CN113358615A

CN113358615A - Application of melane and fluorescein sodium double staining method in living cell imaging

Info

Publication number: CN113358615A
Application number: CN202110608215.1A
Authority: CN
Inventors: 张慧敏
Original assignee: Individual
Current assignee: Zhang Huimin
Priority date: 2021-06-01
Filing date: 2021-06-01
Publication date: 2021-09-07
Anticipated expiration: 2041-06-01
Also published as: CN113358615B

Abstract

The invention provides a fluorescent staining method for imaging living cells, which is characterized in that cells are doubly stained by sodium fluorescein and methylene blue, clear fluorescent cell images are detected and displayed by a fluorescent microscope, and the cell morphology and the cell nucleus morphology can be observed by the acquired images.

Description

Application of melane and fluorescein sodium double staining method in living cell imaging

Technical Field

The invention relates to a cell staining method, in particular to a method for double staining by using a fluorescent biomarker, belonging to the technical field of biology.

Background

The combination of fluorescence staining method and fluorescence microscopy makes fluorescence imaging widely used in bioactive substance detection and cell imaging, wherein the cell imaging technology is an important research means in the field of life science technology. Compared with other technologies, fluorescent staining has the advantages of high sensitivity, high selectivity, simple operation, sensitive reaction and the like, and is a highly sensitive visual analysis technology widely applied to living cell analysis at present.

Fluorescence is a cold luminescence phenomenon known as "photoluminescence". When a certain normal temperature substance is irradiated by incident light with a specific wavelength, the substance enters an excited state after absorbing light energy, and emergent light with the property longer than the wavelength of the incident light is emitted immediately, and the emergent light with the property is called fluorescence.

The fluorescence spectrum includes both an excitation spectrum and an emission spectrum. The excitation spectrum refers to the relationship between a certain emission spectrum line of a fluorescent substance and the intensity of a spectrum band or the luminous efficiency of the substance and the wavelength of excitation light under the excitation of different wavelengths of light; the emission spectrum refers to the intensity change of the luminous intensity of different wavelengths of a fluorescent substance under the excitation of certain exciting light. Each fluorescent substance has an excitation spectrum and an emission spectrum, and its optimum excitation band and emission band. The current research on fluorescence is mainly to study the excitation spectrum and emission spectrum of fluorescent substances to find their optimum excitation band and emission band, such as 5-aminolevulinic acid (5-ALA) which is widely used clinically.

Fluorescence detection of malignant lesions based on 5-ALA is currently applied to brain surgery, urology surgery and gastrointestinal surgery in clinic, wherein fluorescence detection of 5-ALA-induced protoporphyrin IX (PP IX) is recently a promising intraoperative method for detecting malignant lesions. In order to improve the detection accuracy of 5-ALA fluorescence under strong autofluorescence conditions, several spectroscopic methods are available (Valdes, P.A. et al (2011) neurosurg.115, 11-17; Xu, H. & Rice, B.W. (2009) Journal of biological optics 14,064011; Harada, K.et al. (2013) International Journal of Molecular Sciences 14, 23140-23152; Koizumi, N.et al. (2013) Ann.Surg.Oncol.20, 3541-3548; Kondo, Y.et al. (2014) int.J.Oncol.45, 41-46). Although some studies report the effectiveness of the 5-ALA fluorescence detection method in clinical applications, detection errors often occur due to the strong background of autofluorescence of chromophores.

Some fluorescent biomarkers have been studied, and in addition to the above-mentioned 5-ALA, sodium Fluorescein (FS) and indocyanine green (ICG) are currently used more widely.

Fluorescein sodium (Fluorescein) is a living fluorescent dye, and an aqueous solution thereof can mark the cell soma outline for improving the visualization of tumor tissues, and has no specificity to tumor cells. When excited by light with the wavelength of 460-690 nm, the dye emits fluorescence radiation with the wavelength of 540-690 nm, and has wide application in medicine, particularly in brain tumor surgery (Copeman SM, Coke F, Gouldesbrough C., Br Med J. (1929)2: 233-42; Hamameoglu MK. et al., Clin neuro (2016)143: 39-45; O' goshi K, Serup J., Ski Res Technol. (2006)12: 155-61; Koc K. et al., Br J neuro g (2008)22: 99-103; Hara T.et al., Am J Ophthalmol (1998)126: 560-4; Kuroil T.T.1998) Neurg et al (1998) 50-8).

