A staining method for live-cell imaging

Abstract

The present invention provides a fluorescent staining method for live-cell imaging. First, cells are double stained through the first and second fluorescent biomarker. Then, the clear fluorescent cell image is shown under a fluorescent microscope, and we can observe the nucleus form while observing the cell form through the obtained image.

Claims

1. A staining method for live-cell imaging, characterized in that said method includes the following steps: simultaneously or separately (i) using the first fluorescent biomarker to stain the target cell; (ii) using the second fluorescent biomarker to stain the target cells; (iii) using a fluorescent microscope to obtain the fluorescent image of the target cells, which can observe the nucleus form while observing the cell form.

2. The method according to claim 1, characterized in that the first fluorescent biomarker has a 460-800 nm excitation wavelength, and the second fluorescent biomarker has a 350-670 nm excitation wavelength.

3. The method according to claim 1, characterized in that the maximum value of the emission spectrum of the second fluorescent biomarker is at least 50 nm different from the maximum value of the excitation spectrum of the said first fluorescent biomarker.

4. The method according to claim 1, characterized in that the first biomarker emits light and the second biomarker absorbs light when using a fluorescent microscope to obtain the fluorescent images of the target cells.

5. The method according to claim 4, characterized in that the first fluorescent biomarker is selected from fluorescein sodium, 5-aminolevulinic acid, and indocyanine green; the second biomarker is selected from methylene blue, acriflavine and crystal violet.

6. The method according to claim 5, characterized in that the first biomarker is fluorescein sodium, and the second biomarker is methylene blue.

7. The method according to claim 5, characterized in that the first biomarker is 5-aminolevulinic acid, and the second biomarker is acriflavine.

8. The method according to claim 5, characterized in that the first biomarker is indocyanine green, and the second biomarker is crystal violet.

9. The method according to claim 1, characterized in that the method is applied to live tissue staining.

10. The method according to claim 1, characterized in that the live cells include tumor cells.

11. A composition for live cell staining, characterized in that said composition includes the first biomarker and the second biomarker according to one of preceding claims.

12. The composition according to claim 11, characterized in that the first biomarker is fluorescein sodium, and the second biomarker is methylene blue.

13. The composition according to claim 11, characterized in that the first biomarker is 5-aminolevulinic acid, and the second biomarker is acriflavine.

14. The composition according to claim 11, characterized in that the first biomarker is indocyanine green, and the second biomarker is crystal violet.

15. The composition according to claim 11, characterized in that said composition further also includes stabilizers, antioxidants, protectives, preservatives, and pH regulators.

16. A method for diagnosing tumor in a mammal comprising the step of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 shows the imaging effect comparison of the mouse kidneys stained by only methylene blue, only fluorescein sodium, and dual-stained by methylene blue and fluorescein sodium. FIG. 1A shows the result of the mouse kidneys only stained by fluorescein sodium and imaged at 470 nm wavelength; FIG. 1B shows the result of the mouse kidneys only stained by methylene blue and imaged at 470 nm wavelength. FIG. 1C shows the mouse kidneys only stained by methylene blue and imaged at 660 nm wavelength. FIG. 1D shows the mouse kidneys dual stained by fluorescein sodium and methylene blue and imaged at 470 nm wavelength. FIG. 1E shows the control of hematoxylin and eosin (HE) stain.

[0057] FIG. 2 shows the imaging effect comparison of the mouse livers stained by only methylene blue, only fluorescein sodium, and dual-stained by methylene blue and fluorescein sodium. FIG. 2A shows the result of the mouse livers only stained by fluorescein sodium and imaged at 470 nm wavelength; FIG. 2B shows the result of the mouse livers only stained by methylene blue and imaged at 470 nm wavelength. FIG. 2C shows the mouse livers dual stained by fluorescein sodium and methylene blue and imaged at 470 nm wavelength. FIG. 2D shows the control of hematoxylin and eosin (HE) stain.

