CN113358615B - Application of Melan and sodium fluorescein double staining method in living cell imaging - Google Patents
Application of Melan and sodium fluorescein double staining method in living cell imaging Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention provides a fluorescence staining method for living cell imaging, which is characterized in that cells are doubly stained by fluorescein sodium and merland, a fluorescence microscope is used for detecting and displaying clear fluorescent cell images, and the obtained images can observe cell morphology and cell nucleus morphology at the same time.
Description
Technical Field
The invention relates to a cell staining method, in particular to a method for double staining by using fluorescent biomarkers, and belongs to the technical field of biology.
Background
The combination of fluorescent staining methods with fluorescent microscopy has led to a wide application of fluorescent imaging in bioactive substance detection and cell imaging, which is an important research tool in the field of life sciences technology. Compared with other technologies, the fluorescent staining has the advantages of high sensitivity, high selectivity, simple operation, sensitive reaction and the like, and is a high-sensitivity visual analysis technology widely applied to living cell analysis at present.
Fluorescence is a cold luminescence phenomenon of "photoluminescence". When a certain normal temperature substance is irradiated with incident light of a specific wavelength, light energy is absorbed and enters an excited state, and emitted light longer than the wavelength of the incident light is emitted immediately, the emitted light having such a property is called fluorescence.
The fluorescence spectrum includes two kinds of excitation spectrum and emission spectrum. The excitation spectrum refers to the relation between the intensity of a certain luminous spectrum line and a band of a fluorescent substance or the luminous efficiency and the wavelength of excitation light under the excitation of light with different wavelengths; the emission spectrum refers to the intensity change of the luminous intensity of the fluorescent substance with different wavelengths under the excitation of a certain excitation light. Each fluorescent substance has an excitation spectrum and an emission spectrum, and its most suitable 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 most suitable excitation and emission bands, such as 5-aminolevulinic acid (5-ALA), which is widely used in clinic.
Fluorescence detection of malignant lesions based on 5-ALA is currently used clinically in brain surgery, urology surgery, gastrointestinal surgery, where 5-ALA-induced fluorescence detection of protoporphyrin IX (PP IX) has recently become a promising method for intraoperative detection of malignant lesions. In order to improve the detection accuracy of 5-ALA fluorescence under strong autofluorescence conditions, there are several spectroscopic analysis methods (Valdes, P.A. et al (2011) Neurosurg.115,11-17; xu, H. & Rice, B.W. (2009) Journal of biomedical 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 chromophore autofluorescence.
Some fluorescent biomarkers have been studied, and in addition to the above 5-ALA, sodium Fluorescein (FS) and indocyanine green (ICG) are currently used more widely.
Sodium Fluorescein (Fluorescein) is a vital fluorescent dye whose aqueous solution can mark the cell body contours for improving visualization of tumor tissue, and which is not specific for tumor cells. Such dyes, when excited by light having a wavelength in the range 460-500nm, emit fluorescent radiation having a wavelength in the range 540-690nm, have a wide range of medical applications, particularly in brain tumor surgery (Copeman SM, coke F, gouldesbrough C., br Med J. (1929) 2:233-42;Hamamcioglu MK.et al, clin Neurol Neurosurg (2016) 143:39-45;O'goshi K,Serup J, ski Res technology (2006) 12:155-61; koc K.et., br J Neurosurg (2008) 22:99-103; hara T.et., am J Ophthalmol. (1998) 126:560-4;Kuroiwa T.et al, surg Neurol (1998) 50:41-8).
Indocyanine green (ICG) is an amphiphilic small molecule (< 800 daltons), a near infrared spectroscopy (NIR) fluorophore (peak excitation = 805nm, peak emission = 835 nm) that when injected intravenously, typically remains in the blood vessel, binding mainly to albumin and other plasma proteins, near infrared imaging to delineate the vascular system. In recent years, ICG has been found to accumulate in tumors in rats and human patient tumor tissues, with a significant contrast between tumor and background, and has proven to be of practical value in marking tumor tissues (Cho SS.et al, (2019) front. Surg.6:11;Hansen DA et al, surg neurol (1993) 40:451-6;Haglund MM.et al, neuroberger (1994) 35:930-40; haglund MM, et al, neuroberger (1996) 38:308-17; madajewski B, et al, clin Cancer Res. (2012) 18:5741-51;30.Jiang JX., et al, am J Nucl Med Mol imaging (2015) 5:390-400; zeh R.et al, PLoS E. (2017) 12:0184).