Indocyanine green (ICG) is an amphiphilic small molecule (<800 daltons), a near infrared spectroscopy (NIR) fluorophore (peak excitation 805nm, peak emission 835nm), which, when injected intravenously, is usually left in the blood vessel, mainly in association with albumin and other plasma proteins, and near infrared imaging is performed to delineate the vasculature. In recent years, the study of ICG in tumor tissue of rats and human patients has also found that ICG accumulates in tumors, with significant contrast between tumor and background, and that ICG has proven to be of practical value in labeling tumor tissue (Cho SS. et al, (2019) front. Surg.6: 11; Hansen DA. et al, Surg neuron. (1993)40: 451-6; Haglund MM. et al, neurosurger. (1994)35: 930-40; Haglund MM.. et al, neurosurger. (1996)38: 308-17; Madajewski B. et al, Clin Cancer Res. (2012: 5741-51; 30.Jiang JX., Im J. cl. Mol. aging (635: 20152; PLD.: 034: 8252).

Methylene Blue (MB), also known as Methylene Blue, is a non-toxic alternative dye approved by the U.S. food and drug administration for the treatment of methemoglobinemia. Methylene blue is another near infrared fluorophore currently available in human clinical trials in addition to ICG, which has a peak emission at 700nm and fluoresces in a different near infrared band than sodium fluorescein and ICG.

Despite the extensive research and application of fluorescent dyes, the staining effect of these fluorescent dyes is still deficient, for example, sodium fluorescein and ICG are not specific; 5ALA is more specific than fluorescein sodium, but it is not as sensitive and weakly contrasted with the surrounding normal tissue (Okuda T.et al, J Clin Neurosci (2012)19: 1719-22; Acerbi F.et al, Neurosurg.Focus (2014)36: E5; Okuda T.et al, J Clin Neurosci (2010)17: 118-; several studies have attempted dual injection of 5-ALA and fluorescein, enhancing detection of tumor tissue by increasing the contrast between tumor tissue that uptake 5-ALA and peritumoral regions that uptake fluorescein (Suero Molina e.et al, J Neurosurg. (2018)128: 399-.

The fluorescence staining agent is widely applied in clinic, and a fluorescence imaging is formed by the absorption and conversion of the cell mass to the staining agent and the combination of the cold luminescence phenomenon of fluorescence photoluminescence, so that the observation of the whole tissue is facilitated. When live cells are observed in a fluorescence microscope, these stains are only absorbed by the cell body or the cell nucleus when they are absorbed by a single cell. When living cells are subjected to fluorescent staining and fluorescent microscopic observation, the nuclear morphology can be observed but cannot be observed by acridine yellow staining imaging, and the nuclear morphology can be observed but cannot be observed by fluorescein sodium staining imaging. When microscopic observation is carried out, only the nuclear morphology or the cell morphology can be observed, and the requirement that the nuclear morphology and the cell morphology need to be observed simultaneously when the cells are observed is not met.

In summary, the current fluorescence staining method commonly used for living cell imaging analysis has the disadvantages of cell fixation, tedious use process, long staining process time, no specificity, insufficient contrast, incapability of clearly observing cell morphology and cell nucleus morphology simultaneously and the like in practical application. Therefore, improving the selectivity and accuracy of visualization analysis is a problem that needs to be solved urgently at present.

Disclosure of Invention

The main object of the present invention is to provide a novel staining method for double staining of living cells with fluorescent biomarkers to overcome the disadvantages of the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a method for staining living cells, which comprises the following steps: simultaneously or separately (i) staining the target cells with a first fluorescent biomarker; (ii) staining the target cells with a second fluorescent biomarker; (iii) and acquiring a fluorescence image of the target cell by using a fluorescence microscope, wherein the cell morphology can be observed simultaneously with the nucleus morphology.

Preferably, the first fluorescent biomarker has an excitation wavelength of 460-800 nm.

Preferably, the second fluorescent biomarker has an excitation wavelength of 350-670 nm.

Preferably, the emission spectrum maximum of the second fluorescent biomarker differs from the emission spectrum maximum of the first fluorescent biomarker by at least 50 nm.

Preferably, when a fluorescence image of the target cell is acquired using a fluorescence microscope, the first fluorescent biomarker emits light while the second fluorescent biomarker absorbs light.

Preferably, the first fluorescent biomarker is selected from the group consisting of sodium fluorescein, 5-aminolevulinic acid and indocyanine green.

