A fluorescent probe targeting endoplasmic reticulum and preparation method and application thereof

Abstract

The application provides a fluorescence probe targeting endoplasmic reticulum and a preparation method and application thereof, and the fluorescence probe is an azido phosphorus corrole complex. F The complex has good solubility and fat solubility (LogP=1.08), a relatively high fluorescence quantum yield (Phi 2 Red light illumination for 1h, and basically no photobleaching phenomenon is observed), in addition, the complex can quickly penetrate the cell membrane and be taken up by cells (uptake time is within 30min), and is highly specific to the endoplasmic reticulum in cells (Pierce coefficient is as high as 0.92), and the labeling condition is simple and mild, and residues are easy to remove, and is a very potential endoplasmic reticulum fluorescence probe.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, specifically to a fluorescent probe targeting the endoplasmic reticulum, its preparation method, and its application. Background Technology

[0002] The endoplasmic reticulum is the largest organelle in eukaryotic cells and plays an important role in a variety of physiological functions, including biosynthesis, sensing, signal transduction, protein folding, post-translational modification, and regulation of calcium ion homeostasis (Z. Feng, H. Wang, B. Xu et al.). J. Am. Chem. Soc. 2018, 140 , 9566; J. Boelens, S. Lust, BW Vanhoecke et al. In Vivo 2007, 21 , 215; E. Buytaert, G. Callewaert,J. Grooten, P. Agostinis et al. FASEB J. ,2006, 20 Interfering with the endoplasmic reticulum's (ER) protein folding ability leads to ER stress, ultimately activating apoptosis signaling pathways and causing cell death. Studies have shown that selectively disrupting ER function in tumor cells is a promising new strategy for anti-tumor therapy (J. Boelens, S. Lust, BWVanhoecke et al.). In Vivo 2007, 21 , 215; SJM Healy, AM Gorman, A.Samali et al. Eur. J. Pharmacol. 2009, 625 , 234; JS Nam, MG Kang, THKwon et al. J. Am. Chem. Soc. 2016, 138 Therefore, monitoring the endoplasmic reticulum (ER) is of great significance for the diagnosis and treatment of ER-related diseases. However, due to the complexity of cell signaling and the lack of obvious acid-base or electrical properties of the ER, there is a lack of effective strategies for targeting the ER, resulting in very few small molecule probes targeting the ER being developed and explored (DR Chisholm, JG Hughes, A. Whiting et al.). Org. Biomol. Chem. 2020 18, 9231; JR Cubillos-Ruiz, SE Bettigole, LHGlimcher Cell 2017, 168, 692).

[0003] Corrole, an analogue of porphyrin, is an important compound in the tetrapyrrole macrocyclic family. Corrole has one less methylene group than porphyrin, resulting in an 18π conjugated electron system. Therefore, its ring cavity is more constricted than that of porphyrin, and it possesses a trianion structure, which increases the covalent degree of the coordinated metal-N bond, making it more robust and less prone to hydrolysis or demetallization, thus endowing it with high stability and low toxicity. Furthermore, the photophysical and photochemical properties of corrole molecules can be flexibly controlled by changing the central metal coordinated with it and the substituents on the ring. Studies have shown that because the corrole skeleton is more rigid than the porphyrin skeleton, internal conversion is more difficult to occur, resulting in stronger fluorescence and a higher fluorescence quantum yield (I. Aviv-Harel, Z. Gross). Coord. Chem. Rev. 2011 , 255 (717). It is worth noting that phosphorus-based carbide exhibits excellent biocompatibility due to its non-toxicity and relative stability. Furthermore, compared to carbide with other elements as the central metal, phosphorus-based carbide generally possesses a higher fluorescence quantum yield, making it well-suited for optical imaging research (A. Mahammed, Z. Gross). Coord. Chem. Rev. 2019. 379 , 121; M. Naitana, S. Nardis, R. Paolesse Chem. Eur. J. 2017, 23 ,905; J. Vestfrid, R. Kothari, Z. Gross Inorg. Chem. 2016, 55 , 6061).