Methylene Blue (MB), a dye approved by the U.S. food and drug administration for the treatment of methemoglobinemia, is a nontoxic alternative dye. Melan is currently another near infrared fluorophore available in human clinical trials other than ICG, which has a peak emission at 700nm, and fluoresces in a different near infrared band than sodium fluorescein and ICG.
Although the research and application of fluorescent dyes are very extensive at present, these fluorescent dyes still have drawbacks in their staining effect, for example, sodium fluorescein and ICG are not specific; 5ALA is more specific than fluorescein sodium, but is less sensitive, weakly contrasted with surrounding normal tissues (Okuda T.et al., (2012) 19:1719-22;Acerbi F.et al, neurosprig.focus. (2014) 36:E5;Okuda T.et al, J Clin neurospri. (2010) 17:118-121;35.Chen B.et al, int J Med Sci. (2012) 9:708-714;Francaviglia N.et al, surg Neurol Int. (2017) 8:145;Bowden SG.et al, neurosprerger. (2018) 82:719-27); several studies attempted double injection of 5-ALA and fluorescein, with enhanced detection of tumor tissue by increasing the contrast between tumor tissue uptake of 5-ALA and peri-tumor regions uptake of fluorescein (Suero Molina E.et al, J Neurosurg. (2018) 128:399-405), improved efficacy, but still insufficient to meet clinical needs.
The fluorescent dye is widely applied clinically, and a fluorescent imaging is formed by combining the cold luminescence phenomenon of fluorescence photoluminescence through the absorption and conversion of cell aggregates to the dye, so that the observation of the whole tissue is convenient. When living cells are observed by fluorescence microscopy, these stains are only absorbed by the cell body or by the nucleus, since they are currently absorbed by single cells. When living cells are subjected to fluorescent staining and fluorescent microscopic observation, the cell nucleus morphology can be observed through acridine yellow staining imaging, but the cell morphology cannot be observed, and the cell morphology can be observed through sodium fluorescein staining imaging, but the cell nucleus morphology cannot be observed. When microscopic observation is carried out, only the cell nucleus morphology or the cell morphology can be observed, and the requirement that the cell nucleus and the cell morphology are simultaneously required to be observed when the cell is observed is not met.
In summary, the fluorescent staining method commonly used for living cell imaging analysis at present has the defects that cells need to be fixed in practical application, the use process is complicated, the staining process is long in time, no specificity exists, the contrast ratio is insufficient, the cell morphology and the cell nucleus morphology cannot be clearly observed at the same time, and the like. Therefore, improving the selectivity and accuracy of visual analysis is a currently urgent need to be addressed.
Disclosure of Invention
The main object of the present invention is to provide a new staining method, which uses fluorescent biomarker to double stain living cells to overcome the defects in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
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) The fluorescence image of the target cells was obtained by a fluorescence microscope, and the method was capable of observing the morphology of the cell and the morphology of the nucleus at the same time.
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 50nm.
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 meran, acridine yellow and crystal violet.
Preferably, the first fluorescent biomarker is sodium fluorescein and the second fluorescent biomarker is methylene blue.
Preferably, the above-mentioned sodium fluorescein concentration is 0.1% -1% and the concentration of the merland is 0.5% -3%.
More preferably, the above-mentioned sodium fluorescein concentration is 0.25% and the concentration of the 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 indocyanine green concentration is 0.05%, and the crystal violet concentration is 0.05%.
Preferably, the method is used for in vivo 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 surgical procedure is a cancer surgical procedure.
In another embodiment, the invention also provides a composition for staining living cells, the composition comprising a first fluorescent biomarker and a second fluorescent biomarker.
Preferably, the second fluorescent biomarker is selected from the group consisting of meran, acridine yellow and crystal violet.
Preferably, the first fluorescent biomarker is sodium fluorescein and the second fluorescent biomarker is methylene blue.
