A multiple fluorescent staining solution and preparation method and use method thereof

Abstract

The present invention discloses a multiple fluorescent staining liquid and a preparation method and a use method thereof, wherein the multiple fluorescent staining liquid is an aqueous solution, comprising the following components: multiple salts, acetic acid, multiple binding proteins, preservatives, solubilizers, membrane disruptors, and multiple fluorescent stains. The present invention chelates fungal detection objects and detection objects of other microorganisms other than fungi together, and only requires one application, one staining, and one color observation to obtain the detection result. The effect after staining is clearer, and the staining boundary of the target object is sharper, which is very beneficial for the identification of small targets, especially bacteria and fungi in microorganisms are relatively small in shape, with clear boundaries and sharp fluorescent staining effects, which is conducive to the identification of such targets.

Description

Technical Field

[0001] The invention belongs to the technical field of multiple fluorescence detection, in particular to gynecological vaginal microecological detection; and specifically relates to a multiple fluorescence staining solution and a preparation method and a use method thereof. Background Art

[0002] The existing gynecological vaginal microecological detection method uses a two-step method to stain the sample, that is, the detection of fungi in each sample must be stained separately. Each sample needs to be stained with two slides, one slide is stained with fungi, and the ultraviolet fluorescence module is used for detection; the other slide is stained with microorganisms and other formed elements other than fungi, and the blue light fluorescence module is used for detection; therefore, the above detection method has the advantages of long operation time, cumbersome steps, and requires two fluorescence modules, which has high requirements for the configuration of imaging equipment.

[0003] In addition, similar products currently do not have high staining clarity for target objects, especially for small targets (such as bacteria, fungi, etc.), the imaging contours are not clear, which will cause errors (omissions and misidentifications) during the identification (manual and automatic) process; the current traditional non-staining method (saline film testing) has a maximum recognition accuracy of only 75%-80% due to the limitations of the technology itself. Summary of the invention

[0004] The present invention aims at the above-mentioned problems existing in the existing gynecological vaginal microecological detection method and provides a multiple fluorescent staining solution.

[0005] The present invention adopts the following technical scheme: a multiple fluorescent staining solution, comprising the following components per liter: 105.5-365mM strong base, 0.01-50g acid, 5-250g binding protein, 0.01-0.2L preservative, 0.1-50L solubilizer, 0.001-20L membrane disrupting agent and 0.0194-180mg fluorescent stain, and the balance is sterile distilled water.

[0006] It is further defined that the strong base includes one or more of NaCl 100-200 mM, KCl 1-5 mM, Na2HPO4 0.5-2 mM and KH2PO4 5-158 mM.

[0007] It is further defined that the binding protein comprises the following components per liter: one or more of 2-100 g of mouse anti-monoclonal antibody, 1-50 g of goat anti-monoclonal antibody and 1-50 g of rabbit anti-monoclonal antibody.

[0008] In a further embodiment, the acid is acetic acid.

[0009] It is further defined that the preservative is a food or pharmaceutical preservative, preferably Proclin 300.

[0010] It is further defined that the solubilizing agent is an industrial organic solvent, preferably dimethyl sulfoxide.

[0011] It is further defined that the membrane disrupting agent is a surfactant for regulating the fluidity of biomembrane molecules, preferably one of glycerol, propylene glycol or Tween 20.

[0012] It is further defined that the fluorescent dye comprises the following components per liter: a mixture of one or more of Hochest 0.01-10 mg, 4,4'-bis(2-benzoxazolyl)stilbene 0.001-10 mg, acridine orange 0.001-10 mg, carboxyfluorescein diacetate succinimidyl ester 0.001-50 mg, Sybr Green I 0.001-10 mg, Sybr Green II 0.001-10 mg, cell membrane fluorescent probe 0.0003-40 mg, fluorescein isothiocyanate 0.001-10 mg, phycoerythrin 0.001-10 mg, peridinin-chlorophyll-protein complex 0.001-10 mg, propidium iodide 0.001-10 mg and allophycocyanin 0.001-10 mg.