Preferably, the second fluorescent biomarker is selected from the group consisting of methylene blue, acridine yellow and crystal violet.

Preferably, the first fluorescent biomarker is sodium fluorescein, and the second fluorescent biomarker is methylene blue.

Preferably, the concentration of the fluorescein sodium is 0.1% -1%, and the concentration of the methylene blue is 0.5% -3%.

More preferably, the concentration of sodium fluorescein is 0.25% and the concentration of methylene blue is 1%.

Preferably, the first fluorescent biomarker is 5-aminolevulinic acid, and the second fluorescent biomarker is acridine yellow.

More preferably, the concentration of 5-aminolevulinic acid is 0.05%, and the concentration of acridine yellow is 1%.

Preferably, the first fluorescent biomarker is indocyanine green, and the second fluorescent biomarker is crystal violet.

More preferably, the concentration of indocyanine green is 0.05%, and the concentration of crystal violet is 0.05%.

Preferably, the method is used for vital tissue staining.

Preferably, the living cells referred to in the present invention comprise cancer cells.

More preferably, the first fluorescent biomarker and the second fluorescent biomarker are administered to the affected area prior to performing the surgical procedure.

More preferably, the surgery is cancer surgery.

In another embodiment, the present invention also provides a composition for staining living cells, the composition comprising a first fluorescent biomarker and a second fluorescent biomarker.

More preferably, the composition further comprises a stabilizer, an antioxidant, a protective agent, a preservative, and a pH adjuster.

The compositions of the present invention are also formulated with one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active agents into preparations which can be used pharmaceutically. The appropriate formulation depends on the route of administration selected.

The stabilizer can be one or more selected from sorbitol ester, polyoxyethylene hydrogenated castor oil, and polyvinyl alcohol.

The antioxidant can be one or more selected from sodium sulfite, potassium sulfite, sodium sulfate, potassium sulfate, citric acid, dibutyl hydroxy toluene, tert-butyl hydroquinone, and citric acid.

The protective agent can be one or more selected from hydroxypropyl methylcellulose, medical sodium hyaluronate, polyacrylamide, carbomer, xylitol, glucose and alkyl glycoside.

The pH regulator may be one or more selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethanolamine, hydrochloric acid and phosphoric acid.

The preservative can be one or more of benzalkonium chloride, benzalkonium bromide, chlorobutanol and sorbitol.

The aforementioned compositions of the present invention may be sold by other alternative names, such as kits, kits or systems. The two substances in the composition can be mixed together and packaged or can be independently and separately packaged.

The invention discovers that the imaging effect can be obviously improved by carrying out imaging by a fluorescein sodium and methylene blue double-staining method, the fluorescein sodium improves the contrast with the background, and simultaneously methylene blue can well mark the cell nucleus, so that the cell nucleus can be observed clearly while the cell morphology is observed, and particularly important information such as the nuclear-to-cytoplasmic ratio change of tumor cells and the like can be observed clearly, so that normal tissues and tumor tissues can be judged clearly, the visualization of the boundary of tumor tissues and non-tumor tissues can be realized, and the clinical requirement can be met. And at present, both dyes are approved to be used clinically, and the safety is ensured.

In a preferred embodiment of the invention, the use of two different fluorescent wavelength dyes is exemplified. It is to be understood that three or more different stains may be used in combination for tissue staining as long as the targets they recognize are different from each other and have different fluorescence wavelengths.

Further, the fluorescent biomarker may also be selected from the following dyes: fluorescein Isothiocyanate (FITC), phycoerythrin (phyerythrin), phycocyanin (phycyanin), allophycocyanin (allophycocyanin), o-phthaldehyde (ophthadhehyde), rhodamine (rhodamine), and AlexaFluor series dyes, DAPI, Hoechst 33342 thiazole orange, acridine orange, and the like.

In practical application, the staining method of the invention can be applied not only to pathological tissue sections, but also to staining of living tissues and cells cultured in vitro, and can be used for direct contact staining of cells of tissues to be imaged, cells on microcarriers or cells on smears and the stain of the invention, and further applied to clinical applications such as surgery, diagnosis, drug delivery and the like.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a fluorescent biomarker double staining method for living cell imaging, which has the advantages of rapidness, high efficiency, safety and the like, can observe the cell morphology and the cell nucleus morphology simultaneously, can clearly distinguish normal cells and tumor cells, and has important significance for the development of the fields of tissue staining, cell morphology research, image-guided surgery and the like.