Preferably, the above-mentioned sodium fluorescein concentration is 0.1% -1% and the concentration of the merland is 0.5% -3%.
More preferably, the above-mentioned sodium fluorescein concentration is 0.25% and the concentration of the 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 indocyanine green concentration is 0.05%, and the crystal violet concentration is 0.05%.
More preferably, the above composition further comprises a stabilizer, an antioxidant, a protectant, a preservative, 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 is 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 protecting 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 selected from benzalkonium chloride, benzalkonium bromide, chlorobutanol and sorbitol.
The aforementioned compositions of the present invention may be sold under alternative names, such as kits, or systems. The two materials in the composition can be mixed together for packaging, or can be independently and separately packaged.
The invention discovers that the imaging effect can be obviously improved by adopting the double-staining method of fluorescein sodium and methylene blue, the contrast ratio between the fluorescein sodium and the background is improved, and simultaneously, the methylene blue can well mark the cell nucleus, so that the important information such as the cell nucleus, especially the nuclear-to-cytoplasmic ratio change of tumor cells, can be clearly observed while the cell morphology is observed, and therefore, normal tissues and tumor tissues can be clearly judged, and the visualization of the boundaries of tumor and non-tumor tissues is achieved, so as to meet clinical requirements. And at present, both dyes are approved for clinical use, and the safety is ensured.
In a preferred embodiment of the invention, the use of two dyes of different fluorescence wavelengths is illustrated. It should be understood that three or more different dyes may be used in combination for tissue staining, provided that 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 (phycocyanohrin), phycocyanin (phycocyanocyanin), allophycocyanin (allophycocyanin), phthaldehyde (ophtalmide), rhodamine (rhodoamine), alexaFluor-series dye, DAPI, hoechst 33342 thiazole orange, acridine orange, etc.
In practical application, the staining method of the invention can be applied to pathological tissue sections, living tissues and cells cultured in vitro, and cells of tissues to be imaged, cells on microcarriers or cells on smears can be directly contacted with the dye of the invention for staining, so that the staining method is 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 at the same time, can clearly distinguish normal cells and tumor cells, and has important significance for development in the fields of tissue staining, cell morphology research, image-guided surgery and the like.
Drawings
Fig. 1: the kidneys of mice were aligned for imaging effects on the two-stain method using methylene blue alone, sodium fluorescein alone, and sodium methylene blue-sodium fluorescein alone. Wherein FIG. 1A is the imaging results at 470nm wavelength using sodium fluorescein staining alone; FIG. 1B is a graph showing imaging results at 470nm using Melan staining alone; FIG. 1C is the imaging results at 660nm using Melan staining alone; FIG. 1D is the results of imaging sodium fluorescein and Melan double staining at 470nm wavelength; FIG. 1E is a HE staining control.
Fig. 2: mouse livers were aligned for imaging effect on the two-stain method using methylene blue alone, sodium fluorescein alone, and sodium methylene blue-fluorescein alone. Wherein figure 2A is the imaging results at 470nm wavelength using sodium fluorescein staining alone; FIG. 2B is a graph showing imaging results at 470nm using Melan staining alone; FIG. 2C is the results of imaging sodium fluorescein and Melan double staining at 470nm wavelength; fig. 2D is a HE staining control.
Fig. 3: pig kidneys were compared using different concentrations of fluorescein sodium and Melan double staining imaging effects. Wherein FIG. 3A is the imaging result at 470nm for a 0.1% sodium fluorescein +0.5% merland combination stain; FIG. 3B is an imaging result of 0.25% sodium fluorescein+1% Melan combination staining at 470nm wavelength; FIG. 3C is a graph showing the imaging results of a 0.5% sodium fluorescein +2% merland combination stain at a wavelength of 470 nm; FIG. 3D is the imaging results of 1% sodium fluorescein+3% meran combinatorial staining at 470nm wavelength.
Fig. 4: imaging results of porcine liver using double staining with 5-ALA and acridine yellow.
Fig. 5: pig liver was imaged using double staining of meran and ICG.