[0013] The beneficial effects of the present invention are as follows: the present invention combines fungal detection objects and other microbial detection objects other than fungi together, so only one application, one staining, and one color observation are required to obtain the detection result, and the effect after staining is clearer, and the staining boundary of the target object is sharper, which is very beneficial for the identification of small targets, especially bacteria and fungi among microorganisms are relatively small in morphology, with clear boundaries and sharp fluorescent staining effects, which is beneficial for the identification of such targets, and is also more conducive to the implementation of "artificial intelligence recognition" and the automation of gynecological vaginal microecological detection. The use of the above-mentioned multiple fluorescent staining solution has a short operation time, simple operation steps, improved experimental efficiency, and reduced configuration requirements for detection equipment;

[0014] The multiple fluorescent staining solution provided by the present invention can fluorescently stain or mark all tangible microorganisms and tissue cells, fully present the microecological conditions of the detection environment, and facilitate the judgment of clinical detection results.

[0015] The present invention also discloses a method for preparing a multiple fluorescent staining solution, comprising the following steps:

[0016] Mix strong alkali, acetic acid and sterile distilled water to prepare salt mother solution, stir for 20-35 minutes, filter through 0.22μm water system membrane, and store at 2-5℃ away from light;

[0017] Prepare the binding protein stock solution, filter it through a 0.22μm water system membrane, and store it in aliquots at -20~-17℃ in the dark;

[0018] Prepare fluorescent dye stock solution, filter through 0.22μm lipid-soluble membrane, and store at 2-5℃ away from light;

[0019] The salt mother solution, binding protein mother solution, fluorescent dye mother solution, preservative, solubilizer and membrane-breaking agent are mixed, and sterile distilled water is used as a diluent to dilute to obtain a multiple fluorescent dye solution, which is stored at room temperature away from light.

[0020] The beneficial effects of the present invention are as follows: the preparation method is simple, chelation can be achieved at room temperature and only requires stirring, and the product only needs to be stored at room temperature, and the storage conditions are friendly.

[0021] The present invention also discloses a method for using the multiple fluorescent staining solution, comprising the following steps:

[0022] Submerge the sampling end of the sampling swab with saline and stir for 1-2 minutes;

[0023] Apply a single layer of sample film on the glass slide by placing the sampling end of the sampling swab;

[0024] After curing the sample, add the multi-fluorescent staining solution to completely cover the sample, and rinse the sample after 30-45 seconds;

[0025] The rinsed samples are dried and then placed under a microscope with a blue light fluorescence module for observation.

[0026] The beneficial effects of the present invention are: the method of use is simple, the operability is strong, and the factors affecting the test results are reduced, so the test results are highly accurate, and the use method can reduce the amount of physiological saline used. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a staining image of pathogen spores under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2;

[0028] Figure 2 This is a staining image of pathogen spores under a microscope after staining the sample with an existing fluorescent staining solution;

[0029] Figure 3 This is a staining image of pathogen clue cells under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2;

[0030] Figure 4 This is a staining image of pathogen clue cells under a microscope after staining the sample with an existing fluorescent staining solution;

[0031] Figure 5 The staining images of epithelial cells, leukocytes and lactobacilli under a microscope after the sample was stained with the multiple fluorescent staining solution prepared in Example 2;

[0032] Figure 6 This is a staining image of epithelial cells infected with Gardnerella under a microscope after the sample was stained with the multiple fluorescent staining solution prepared in Example 2;

[0033] Figure 7 This is a staining image of clue cell fungal spores under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2;

[0034] Figure 8 This is a staining image of hyphae under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2;

[0035] Fig. 9 The staining image of Trichomonas under a microscope after staining the sample with the multiple fluorescent staining solution prepared in Example 2;

[0036] Fig.10 This is a microscope image of a sample stained with the multiple fluorescent staining solution prepared in Example 2 in Example 5;

[0037] Figure 11-14 These are multiple local staining images under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2 in Example 5;