Drawings

FIG. 1: the mouse kidney is compared with the imaging effect of the Meilan single staining method, the fluorescein sodium single staining method and the Meilan-fluorescein sodium double staining method. Wherein FIG. 1A is the results of imaging at 470nm wavelength using sodium fluorescein staining alone; FIG. 1B is the result of imaging at 470nm wavelength using methylene blue staining alone; FIG. 1C is the results of imaging using methylene blue staining alone at a wavelength of 660 nm; FIG. 1D shows the results of fluorescein sodium and Meilan double staining at 470 nm; figure 1E is HE staining control.

FIG. 2: the imaging effect of mouse livers is compared by a single-use Meilan staining method, a single-use sodium fluorescein staining method and a Meilan-sodium fluorescein staining method. Wherein FIG. 2A is the results of imaging at 470nm wavelength using sodium fluorescein staining alone; FIG. 2B is the result of imaging at 470nm wavelength using methylene blue staining alone; FIG. 2C is the results of fluorescein sodium and Meilan double staining imaged at 470 nm; figure 2D is HE staining control.

FIG. 3: the pig kidneys were compared using double staining imaging results of different concentrations of fluorescein sodium and methylene blue. Wherein FIG. 3A is the results of imaging a 0.1% sodium fluorescein + 0.5% methylene combined stain at 470nm wavelength; FIG. 3B is the results of imaging of a 0.25% sodium fluorescein + 1% methylene blue combination stain at 470 nm; FIG. 3C is the results of imaging of a 0.5% sodium fluorescein + 2% methylene combined stain at 470nm wavelength; FIG. 3D is the results of combined staining with 1% sodium fluorescein + 3% methylene blue at 470 nm.

FIG. 4: imaging results of pig liver with 5-ALA and acridine yellow double staining.

FIG. 5: imaging of pig liver with dual staining of methylene blue and ICG.

FIG. 6: imaging results of pig kidneys using dual staining with sodium fluorescein and crystal violet.

Detailed Description

The present invention will be further described with reference to the following embodiments and drawings, and the present invention is not limited to the following embodiments. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The experimental procedures in the following examples, in which specific conditions are not specified, are all common knowledge and general knowledge of those skilled in the art, or conditions recommended by the manufacturer. All materials and reagents used in the examples are commercially available products unless otherwise specified.

Mouse

Mice were of the C57BL6 strain, purchased from the south large animal center.

Fluorescence image analysis

Fluorescence images were obtained by MCI microscopy (DiveScope), stained and visualized at 470nm, and randomly selected regions of interest were imaged.

Preparation of dyeing liquor

0.1% fluorescein sodium (florescein): weighing 0.01g of fluorescein sodium powder, filling the fluorescein sodium powder into a light-proof test tube, adding 10mL of physiological saline, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

0.25% fluorescein sodium (florescein): weighing 0.025g of fluorescein sodium powder, filling the fluorescein sodium powder into a light-proof test tube, adding 10mL of saline cleaning solution, shaking up, wrapping the prepared solution with tinfoil paper, and storing in a dark place.

0.5% fluorescein sodium (florescein): weighing 0.05g of fluorescein sodium powder, filling the fluorescein sodium powder into a light-proof test tube, adding 10mL of physiological saline, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

1% fluorescein sodium (florescein): weighing 0.1g of fluorescein sodium powder, filling the fluorescein sodium powder into a light-proof test tube, adding 10mL of physiological saline, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

0.5% Methylene blue (Methylene blue): weighing 0.05g of methylene blue powder, filling the methylene blue powder into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

1% Methylene blue (Methylene blue): weighing 0.1g of methylene blue powder, filling the methylene blue powder into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

2% Methylene blue (Methylene blue): weighing 0.2g of methylene blue powder, filling the methylene blue powder into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

3% Methylene blue (Methylene blue): weighing 0.3g of methylene blue powder, filling the methylene blue powder into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking up, wrapping the prepared solution with tinfoil paper, and storing in the dark.

0.05% 5-ALA (5-aminolevulinic acid): weighing 0.005g of 5-ALA powder, filling into a light-proof test tube, adding 10mL of 5% glucose solution, shaking uniformly, wrapping the prepared solution with tinfoil paper, and storing in the dark.

1% acridine yellow (acriflavine): weighing 0.1g of acridine yellow powder, filling the acridine yellow powder into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and storing the solution in the dark.