Fig. 6: imaging results of porcine kidneys with double staining with sodium fluorescein and crystal violet.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, to which the invention is not limited. It is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Variations and advantages that will occur to those skilled in the art are included within the following claims and any equivalents thereof without departing from the spirit and scope of the inventive concept. 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 methods in the following examples, in which specific conditions are not specified, are common knowledge and common knowledge to those skilled in the art, or according to the conditions recommended by the manufacturer. All materials and reagents used in the examples were commercially available products unless otherwise specified.
A mouse
Mice were C57BL6 strain, purchased from the south China large animal center.
Fluorescence image analysis
Fluorescence images were obtained by MCI microscopy (DiveScope), staining the samples, and then observing at 470nm, randomly selecting the region of interest for imaging.
Dyeing liquid preparation
0.1% sodium fluorescein (florescein): weighing 0.01g of fluorescein sodium powder, loading into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
0.25% sodium fluorescein (florescein): weighing 0.025g of fluorescein sodium powder, loading into a light-proof test tube, adding 10mL of saline cleaning solution, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
0.5% sodium fluorescein (florescein): weighing 0.05g of fluorescein sodium powder, loading into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
1% sodium fluorescein (florescein): weighing 0.1g of fluorescein sodium powder, loading into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
0.5% Methylene blue: 0.05g of methylene blue powder is weighed and put into a light-proof test tube, 10mL of 5% sodium bicarbonate solution is added, the solution is uniformly shaken, and then the prepared solution is wrapped by tinfoil paper and stored in a light-proof state.
1% Methylene blue: weighing 0.1g of methylene blue powder, putting into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
2% Methylene blue: weighing 0.2g of methylene blue powder, putting into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
3% Methylene blue: weighing 0.3g of methylene blue powder, putting into a light-proof test tube, adding 10mL of 5% sodium bicarbonate solution, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
0.05%5-ALA (5-aminolevulinic acid): weighing 0.005g of 5-ALA powder, loading into a light-proof test tube, adding 10mL of 5% glucose solution, shaking, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
1% acridine yellow (acriflavine): weighing 0.1g of acridine yellow powder, loading into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
0.05% ICG (indocyanine-green): 0.005g of ICG powder is weighed, filled into a light-proof test tube, 10mL of physiological saline is added, and after shaking, the prepared solution is wrapped by tinfoil paper and stored in a light-proof state.
0.05% crystal violet (crystal violet): weighing 0.005g crystal violet powder, loading into a light-proof test tube, adding 10mL of physiological saline, shaking uniformly, wrapping the prepared solution with tinfoil paper, and preserving in light-proof.
Example 1: tissue staining by sodium fluorescein and Melan double staining
1.1 renal staining of mice
Tissue staining was performed using mice, the staining procedure being as follows:
1. mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital at an anesthetic dose of 8-9mL/g.
2. The body hair at the back of the mouse is removed, the epidermis is cut off, the kidney is exposed, the surface of the kidney is fixed by a blade after the kidney is completely resected and taken out, and the envelope on the surface of the kidney is removed by scissors and forceps under a microscope.
3. Hemostatic with cotton swab, applying 0.25% fluorescein sodium to kidney surface for 2 min, and cleaning with physiological saline for three times; then, the surface was stained with 1% methylene blue staining solution for 2 minutes, and after the lapse of time, the solution was washed three times with physiological saline, and then observed under a microscope at 470 nm.
The results of fig. 1 show that the kidneys of mice were aligned in the effect of staining with meran alone, staining with sodium fluorescein alone, and double staining with meran-sodium fluorescein. Wherein FIG. 1A is the result of imaging at 470nm wavelength using sodium fluorescein alone; FIG. 1B is a graph showing the results of imaging at 470nm using staining of the methylene blue alone, it being seen that the methylene blue cannot be imaged at 470 nm; FIG. 1C is a graph showing imaging results at 660nm using staining with methylene blue alone; FIG. 1D is a graph of the results of sodium fluorescein and Melan double staining at 470nm, clearly showing the outline of the mouse tubular cells and the morphology of the nuclei after double staining with sodium fluorescein and Melan; FIG. 1E is a HE staining control.
1.2 liver staining of mice
Tissue staining was performed using mice, the staining procedure being as follows:
1. mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital at an anesthetic dose of 8-9mL/g.