[0038] Figure 15-18 These are multiple local staining images of pathogen clue cells under a microscope after the sample is stained with the multiple fluorescent staining solution prepared in Example 2 in Example 5;

[0039] Fig.19 This is a picture taken under a microscope after the sample is stained with an existing fluorescent staining solution;

[0040] Figure 20-23 It is a multiple local staining picture of pathogen spores and other visible components under a microscope after staining the sample with existing fluorescent staining solution;

[0041] Figure 24-26 These are multiple local staining images of pathogen clue cells under a microscope after staining the sample with existing fluorescent staining solution. DETAILED DESCRIPTION

[0042] Example 1

[0043] The preparation of a multiple fluorescent staining solution comprises the following steps:

[0044] S1: Weigh or measure the following substances: NaCl 100mM, KCl 1mM, Na2HPO4 0.5mM, KH2PO4 5mM, mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C 1g, rabbit anti-Candida albicans monoclonal antibody A / B 1g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B 1g, mouse anti-Trichomonas vaginalis monoclonal antibody A / B 1g, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B 1g, Proclin 300 0.01L, DMSO 0.1L, Tween 200 0.001L, Hochest 0.01mg, 4,4'-bis(2-benzoxazolyl)stilbene 0.001mg, acridine orange 0.001mg, carboxyfluorescein diacetate succinimidyl ester 0.001mg, Sybr Green I0.001mg, Sybr Green Ⅱ 0.001mg, cell membrane fluorescent probe (DiI) 0.0001mg, cell membrane fluorescent probe (DiO) 0.0001mg, cell membrane fluorescent probe (DiR) 0.0001mg, fluorescein isothiocyanate 0.001mg, phycoerythrin 0.001mg, peridinin-chlorophyll-protein complex 0.001mg, propidium iodide 0.001mg, allophycocyanin 0.001mg and acetic acid 0.01g.

[0045] S2: Preparation of salt mother solution: NaCl, KCl, Na2HPO4, KH2PO4, acetic acid, with sterile distilled water as solvent, were mixed and stirred for 30 minutes, filtered through a 0.22 μm water system membrane to obtain 100X salt mother solution, and stored at 4°C away from light;

[0046] S3: preparing binding protein stock solution: using sterile distilled water as solvent, mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C, rabbit anti-Candida albicans monoclonal antibody A / B, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B, mouse anti-Trichomonas vaginalis monoclonal antibody A / B, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B, mixed and stirred for 30 minutes, filtered through 0.22 μm water system membrane to obtain 100X binding protein stock solution, and stored at -20°C in the dark;

[0047] S4: Prepare fluorescent dye stock solution: Hochest, fluorescent brightener, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probes (DiI, DiO, DiR), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein complex (PerCP), propidium iodide (PI), allophycocyanin (APC), mix with DMSO as solvent and stir for 30 minutes, filter through 0.22 μm lipid-soluble membrane to obtain 1000X fluorescent dye stock solution, and store at 4°C away from light;

[0048] S5: 100X salt stock solution, 100X binding protein stock solution, 1000X fluorescent dye stock solution, Proclin300, DMSO, Tween 20, and sterile distilled water are mixed and filtered through a 0.22 μm lipid-soluble membrane to obtain a multiple fluorescent dye solution, which is stored at room temperature away from light.

[0049] Example 2

[0050] The preparation of a multiple fluorescent staining solution comprises the following steps:

[0051] S1: Weigh or measure the following substances: NaCl 135mM, KCl 2.7mM, Na2HPO41.5mM, KH2PO48mM, mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C 5g, rabbit anti-Candida albicans monoclonal antibody A / B 10g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B 7.5g, mouse anti-Trichomonas vaginalis monoclonal antibody A / B 12g, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B 6.7g, Proclin300 0.05L, DMSO 2.5L, Tween 200.01L, Hochest 0.05mg, 4,4'-bis(2-benzoxazolyl)stilbene 10mg, acridine orange 10mg, carboxyfluorescein diacetate succinimidyl ester 50mg, Sybr Green I10mg, Sybr Green Ⅱ 10mg, cell membrane fluorescent probe (DiI) 10mg, cell membrane fluorescent probe (DiO) 10mg, cell membrane fluorescent probe (DiR) 10mg, fluorescein isothiocyanate 10mg, phycoerythrin 10mg, peridinin-chlorophyll-protein complex 10mg, propidium iodide 10mg, allophycocyanin 10mg and acetic acid 50g;