[0058] FIG. 3 shows the imaging effect comparison of the pig kidneys stained by different fluorescein sodium and methylene blue concentration combinations. FIG. 3A shows the result of the pig kidneys stained by 0.1% fluorescein sodium+0.5% methylene blue and imaged at 470 nm wavelength. FIG. 3B shows the result of the pig kidneys only stained by 0.25% fluorescein sodium+1% methylene blue and imaged at 470 nm wavelength. FIG. 3C shows the pig kidneys stained by 0.5% fluorescein sodium+2% methylene blue and imaged at 470 nm wavelength, FIG. 3D shows pig kidneys stained by 1% fluorescein sodium+3% methylene blue and imaged at 470 nm wavelength.

[0059] FIG. 4 shows the imaging effect of the pig livers stained by 5-ALA and acriflavine.

[0060] FIG. 5 shows the imaging effect of the pig livers stained by methylene blue and ICG.

[0061] FIG. 6 shows the imaging result of the pig kidneys stained by fluorescein sodium and crystal violet.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention is further described by reference to the following examples. It should be understood that the following embodiments are only used to describe the present invention, rather than limiting the scope of the present invention. In the scope of the invention conception, the changes and advantages which a person skilled in the art can think of are included in the present invention, and the claims and any equivalence are in the protection scope of the present invention. In this specification and the claims of the invention, the meaning of “a,” “an and “the includes plural reference unless the context clearly dictates otherwise. The experimental methods that do not indicate the specific conditions in the following examples are general and common knowledge for a person skilled in the art, or according to the conditions recommended by the manufacturer. If there is no specific explanation, all materials and reagents used in the embodiments are commercially available products.

[0063] Mice

[0064] We used C57BL6 strain mice in the experiments, bought from Model Animal Center of Nanjing University.

[0065] Fluorescent Image Analysis

[0066] Fluorescent images were obtained by MCI microscope (DiveScope). We observed a sample at 470 nm wavelength after staining the samples. We randomly selected the region of interest for imaging.

[0067] Preparation of Staining Agents 0.1% Fluorescein sodium: Weight 0.01 g fluorescein sodium powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0068] 0.25% Fluorescein sodium: Weight 0.025 g fluorescein sodium powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0069] 0.5% Fluorescein sodium: Weight 0.05 g fluorescein sodium powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0070] 1% Fluorescein sodium: Weight 0.1 g fluorescein sodium powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0071] 0.5% Methylene blue: Weight 0.05 g methylene blue powder, put it into the light avoidance tube, add 10 mL 5% sodium bicarbonate solution, shake it well, wrap the tube with foil paper, and keep away from light.

[0072] 1% Methylene blue: Weight 0.1 g methylene blue powder, put it into the light avoidance tube, add 10 mL 5% sodium bicarbonate solution, shake it well, wrap the tube with foil paper, and keep away from light.

[0073] 2% Methylene blue: Weight 0.2 g methylene blue powder, put it into the light avoidance tube, add 10 mL 5% sodium bicarbonate solution, shake it well, wrap the tube with foil paper, and keep away from light.

[0074] 3% Methylene blue: Weight 0.3 g methylene blue powder, put it into the light avoidance tube, add 10 mL 5% sodium bicarbonate solution, shake it well, wrap the tube with foil paper, and keep away from light.

[0075] 0.05% 5-ALA (5-aminolevulinic acid): Weight 0.005 g 5-ALA powder, put it into the light avoidance tube, add 10 mL 5% glucose solution, shake it well, wrap the tube with foil paper, and keep away from light.

[0076] 1% Acriflavine: Weight 0.1 g acrilavine powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0077] 0.05% ICG indocyanine-green) Weight 0.005 g ICG powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

[0078] 0.05% Crystal violet: Weight 0.005 g crystal violet powder, put it into the light avoidance tube, add 10 mL saline, shake it well, wrap the tube with foil paper, and keep away from light.