2. The abdominal body hair of the mice is removed, the epidermis is cut off, the liver is exposed, the liver is completely resected and taken out, and the surface of the liver is fixed by a blade.
3. The liver surface is stained with 0.25% fluorescein sodium for 2 minutes, and then washed three times with physiological saline; then, the surface was stained with 1% methylene blue staining solution for 2 minutes, and after the lapse of time, the solution was washed three times with physiological saline, and then observed under a microscope at 470 nm.
Figure 2 is an alignment of the imaging effect of mouse livers on the use of meran staining alone versus sodium fluorescein and meran double staining alone. Wherein figure 2A is the result of imaging at 470nm wavelength using sodium fluorescein alone; FIG. 2B is a graph showing the results of imaging at 470nm using staining of the methylene blue alone, it being seen that the methylene blue cannot be imaged at 470 nm; FIG. 2C is a graph of the results of sodium fluorescein and Melan double staining at 470nm, clearly showing the outline of the mouse liver cells and the morphology of the nuclei after double staining with sodium fluorescein and Melan; fig. 2D is a HE staining control.
As can be seen from fig. 1 and 2, the use of sodium fluorescein and the two-dye method of the meran method greatly improves the imaging effect compared with the use of sodium fluorescein alone or the use of the meran method alone. The sodium fluorescein and the metalan double staining method improve the contrast with the background, can clearly observe the tissue and the cell morphology, and can also observe clear cell nuclei, even cell nucleoli. This information will help doctors to determine tumor and non-tumor tissues, and bring greater value to medical treatment.
Example 2: effect of double dyeing with different concentrations of sodium fluorescein and Melan dyeing
Tissue staining was performed using porcine kidney, the staining procedure was as follows:
1. fresh pig kidneys were purchased and refrigerated in a refrigerator at 4 ℃ for later use.
2. Fresh pig kidneys were cleaned with clear water and the kidney surface coating was removed under a microscope with scissors and forceps.
3. The surfaces of the kidneys are respectively stained with 0.1 percent, 0.25 percent, 0.5 percent and 1 percent of fluorescein sodium for one minute, and then washed three times with normal saline; then, the surfaces of the substrates were stained with 0.5%, 1%, 2% and 3% of a methylene blue staining solution for one minute, and after the time was passed, the substrates were washed three times with a physiological saline solution and then observed under a microscope at 470 nm.
FIG. 3 is an alignment of the imaging effect of porcine kidneys using different concentrations of sodium fluorescein and Melan in combination staining. Wherein FIG. 3A is the imaging result at 470nm for a 0.1% sodium fluorescein +0.5% merland combination stain; FIG. 3B is an imaging result of 0.25% sodium fluorescein+1% Melan combination staining at 470nm wavelength; FIG. 3C is a graph showing the imaging results of a 0.5% sodium fluorescein +2% merland combination stain at a wavelength of 470 nm; FIG. 3D is the imaging results of 1% sodium fluorescein+3% meran combinatorial staining at 470nm wavelength.
As can be seen from FIG. 3, the combination of sodium fluorescein and methylene blue at different concentrations, wherein the concentration of sodium fluorescein is between 0.1% and 1%, the concentration of methylene blue is between 0.5% and 3%, the dyeing effect of the combination of the sodium fluorescein and the methylene blue is similar, and the cell morphology and the cell nuclear structure can be clearly distinguished in the combination within the concentration range.
Example 3: tissue staining by 5-ALA and acridine yellow double staining method
Tissue staining was performed using pig liver, the staining procedure was as follows:
1. fresh pork liver is purchased for a plurality of times and refrigerated in a refrigerator at the temperature of 4 ℃ for standby.
2. Fresh pig livers are cleaned by clean water, and liver surface films are removed by scissors and tweezers under a microscope.
3. The liver surface is stained with 0.05%5-ALA for 3 minutes, and then washed three times with physiological saline; then, the surface was stained with 1% acridine yellow staining solution for 2 minutes, and after the lapse of time, the solution was washed three times with physiological saline, and then observed under a microscope at 635 nm.
FIG. 4 is an image of porcine liver using double staining with 5-ALA and acridine yellow, and the outline of stained liver cells and morphology of nuclei can be clearly seen.