[0004] In summary, by utilizing the photostability, lipophilicity, high fluorescence quantum yield, and excellent biosafety of phosphorus carbole, a highly specific fluorescent probe targeting the endoplasmic reticulum has been developed, which has significant research and practical value for bioimaging, diagnosis, and treatment. Summary of the Invention

[0005] This invention aims to provide a fluorescent probe targeting the endoplasmic reticulum (ER), its preparation method, and its applications. A highly specific fluorescent probe targeting the ER is obtained through molecular design, enabling real-time observation of the ER's localization and morphological changes via fluorescence imaging. This probe possesses advantages such as high selectivity, high stability, strong fluorescence, low toxicity, and rapid cell membrane penetration and uptake by cells.

[0006] The objective of this invention is achieved through the following technical solution: A fluorescent probe targeting the endoplasmic reticulum, wherein the fluorescent probe targeting the endoplasmic reticulum is an azide-based phosphorus carbide complex with the following structure: .

[0007] A method for preparing the above-mentioned fluorescent probe, comprising the following steps: Step I: 4-Nitrobenzaldehyde reacts with sodium azide in the first solvent in a nucleophilic substitution reaction to produce 4-azidobenzaldehyde; Step II: 4-Azidebenzaldehyde and pentafluorodipyrrolidine undergo a condensation reaction in a second solvent with hydrochloric acid as a catalyst, followed by cyclic closure by 2,3-dichloro-5,6-dicyanobenzoquinone in a third solvent to generate carboxylic compounds. Step III: The carbole compound and phosphorus trichloride undergo a coordination reaction in a fourth solvent under nitrogen protection to obtain an azide phosphorus carbole complex, which is the fluorescent probe targeting the endoplasmic reticulum.

[0008] Preferably, in this step, the molar ratio of reactants is 4-nitrobenzaldehyde:sodium azide = 1:2; the first solvent is... N -Methylpyrrolidone; the nucleophilic substitution reaction was carried out at 80±10℃ for 8±2h.

[0009] Preferably, in step II, the molar ratio of reactants is 4-azidobenzaldehyde: pentafluorodipyrrolidine: 2,3-dichloro-5,6-dicyanobenzoquinone = 1: 2: 2; the second solvent is methanol, and the third solvent is dichloromethane; the condensation reaction and oxidative cyclization are both carried out at 25-30°C for 2-3 hours.

[0010] Preferably, in step III, the molar ratio of reactants is carboxylic acid compound: phosphorus trichloride = 1:300~400; the fourth solvent is pyridine; and the coordination reaction is carried out at 125±5℃ for 3±1h.

[0011] Preferably, in step I, the ratio of 4-nitrobenzaldehyde to the first solvent is 1 mmol: 1~2 mL; In step II, the addition ratio of 4-azidobenzaldehyde to the second solvent and the third solvent is 1 mmol: 40 ± 10 mL: 60 ± 10 mL; In step III, the ratio of carboxylic acid compound to the fourth solvent is 1 mmol: 200~250 mL.

[0012] Application of the above fluorescent probes in the detection of endoplasmic reticulum in live cells.

[0013] Preferably, the specific detection method is as follows: cells are incubated with a culture medium containing the fluorescent probe, and then observed and photographed using a laser confocal microscope to detect the localization and morphological changes of the endoplasmic reticulum in real time.

[0014] More preferably, the concentration of the fluorescent probe in the culture medium is 5~10μM; the incubation time is 30±5min; and the parameters of the laser confocal microscope are set to excitation wavelength Ex = 561 nm and emission wavelength Em = 570-720nm.

[0015] Compared with the prior art, the advantages of the present invention are: 1. This invention synthesizes an azide-based phosphorus carbole complex (ER-Cor-P) and explores its application as an endoplasmic reticulum fluorescent probe. This complex exhibits good solubility and lipophilicity (LogP = 1.08), and a high fluorescence quantum yield (Φ). F = 25%), and has excellent photostability (625±2 nm, 0.3 W / cm). 2 After 1 hour of red light irradiation, virtually no photobleaching was observed. Furthermore, the complex can rapidly penetrate the cell membrane and be taken up by the cell (within 30 minutes), and it highly specifically targets the endoplasmic reticulum in the cell (with a Pearson coefficient as high as 0.92). The labeling conditions are simple and mild, and the residue is easy to remove, making it a very promising fluorescent probe for the endoplasmic reticulum.

[0016] 2. The application of the endoplasmic reticulum fluorescent probe ER-Cor-P of this invention is as a detection reagent in the detection of endoplasmic reticulum in live cells. This reagent can target the endoplasmic reticulum with high specificity, and its localization and morphological changes can be detected in real time using laser confocal fluorescence imaging. Attached Figure Description

[0017] Figure 1 It is the structural formula of ER-Cor-P.