[0052] S2: Preparation of salt mother solution: NaCl, KCl, Na2HPO4, KH2PO4, acetic acid, with sterile distilled water as solvent, were mixed and stirred for 35 minutes, filtered through a 0.22 μm water system membrane to obtain 100X salt mother solution, and stored at 4°C away from light;

[0053] S3: preparing binding protein stock solution: mixing mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C, rabbit anti-Candida albicans monoclonal antibody A / B, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B, mouse anti-Trichomonas vaginalis monoclonal antibody A / B, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B with sterile distilled water as solvent for 35 minutes, filtering with 0.22 μm water system membrane to obtain 100X binding protein stock solution, and storing at -20°C in the dark;

[0054] S4: Prepare fluorescent dye stock solution: Hochest, fluorescent brightener, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probes (DiI, DiO, DiR), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein complex (PerCP), propidium iodide (PI), allophycocyanin (APC), mix with DMSO as solvent, stir for 35 minutes, filter through 0.22 μm lipid-soluble membrane to obtain 1000X fluorescent dye stock solution, and store at 4°C away from light;

[0055] S5: 100X salt stock solution, 100X binding protein stock solution, 1000X fluorescent dye stock solution, Proclin300, DMSO, Tween 20, and sterile distilled water are mixed and filtered through a 0.22 μm lipid-soluble membrane to obtain a multiple fluorescent dye solution, which is stored at room temperature away from light.

[0056] Example 3

[0057] The preparation of a multiple fluorescent staining solution comprises the following steps:

[0058] S1: Weigh or measure the following substances: NaCl 200mM, KCl 5mM, Na2HPO4 2mM, KH2PO4 158mM, mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C 50g, rabbit anti-Candida albicans monoclonal antibody A / B 50g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B 50g, mouse anti-Trichomonas vaginalis monoclonal antibody A / B 50g, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B 50g, Proclin 300 0.2L, DMSO 50L, Tween 20 20L, Hochest 10mg, 4,4'-bis(2-benzoxazolyl)stilbene 0.02mg, acridine orange 0.02mg, carboxyfluorescein diacetate succinimidyl ester 0.05mg, Sybr Green I 0.01mg, Sybr Green Ⅱ 0.01mg, cell membrane fluorescent probe (DiI) 0.001mg, cell membrane fluorescent probe (DiO) 0.001mg, cell membrane fluorescent probe (DiR) 0.001mg, fluorescein isothiocyanate 0.01mg, phycoerythrin 0.01mg, peridinin-chlorophyll-protein complex 0.01mg, propidium iodide 0.01mg, allophycocyanin 0.01mg and acetic acid 0.5g;

[0059] S2: Preparation of salt mother solution: NaCl, KCl, Na2HPO4, KH2PO4, acetic acid, with sterile distilled water as solvent, were mixed and stirred for 20 minutes, filtered through a 0.22 μm water system membrane to obtain 100X salt mother solution, and stored at 4°C away from light;

[0060] S3: preparing binding protein stock solution: using sterile distilled water as solvent, mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C, rabbit anti-Candida albicans monoclonal antibody A / B, mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B, mouse anti-Trichomonas vaginalis monoclonal antibody A / B, mouse anti-herpes simplex virus (type I / II) monoclonal antibody A / B, mixed and stirred for 20 minutes, filtered through 0.22 μm water system membrane to obtain 100X binding protein stock solution, and stored at -20°C in the dark;