Example 4: tissue staining by means of double staining method of Melan and ICG
Tissue staining was performed using pig liver, the staining procedure was as follows:
1. fresh pork liver is purchased for a plurality of times and refrigerated in a refrigerator at the temperature of 4 ℃ for standby.
2. Fresh pig livers are cleaned by clean water, and liver surface films are removed by scissors and tweezers under a microscope.
3. 1% of methylene blue is used for staining the liver surface for 2 minutes, and then the liver surface is washed three times by normal saline; 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 835 nm.
Fig. 5 is an image of pig kidneys with both meran and ICG staining, and the morphology of stained liver cells and nuclei can be clearly seen.
Example 5: tissue staining by sodium fluorescein and crystal violet double staining method
Tissue staining was performed using porcine kidney, the staining procedure was as follows:
1. fresh pig kidneys were purchased and refrigerated in a refrigerator at 4 ℃ for later use.
2. Fresh pig kidneys were cleaned with clear water and the kidney surface coating was removed under a microscope with scissors and forceps.
3. After the kidney surface is stained with 0.25% fluorescein sodium for 2 minutes, the kidney surface is washed three times with physiological 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 shows the results of double staining of porcine kidneys with sodium fluorescein and crystal violet, and clearly shows the outline of stained liver cells and the morphology of the nuclei.
All documents mentioned in this application are incorporated by reference in their entirety. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings, and such equivalent modifications are also within the scope of the present application as defined in the claims.
Claims (7)
1. A staining method for live cell imaging, the method comprising the steps of: simultaneously (i) staining the target cells with sodium fluorescein; (ii) staining the target cells with methylene blue; (iii) Obtaining a fluorescence image of the target cell by using a fluorescence microscope, wherein the method can observe the cell morphology and simultaneously observe the cell nucleus morphology; wherein the sodium fluorescein emits light, the methylene blue absorbs light, the concentration of the sodium fluorescein is 0.1% -1%, and the concentration of the methylene blue is 0.5% -3%.
2. The method of claim 1, wherein the sodium fluorescein concentration is 0.25% and the meran concentration is 1%.
3. The method of claim 1, wherein the method is used for in vivo 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 that emits light and a second fluorescent biomarker that absorbs light, the first fluorescent biomarker being sodium fluorescein, the second fluorescent biomarker being methylene blue, the sodium fluorescein concentration being 0.1% -1%, the methylene blue concentration being 0.5% -3%.
6. The composition of claim 5, wherein the sodium fluorescein concentration is 0.25% and the meran concentration is 1%.
7. The composition of claim 5, further comprising stabilizers, antioxidants, protectants, preservatives, pH modifiers.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006238771A (en) * | 2005-03-02 | 2006-09-14 | Sapporo Breweries Ltd | Evaluation method of microbial cell activity by flow cytometric analysis |
| CN104031361A (en) * | 2013-03-07 | 2014-09-10 | 中国人民解放军第二军医大学 | Temperature sensitive hydrogel, and preparation method and application thereof |
| WO2016078207A1 (en) * | 2014-11-18 | 2016-05-26 | 聂飚 | Fluorescent conjugate compound and uses thereof |
| CN108118077A (en) * | 2016-11-30 | 2018-06-05 | 中南大学 | The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide |
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| JP4004385B2 (en) * | 2002-11-07 | 2007-11-07 | 独立行政法人科学技術振興機構 | Cancer detection method by double staining |
| CN104350376A (en) * | 2012-05-18 | 2015-02-11 | 古斯塔夫·鲁西Igr研究所 | Characterization of biological tissues at cellular level using red and far-red fluorescent dyes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006238771A (en) * | 2005-03-02 | 2006-09-14 | Sapporo Breweries Ltd | Evaluation method of microbial cell activity by flow cytometric analysis |
| CN104031361A (en) * | 2013-03-07 | 2014-09-10 | 中国人民解放军第二军医大学 | Temperature sensitive hydrogel, and preparation method and application thereof |
| WO2016078207A1 (en) * | 2014-11-18 | 2016-05-26 | 聂飚 | Fluorescent conjugate compound and uses thereof |
| CN108118077A (en) * | 2016-11-30 | 2018-06-05 | 中南大学 | The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide |
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