[0018] Figure 2 This is the synthetic route for ER-Cor-P.

[0019] Figure 3The images show the UV absorption spectrum (a) and fluorescence emission spectrum (b) of ER-Cor-P in DMF solvent.

[0020] Figure 4 This is the change in the UV spectrum of ER-Cor-P in DMF solvent during photobleaching.

[0021] Figure 5 The UV absorption spectra of ER-Cor-P in the 1-octanol phase before and after reaching partition equilibrium in 1-octanol / PBS buffer at different concentrations (20 μM, 10 μM, 5 μM) are shown.

[0022] Figure 6 The survival rate of cells in ER-Cor-P at different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) under dark conditions.

[0023] Figure 7A This study investigates the uptake time of ER-Cor-P cells.

[0024] Figure 7B This describes the distribution of ER-Cor-P in cells after uptake.

[0025] Figure 8 The images show laser confocal fluorescence imaging of HepG2 cells simultaneously co-stained with 5 μM probe ER-Cor-P and commercial subcellular dyes Lyso-Tracker Green (1 μM), Mito-Tracker Green (250 nM), and ER-Tracker Green (2 μM), respectively. (a) shows the fluorescence signal of commercial Tracker Green in the green channel; (b) shows the fluorescence signal of probe ER-Cor-P in the red channel; (c) shows the bright field image; (d) shows the superposition of the fluorescence signals of probe ER-Cor-P and commercial Tracker Green; (e) shows a magnified view; and (f) represents the correlation between the fluorescence signals of probe ER-Cor-P and commercial Tracker Green.

[0026] Figure 9 It is a probe ER-Cor-P 1 HNMR spectrum.

[0027] Figure 10 It is a probe ER-Cor-P 19 FNMR spectrum.

[0028] Figure 11 It is a probe ER-Cor-P 31PNMR spectrum.

[0029] Figure 12 This is a high-resolution mass spectrum of the probe ER-Cor-P. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] Example 1: Synthesis process of ER-Cor-P Step 1: Dissolve 4-nitrobenzaldehyde (20 mmol, 3.02 g) in... N Sodium azide (40 mmol, 2.6 g) was added to 30 mL of methylpyrrolidone and heated at 80 °C. o The reaction was stirred at C for 8 h. After the 4-nitrobenzaldehyde was completely consumed by TLC, 100 mL of diethyl ether and 20 mL of water were added for extraction. The combined organic phases were dried over anhydrous Na2SO4, the solvent was removed by vacuum distillation, and the mixture was purified by column chromatography using 300-400 mesh silica gel with hexane:dichloromethane = 2:1 as the eluent, yielding 2.86 g of 4-azidobenzaldehyde (97% yield).

[0032] Step 2: Pentafluorodipyrrole (2 mmol, 624 mg) and 4-azidobenzaldehyde (1 mmol, 150 mg) were fully dissolved in methanol (40 mL), and 36% concentrated hydrochloric acid (4 mL) was added. The mixture was stirred at room temperature for 3 h, and the reaction solution was extracted with dichloromethane (60 mL). The solution was dried with anhydrous Na2SO4 and filtered to obtain a solution containing the intermediate product. Then, 2,3-dichloro-5,6-dicyanobenzoquinone (2 mmol, 450 mg) was added, and the mixture was stirred at room temperature for 2 h. The solvent was removed by vacuum distillation, and the product was purified by silica gel column chromatography with a 300-400 mesh column. The developing solvent was n-hexane:dichloromethane = 10:1, yielding 105 mg of azidocarbole (yield 14.1%).

[0033] Step 3: Dissolve 50 mg of azide-based carbole in ultradry pyridine (15 mL), add 2 mL of phosphorus trichloride (23 mmol), and react under nitrogen protection at 125 °C for 3 h. Cool to room temperature, add 1 mL of methanol, stir at room temperature for 15 min, remove the solvent by vacuum distillation, and purify by silica gel column chromatography with dichloromethane (DCM) as the eluent to obtain 39 mg of azide-based phosphorus carbole complex (70% yield). 1 H NMR (500 MHz, Methanol- d 4 ) δ 9.46 (s, 2H), 9.01 (s, 5H), 8.93 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.90(d, J = 7.5 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 3.30 (s, 3H). 19 F NMR (471 MHz, Methanol-) d 4 ) δ -140.2--140.3 (m, 4F), -155.9--156.1 (m, 2F), -164.9--165.2(m, 4F). 31 P NMR (202 MHz, Methanol- d 4 ) δ -177.85. HRMS-ESI in methanol (m / z):calcd for C 39 H 19 F 10 N7O2P [M+H] + : 838.1173, found: 838.1296. The NMR spectrum and high-resolution mass spectrum of the probe ER-Cor-P are attached. Figure 9-12 As shown.