[0061] S4: Prepare fluorescent dye stock solution: Hochest, fluorescent brightener, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probes (DiI, DiO, DiR), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein complex (PerCP), propidium iodide (PI), allophycocyanin (APC), mix with DMSO as solvent, stir for 20 minutes, filter through 0.22 μm lipid-soluble membrane to obtain 1000X fluorescent dye stock solution, and store at 4°C away from light;

[0062] S5: 100X salt stock solution, 100X binding protein stock solution, 1000X fluorescent dye stock solution, Proclin300, DMSO, Tween 20, and sterile distilled water are mixed and filtered through a 0.22 μm lipid-soluble membrane to obtain a multiple fluorescent dye solution, which is stored at room temperature away from light.

[0063] Example 4

[0064] In this embodiment, the following operation is performed on a patient who comes for examination.

[0065] This embodiment discloses a method for using the multiple fluorescent staining solution prepared in Example 2, comprising the following steps:

[0066] H1: Add an appropriate amount of saline to the sampling swab (such as vaginal sampling swab), preferably just covering the sampling end of the swab, stir to mix, gently place the sampling end of the swab on a glass slide, and apply a single layer of sample film;

[0067] H2: air-dry at room temperature or bake at 60°C, fix the sample (methanol for 2 minutes or fire-bake for 3-4 times), add a drop (about 50 μL) of the sample prepared in Example 2, use a bamboo stick to adjust the staining solution to cover the entire sample layer, stain at room temperature for 30 seconds, and rinse with distilled water;

[0068] H3: Dry the slide at room temperature, place the slide under a microscope, and observe under the blue light fluorescence module; the staining results are as follows: Figure 1 , Figure 3 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Fig. 9 shown.

[0069] Comparative Example 1

[0070] The samples from the same patient in Example 4 were tested using the existing two-step test reagents on the market. The results are as follows: Figure 2 , Figure 4 .

[0071] Since diseases caused by pathogen spores and pathogen clue cells account for more than 90% of gynecological vaginal inflammation, the focus is on comparing the detection effects of these two diseases. Pathogen spores are fungi, and pathogen clue cells are squamous epithelial cells infected by bacteria, among which the bacteria are mainly Gardnerella.

[0072] Depend on Figure 1 and Figure 2 By comparison, Figure 1 The pathogen spores are stained red, such as Figure 1 The arrows in the figure point to clear boundaries and high brightness, making them easy to identify and distinguish. Figure 2 Pathogen spores and other visible components are stained green ( Figure 2 The middle arrow indicates that the boundary is fuzzy and the brightness is low, making it difficult to identify and distinguish;

[0073] Depend on Figure 3 and Figure 4 By comparison, Figure 3 The pathogen clue cells are dyed red, and the brightness is obviously different from the background purple bacteria, which is easy to identify and distinguish; Figure 4 The pathogen clue cells are stained yellow-green, and the background bacteria are also completely stained, making them difficult to identify and distinguish.

[0074] Figure 5 Arrow No. 1 in the middle points to epithelial cells, arrow No. 2 points to white blood cells, and arrow No. 3 points to lactobacilli;

[0075] Figure 6 The middle arrows point to epithelial cells infected with Gardnerella; Figure 7 The middle arrow points to the clue cell fungal spore;

[0076] Figure 8 The arrow in the middle points to hyphae. Fig. 9 The middle arrow points to Trichomonas.

[0077] Example 5

[0078] The method of use in Example 4 was used to detect samples from healthy people and patients using the multiple fluorescent staining solution prepared in Example 1. Figure 10-18 As shown by Fig.10 (Samples from healthy people) It can be seen that epithelial cells and white blood cells are clearly stained; normal lactobacilli are stained in a distinct red color, which is beneficial for clinical judgment of the microecological system; the background is clean and there are few noise signals.