0.05% ICG (indocyanine-green): weighing 0.005g of ICG powder, filling the ICG powder into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and storing the solution in the dark.

0.05% crystal violet (crystal violet): weighing 0.005g of crystal violet powder, filling the crystal violet powder into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and storing the solution in the dark.

Example 1: tissue staining by fluorescein sodium and methylene blue double staining method

1.1 staining of mouse Kidney

Mice were used for tissue staining, the staining procedure was as follows:

1. mice were anesthetized with 1% sodium pentobarbital by intraperitoneal injection at a dose of 8-9 mL/g.

2. The body hair on the back side of the mouse is removed, the epidermis is cut open, the kidney is exposed, the kidney is completely cut out and taken out, the surface of the kidney is fixed by a blade, and the surface capsule of the kidney is removed by scissors and tweezers under a microscope.

3. Stopping bleeding with cotton swab, applying 0.25% fluorescein sodium to the surface of kidney for 2 min, and washing with normal saline for three times; then, the surface was stained with 1% methylene blue staining solution for 2 minutes, washed three times with physiological saline after the lapse of time, and then observed under a microscope at a wavelength of 470 nm.

The results in fig. 1 show the comparison of the imaging effect of mouse kidney in melan staining alone, fluorescein sodium staining alone and melan-fluorescein sodium double staining. Wherein FIG. 1A is the result of imaging a stain with sodium fluorescein alone at a wavelength of 470 nm; FIG. 1B is the result of imaging of stain using methylene blue alone at 470nm wavelength, it can be seen that methylene blue is not imaged at 470nm wavelength; FIG. 1C is the result of imaging of staining with methylene blue alone at a wavelength of 660 nm; FIG. 1D shows the result of imaging the double staining of fluorescein sodium and Melland at 470nm, which clearly shows the tubular cell contour and the nuclear morphology of mice double stained with fluorescein sodium and Melland; figure 1E is HE staining control.

1.2 mouse liver staining

Mice were used for tissue staining, the staining procedure was as follows:

2. The abdominal hair of the mouse was removed, the epidermis was cut open, the liver was exposed, and the liver was completely excised and removed, and then the liver surface was fixed with a razor blade.

3. After the liver surface was stained with 0.25% fluorescein sodium for 2 minutes, it was washed three times with physiological saline; then, the surface was stained with 1% methylene blue staining solution for 2 minutes, washed three times with physiological saline after the lapse of time, and then observed under a microscope at a wavelength of 470 nm.

FIG. 2 is a graph comparing the effect of mouse liver imaging using Meilan staining alone with the dual sodium fluorescein and Meilan staining alone. Wherein FIG. 2A is the result of imaging a stain with sodium fluorescein alone at a wavelength of 470 nm; FIG. 2B is the result of imaging of stain using methylene blue alone at 470nm wavelength, where it can be seen that methylene blue is not imaged at 470nm wavelength; FIG. 2C shows the result of imaging the double-staining of fluorescein sodium and Melan at 470nm, which clearly shows the outline of the mouse liver cells and the morphology of the cell nucleus; figure 2D is HE staining control.

As can be seen from FIGS. 1 and 2, the double staining method using fluorescein sodium and methylene blue has a much improved imaging effect compared with the method using fluorescein sodium alone or methylene blue alone. The sodium fluorescein and methylene blue double staining method improves the contrast with the background, can clearly observe the tissue and cell morphology, and can also observe clear cell nucleus, even cell nucleolus. The information can help doctors judge tumor and non-tumor tissues, and brings more value to medical treatment.

Example 2: effect of double staining with different concentrations of fluorescein sodium and methylene blue

Tissue staining was performed using pig kidneys, and the staining procedure was as follows:

1. purchasing a plurality of fresh pig kidneys, and refrigerating the kidneys in a refrigerator at 4 ℃ for later use.

2. The fresh pig kidney is cleaned by clear water, and the surface capsule of the kidney is removed by scissors and tweezers under a microscope.

3. Respectively applying 0.1%, 0.25%, 0.5% and 1% fluorescein sodium on the surface of the kidney for one minute, and then cleaning the kidney with normal saline for three times; then, the surface of the specimen is coated and dyed by 0.5 percent, 1 percent, 2 percent and 3 percent methylene blue staining solution for one minute, and the specimen is washed by physiological saline three times after the time is up and then is observed under a microscope at 470nm wavelength.