EXAMPLE 1. DUAL STAINING TISSUE WITH FLUORESCEIN SODIUM AND METHYLENE BLUE

[0079] 1.1 Mouse kidney staining

[0080] Mice are for tissue staining, and the staining process is as follows: [0081] 1. Mice have injected 1% pentobarbital sodium into the abdominal cavity for anesthesia, and the anesthesia dose is 8-9 ml/g. [0082] 2. Remove the body fur of the mouse back, cut the epidermis, and expose the kidneys. After removing the kidneys, fix the kidney surface with a blade, and remove the basement membrane on the kidney surface under the microscope with scissors and mules. [0083] 3. Use a cotton swab to stop bleeding, apply 0.25% fluorescein sodium to the kidney surface for 2 minutes, wash it with saline three times, and apply 1% methylene blue to the surface staining for 2 minutes. After 2 minutes, we used saline to wash three times, and observed the sample at 470 nm wavelength under the microscope.

[0084] FIG. 1 shows the imaging effect comparison of the mouse kidneys stained by only methylene blue, only fluorescein sodium, and dual-stained by methylene blue and fluorescein sodium. FIG. 1A shows the result of the mouse kidneys only stained by fluorescein sodium and imaged at 470 nm wavelength; FIG. 1B shows the result of the mouse kidneys only stained by methylene blue and imaged at 470 nm wavelength. The samples only stained by methylene blue cannot be imaged at 470 nm wavelength. FIG. 1C shows the mouse kidneys only stained by methylene blue and imaged at 660 nm wavelength. FIG. 1D shows the mouse kidneys dual stained by fluorescein sodium and methylene blue and imaged at 470 nm wavelength. We can clearly see the cell contour and the nucleus form of the mouse renal tubules stained by fluorescein sodium and methylene blue. FIG. 1E shows the control of hematoxylin and eosin (HE) stain.

[0085] 1.2 Mouse liver staining

[0086] Mice are used for tissue staining, and the staining process is as follows: [0087] 1. Mice have injected 1% pentobarbital sodium into the abdominal cavity for anesthesia, and the anesthesia dose is 8-9 ml/g. [0088] 2. Remove the body fur of the mouse back, cut the epidermis, and expose the liver. After removing the liver, fix the live surface with a blade. [0089] 3. Use a cotton swab to stop bleeding, apply 0.25% fluorescein sodium to the kidney surface for 2 minutes, wash it with saline three times, and apply 1% methylene blue to the surface staining for 2 minutes. After 2 minutes, we used saline to wash three times, and observed the sample at 470 nm wavelength under the microscope.

[0090] FIG. 2 shows the imaging effect comparison of the mouse livers stained by only methylene blue, only fluorescein sodium, and dual-stained by methylene blue and fluorescein sodium. FIG. 2A shows the result of the mouse livers only stained by fluorescein sodium and imaged at 470 nm wavelength; FIG. 2B shows the result of the mouse livers only stained by methylene blue and imaged at 470 nm wavelength. The samples only stained by methylene blue cannot be imaged at 470 nm wavelength. FIG. 2C shows the mouse livers dual stained by fluorescein sodium and methylene blue and imaged at 470 nm wavelength. We can clearly see the cell contour and the nucleus form of the livers stained by fluorescein sodium and methylene blue. FIG. 2D shows the control of hematoxylin and eosin (HE) stain.

[0091] Based on FIGS. 1 and 2, the imaging effect of dual staining with fluorescein sodium and methylene blue is greatly improved than the imaging effect of only staining with fluorescein sodium or methylene blue. The dual staining with fluorescein sodium and methylene blue improved the contrast to the background. We can clearly observe the tissue and. cell forms, and at the same time, we can also observe the clear nucleus and even the nucleolus. This information will help doctors to examine tumor and non-tumor tissue, bringing more excellent value to medical care.

EXAMPLE 2. STAINING EFFECT IN DIFFERENT CONCENTRATIONS OF FLUORESCEIN SODIUM AND METHYLENE BLUE

[0092] Pig kidneys are used for tissue staining, and the staining process is as follows: [0093] 1. Several fresh pig kidneys are purchased and refrigerated at 4° C. in the refrigerator. [0094] 2. Wash the fresh pig kidneys with water, and remove the basement membrane on the kidney surface with scissors and mules under a microscope. [0095] 3. Apply separately 0.1%, 0.25%, 0.5%, 1% fluorescein sodium to the kidney surface for 1 minute, wash it with saline three times, and then apply separately 0.5%, 1%, 2%, 3% methylene blue to the surface staining for 1 minute. After 1 minute, we used saline to wash three times, and observed the sample at 470 nm wavelength under the microscope.

