3. It was found that without the Tween enhancement to specifically recognize fluorescence (left in FIG. 3), only 1 cell with very weak fluorescence and incomplete profile was visible, and after signal amplification (right in FIG. 3), the complete profile of this cell could be visualized, while the other cells did not visualize their fluorescence profile, see the white circle with a scale of about 10 μm in the box of the graph (no observed green fluorescence of FAM). After the Tween enhanced specific recognition fluorescence is carried out (the lower left part of the figure 3), the visible fluorescent cells with complete outlines are obviously increased, but the fluorescent signals are still weaker, and the brightness of the cell outlines is still lower; at the same time, there are still cells with very weak fluorescence, incomplete contours (2 cells within the circle). After signal amplification, the fluorescence intensity of the cells is increased significantly, all cells show a high-intensity, visualized and complete cell outline, and no cells that can not be visualized by fluorescence exist (lower right of fig. 3).

4. As shown in FIG. 4, when Tween-80 in example 2 was replaced with Tween-20 and Tween-60, respectively, it was found that: the latter two have extremely weak cellular fluorescence, and after signal amplification, some of the cells still have fluorescence that cannot be observed (white circles in the lower left and middle panels of FIG. 4, no observed green fluorescence of FAM), and Tween-80 enhanced cellular fluorescence after signal amplification, all fluorescent cells can be observed "clearly" (lower right in FIG. 4), and therefore Tween-80 is preferred.

5. As shown in FIG. 5, by adjusting the concentration of Tween-80 in example 2, it was found that the fluorescence signal was stronger as the concentration of Tween-80 in the cell fluid was increased; the fluorescence of the cells in the image of 7.5mmol/L (Tween-80 concentration in the cell fluid) is very weak (upper left of FIG. 5), while the fluorescence of the cells at 13mmol/L is significantly stronger than the former (lower left of FIG. 5), and when the concentration is increased, turbidity appears in the cell suspension, which is caused by poor solubility of Tween-80 in the aqueous solution. In the imaging of 13mmol/L enhanced fluorescence all cells could be observed "clearly" (lower right in FIG. 5), while in the imaging of 7.5mmol/L enhanced fluorescence still some cells could not be observed (upper right in FIG. 5)

6. As shown in FIG. 6, when the cell membrane fluorescence specifically recognized was not enhanced with absolute ethanol (upper left), the cell fluorescence was weak, and there was very weak cell fluorescence (shown by a square in the figure) which was difficult to observe, or even fluorescence which was almost not observed (shown by a circle in the figure). After digital amplification of the signal (top right), most of the cell profile can be clearly and completely observed, but cellular fluorescence, which was not substantially observed before amplification, can now be observed, but the cell profile is still incomplete. When the absolute ethanol enhancement was performed (lower left), the cell fluorescence was significantly enhanced, and after digital signal amplification, the contours of all cells were completely represented and clearly visible.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (10)

1. A method for visualizing rare single-cell identification profiles, comprising the steps of,

s1: performing cell membrane fluorescent staining for specific recognition on a cell membrane marker of a rare single cell to obtain a cell sap I with a solvent of water or a water solution;

s2: adding a fluorescence enhancement reagent into the cell sap I to obtain cell sap II; the fluorescence enhancement reagent is an organic phase solution of a surfactant or liquid organic alcohol;

s3: performing microscopic imaging on the cell sap II;

s4: and converting the photo of the microscopic imaging into a digital photo, and amplifying the digital signal in the digital photo to obtain a visualized cell contour map.

2. The method for visualizing the rare single-cell identification profile of claim 1, wherein said step S1 is preceded by an operation of immobilizing said rare single cell.

3. The method for visualizing the recognition profile of a rare single cell as in claim 1, wherein said step S1 is preceded by an operation of blocking non-specific sites of the cell membrane of said rare single cell.

4. The method for visualizing the rare single-cell recognition profile of claim 1, wherein in step S2, the fluorescence enhancing reagent is added directly to the cytosol I or added dropwise to the cytosol I on a carrier.

5. The method for visualization of a rare single cell recognition profile of claim 1, wherein the surfactant is selected from the group consisting of cationic, anionic, or nonionic surfactants.

6. The method for visualization of a rare single-cell recognition profile of claim 1, wherein the concentration of surfactant in cytosol II is 1-16 mmol/L.

7. The method for visualization of a rare single cell identification profile of claim 1, wherein the liquid organic alcohol is selected from methanol, ethanol, propanol, or isopropanol.

8. The method for visualizing the rare single-cell recognition profile of claim 1, wherein in step S4, the programmed program is used to convert the micro-imaged picture into a digital picture, and the digital picture is subjected to specific digital signal amplification or non-specific digital signal amplification.

9. The method for visualization of the rare single cell recognition profile of claim 8, wherein the non-specific digital signal amplification is amplification of all digital signals in the photograph at a magnification that is standard for not allowing the substrate fluorescence to be observed, while allowing the cell profile to be clearly visible.

10. The method for visualization of rare single cell identification profiles of claim 8 wherein said specific digital signal amplification is a signal with a signal to noise ratio greater than 3, with magnification on the basis of clear visualization of cell contours.

Images