FIG. 3 is a comparison of the imaging effect of porcine kidney using dual combination staining of different concentrations of fluorescein sodium and methylene blue. Wherein FIG. 3A is the results of imaging a 0.1% sodium fluorescein + 0.5% methylene combined stain at 470nm wavelength; FIG. 3B is the results of imaging of a 0.25% sodium fluorescein + 1% methylene blue combination stain at 470 nm; FIG. 3C is the results of imaging of a 0.5% sodium fluorescein + 2% methylene combined stain at 470nm wavelength; FIG. 3D is the results of combined staining with 1% sodium fluorescein + 3% methylene blue at 470 nm.

As can be seen from FIG. 3, the combination of sodium fluorescein and methylene blue in different concentrations, wherein the concentration of sodium fluorescein is between 0.1% and 1%, and the concentration of methylene blue is between 0.5% and 3%, the staining effect of the combination is similar between the concentrations, and the combination within the concentration range can clearly distinguish the cell morphology and the cell nucleus structure.

Example 3: tissue staining by 5-ALA and acridine yellow double staining method

Tissue staining was performed using pig liver, and the staining procedure was as follows:

1. purchasing a plurality of fresh pig livers, and refrigerating the fresh pig livers in a refrigerator at 4 ℃ for later use.

2. The fresh pig liver is cleaned by clear water, and the envelope on the surface of the liver is removed by scissors and tweezers under a microscope.

3. After the liver surface is applied and stained with 0.05 percent of 5-ALA for 3 minutes, the liver surface is washed with physiological saline for three times; then, the surface of the sample was applied with 1% acridine yellow staining solution for 2 minutes, and after the application of the staining solution, the sample was washed three times with physiological saline and then observed under a microscope at a wavelength of 635 nm.

FIG. 4 is an image of pig liver with double staining of 5-ALA and acridine yellow, which clearly shows the outline of the stained liver cells and the morphology of the nuclei.

Example 4: tissue staining by the Meilan and ICG double staining method

3. After the liver surface is applied and stained with 1% methylene blue for 2 minutes, the liver surface is washed with physiological saline for three times; the surface was then stained with 0.05% ICG staining solution for 2 minutes, washed three times with physiological saline after the lapse of time, and then observed under a microscope at a wavelength of 835 nm.

FIG. 5 is an image of porcine kidney using a double staining of Meilan and ICG, where the stained liver cell outline and the morphology of the nuclei can be clearly seen.

Example 5: tissue staining by fluorescein sodium and crystal violet double staining method

3. After the surface of the kidney is coated and dyed for 2 minutes by 0.25 percent of fluorescein sodium, the kidney is washed three times by normal saline; then, the surface was stained with 0.05% crystal violet staining solution for 3 minutes, washed three times with physiological saline after the lapse of time, and then observed under a microscope at a wavelength of 525 nm.

Fig. 6 is an image of pig kidney double-stained with fluorescein sodium and crystal violet, wherein the stained liver cell outline and the morphology of the cell nucleus can be clearly seen.

All documents referred to herein are incorporated by reference in their entirety. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above teachings of the present invention, and such equivalent modifications also fall within the scope of the appended claims.

Claims

1. A staining method for imaging living cells, characterized in that it comprises the following steps: simultaneously or separately (i) staining the target cells with sodium fluorescein; (ii) staining the target cells with methylene blue; (iii) acquiring a fluorescence image of the target cell by using a fluorescence microscope, wherein the method can observe cell morphology and cell nucleus morphology simultaneously; wherein the concentration of the fluorescein sodium is 0.1-1%, and the concentration of the methylene blue is 0.5-3%.

2. The method of claim 1, wherein the concentration of sodium fluorescein is 0.25% and the concentration of methylene is 1%.

3. The method of claim 1, wherein the method is used for vital tissue staining.

4. The method of claim 1, wherein the living cells comprise cancer cells.

5. A composition for staining living cells, comprising a first fluorescent biomarker of claim 1 and a second fluorescent biomarker.

6. The composition of claim 5, wherein the first fluorescent biomarker is sodium fluorescein and the second fluorescent biomarker is methylene blue.

7. The composition of claim 6, wherein the concentration of sodium fluorescein is 0.1% -1% and the concentration of methylene blue is 0.5% -3%.

8. The composition of claim 7, wherein the concentration of sodium fluorescein is 0.25% and the concentration of methylene is 1%.

9. The composition of claim 5, wherein the composition further comprises stabilizers, antioxidants, protectants, preservatives, pH adjusting agents.