[0096] FIG. 3 shows the imaging effect comparison of the pig kidneys stained by different fluorescein sodium and methylene blue concentration combinations. Among them, FIG. 3A shows the result of the pig kidneys stained by 0.1% fluorescein sodium+0.5% methylene blue and imaged at 470 nm wavelength. FIG. 3B shows the result of the pig kidneys only stained by 0.25% fluorescein sodium+1% methylene blue and imaged at 470 nm wavelength, FIG. 3C shows the pig kidneys stained by 0.5% fluorescein sodium+2% methylene blue and imaged at 470 nm wavelength. FIG. 3D shows pig kidneys stained by 1% fluorescein sodium+3% methylene blue and imaged at 470 nm wavelength.

[0097] Based on FIG. 3, different concentrations of fluorescein sodium and methylene blue combination, wherein the concentration of fluorescein sodium is 01%-1%, and the concentration of methylene blue is 0.5%-3%. The combination of the two in the concentration range has a similar staining effect. The combination within this concentration range can clearly distinguish the cell form and the nucleus structure.

EXAMPLE 3. DUAL STAINING TISSUE WITH 5-ALA AND ACRIFLAVINE

[0098] Pig livers are used for tissue staining, and the staining process is as follows: [0099] 1. Several fresh pig livers are purchased and refrigerated at 4° C. in the refrigerator, [0100] 2. Wash the fresh pig livers with water, and remove the basement membrane on the liver surface with scissors and mules under a microscope. [0101] 3. Apply 0.05% 5-ALA to the liver surface for 3 minutes, wash it with saline three times, and then apply 1% acriflavine to the surface staining for 2 minutes. After 2 minutes, we used saline to wash three times, and observed the sample at 635 nm wavelength under the microscope.

[0102] FIG. 4 shows the imaging effect of the pig livers stained by 5-ALA and acriflavine. We can clearly see the contour and the nucleus form of the stained liver cells.

EXAMPLE 4. DUAL STAINING TISSUE WITH METHYLENE BLUE AND ICG

[0103] Pig livers are used for tissue staining, and the staining process is as follows: [0104] 1. Several fresh pig livers are purchased and refrigerated at 4° C. in the refrigerator. [0105] 2. Wash the fresh pig livers with water, and remove the basement membrane on the liver surface with scissors and mules under a microscope. [0106] 3. Apply 1% methylene blue to the liver surface for 2 minutes, wash it with saline three times, and then apply 0.05% ICG to the surface staining for 2 minutes. After 2 minutes, we used saline to wash three times, and observed the sample at 835 nm wavelength under the microscope.

[0107] FIG. 5 shows the imaging effect of the pig livers stained by methylene blue and ICG. We can clearly see the contour and the nucleus form of the stained liver cells.

EXAMPLE 5. DUAL STAINING TISSUE WITH FLUORESCEIN SODIUM AND CRYSTAL VIOLET

[0108] Pig kidneys are used for tissue staining, and the staining process is as follows: [0109] 1. Several fresh pig kidneys are purchased and refrigerated at 4° C. in the refrigerator. [0110] 2. Wash the fresh pig kidneys with water, and remove the basement membrane on the kidney surface with scissors and mules under a microscope. [0111] 3. Apply 0.25% fluorescein sodium to the kidney surface for 2 minutes, wash it with saline three times, and then apply 0.05% crystal violet to the surface staining for 3 minutes. After 3 minutes, we used saline to wash three times, and observed the sample at 525 nm wavelength under the microscope.

[0112] FIG. 6 shows the imaging result of the pig kidneys stained by fluorescein sodium and crystal violet. We can clearly see the contour and the nucleus form of the stained liver cells.

[0113] All documents mentioned in the present invention are referred to in the full text of this application. In addition, after reading the content mentioned above about the present invention, a person skilled in the art can make various changes or modifications to the present invention. The equivalent amendments also fall in the scope of this application claims.