[0079] Figure 11-14 (Sample from a patient with fungal infection) shows the staining of pathogen spores. Figure 11-14 It can be seen that the pathogen spores (the arrow in the picture points to the red spores, which are easy to identify) are dyed red and are very bright and easy to identify and distinguish; Figure 15-18 (Bacterial infection patients) for the staining of pathogen clue cells, Figure 15-18 It can be seen that the pathogen clue cells are dyed red, and the brightness is obviously different from the background bacteria, which is very easy to identify and distinguish.

[0080] Comparative Example 2

[0081] The samples from the same patients in Example 5 were tested using the existing two-step test reagents on the market. The results are as follows: Figure 19-26 As shown by Fig.19 It can be seen that epithelial cells, leukocytes, and lactobacilli are all stained green, with no color distinction, which is not conducive to clinical observation and distinction; background bacteria are also all stained, with strong interference signals.

[0082] Figure 20-23 The staining of pathogen spores and other visible components is determined by Figure 20-23 It can be seen that pathogen spores and other visible components are dyed green and have low brightness, making them difficult to identify and distinguish (the arrows in the figure point to the green spores, which are not easy to identify).

[0083] Figure 24-26 The staining of pathogen clue cells is Figure 24-26 It can be seen that the pathogen clue cells are stained yellow-green, and the background bacteria are also completely stained, making them difficult to identify and distinguish.

[0084] It can be seen that the multiple fluorescent staining solution provided by the present invention can not only complete the detection of bacteria and fungi at one time, but also increase the brightness of fungi and bacteria after being stained, and make the boundaries clear, which is beneficial to clinical judgment.

[0085] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a multiple fluorescent staining solution, characterized in that The following steps are involved: Prepare the salt mother solution by weighing NaCl, KCl, Na2HPO4, KH2PO4 and acetic acid in sterile distilled water as solvent, mix and stir for 35 minutes, filter through 0.22 micron water membrane, and store at 4℃ away from light; Prepare the binding protein stock solution, weigh the binding protein and use sterile distilled water as solvent, mix and stir for 35 minutes, filter with 0.22 micron water system membrane, and store at -20℃ away from light; Prepare a fluorescent dye stock solution, weigh the fluorescent dye and use a solubilizer as a solvent, mix and stir for 35 minutes, filter through a 0.22 micron lipid-soluble membrane, and store at 4°C away from light; and The salt mother solution, the binding protein mother solution, the fluorescent dye mother solution, the preservative, the solubilizer and the membrane-breaking agent are mixed, and the multiple fluorescent dye solution is obtained by diluting with sterile distilled water as a diluent, and stored at room temperature away from light; Wherein, the multiple fluorescent staining solution includes the following components per liter: 0.01 g acetic acid, 100 mM NaCl, 1 mM KCl, 0.5 mM Na2HPO4 and 5 mM KH2PO4; 0.01L Proclin300, as the preservative; 0.1 L dimethyl sulfoxide, as the solubilizing agent; 0.001 L Tween 20, as the membrane-breaking agent; 1g of mouse anti-Gardnerella vaginalis monoclonal antibody A / B / C, 1g of rabbit anti-Candida albicans monoclonal antibody A / B, 1g of mouse anti-Neisseria gonorrhoeae monoclonal antibody A / B, 1g of mouse anti-Trichomonas vaginalis monoclonal antibody A / B, and 1g of type I / II mouse anti-herpes simplex virus monoclonal antibody A / B, as the binding proteins; and As the fluorescent staining agent, 0.01 mg Hochest, 0.001 mg 4,4'-bis(2-benzoxazolyl)stilbene, 0.001 mg acridine orange, 0.001 mg carboxyfluorescein diacetate succinimidyl ester, 0.001 mg Sybr Green I, 0.001 mg Sybr Green II, 0.0001 mg cell membrane fluorescent probe DiI, 0.0001 mg cell membrane fluorescent probe DiO, 0.0001 mg cell membrane fluorescent probe DiR, 0.001 mg fluorescein isothiocyanate, 0.001 mg phycoerythrin, 0.001 mg peridinin-chlorophyll-protein complex, 0.001 mg propidium iodide, and 0.001 mg allophycocyanin were used.

Images