[0034] Example 2: Spectroscopic testing of ER-Cor-P A 10 mM stock solution was prepared by dissolving the azide-based phosphorus carboxylic acid ER-Cor-P of the present invention in dimethyl sulfoxide (DMSO). 3 μL of the ER-Cor-P stock solution was then diluted to 3 mL. N,NA 10 μM solution was prepared in dimethylformamide (DMF) solvent. By investigating the UV absorption spectrum of ER-Cor-P, four distinct absorption peaks with large molar extinction coefficients were observed: 413 nm (5.17), 529 nm (3.86), 570 nm (4.16), and 596 nm (4.41). Furthermore, the fluorescence emission spectrum of ER-Cor-P was also investigated. With an excitation wavelength of 413 nm and a detection range of 550-750 nm, the strongest fluorescence emission of the probe ER-Cor-P was observed at 603 nm. Spectroscopic tests revealed (e.g.) Figure 3 The fluorescent probe ER-Cor-P of this invention has the characteristics of near-infrared emission and large Skorthos shift.

[0035] Example 3: Photostability Spectrum Test of ER-Cor-P A 10 mM stock solution was prepared by dissolving ER-Cor-P in DMSO. 3 μL of the ER-Cor-P stock solution was diluted in 3 mM DMSO to prepare a 10 μM test solution. The test solution was then placed under a red LED lamp (625 ± 2 nm, 0.3 W / m²). 2 After irradiation for 0, 10, 20, 30, 40, 50, and 60 min respectively, the absorption spectrum of the probe in the 300-800 nm range was measured. No obvious photobleaching phenomenon was observed with increasing irradiation time, indicating that the endoplasmic reticulum fluorescent probe ER-Cor-P in this invention has excellent photostability (see...). Figure 4 ).

[0036] Example 4: Fluorescence quantum yield test of ER-Cor-P A 10 mM ER-Cor-P stock solution was diluted with DMSO to prepare a 0.1 μM test solution, at which point the absorbance of the ER-Cor-P test solution was 0.0126. A 10 mM stock solution was prepared by dissolving tetraphenylporphyrin (TPP) in dichloromethane. The TPP stock solution was then diluted with toluene to prepare a 0.1 μM test solution, at which point the absorbance of TPP was 0.0502. Using a standard sample with a reported fluorescence quantum yield of 0.11 in toluene, and an excitation wavelength of 419 nm, the ER-Cor-P test solution was tested using a fluorescence spectrophotometer, yielding a fluorescence quantum yield of 0.25. This indicates that the endoplasmic reticulum probe ER-Cor-P of this invention has a high fluorescence quantum yield.

[0037] Example 5: Lipophilicity test of ER-Cor-P ER-Cor-P was dissolved in 1-octanol saturated with PBS (10 mM, pH 7.4) to prepare 20 μM, 10 μM, and 5 μM solutions, respectively. The UV absorption spectra were measured and recorded as 20 μM-initial, 10 μM-initial, and 5 μM-initial, respectively. Then, equal volumes of PBS (10 mM, pH 7.4) saturated with 1-octanol were added to each solution and mixed thoroughly by vortexing. The solutions were then incubated at 25°C and 200 rpm for 4 h. The resulting solutions were then centrifuged (2500 rpm, 15 min) to separate the two phases. The UV spectra of the upper 1-octanol phase solution were measured (see...). Figure 5 The values ​​were recorded as 20 μM-final, 10 μM-final, and 5 μM-final. The lipophilicity of ER-Cor-P was calculated using formula (1) as LogP = 1.08. This indicates that the endoplasmic reticulum probe of the present invention has certain solubility and lipophilicity, and possesses the potential to penetrate the cell membrane and be taken up by the cell.

[0038] Log P o / w = Log( c o / c w ) = Log[[ c ] final / ([ c ] initial -[ c ] final (1) Example 6: Cytotoxicity test of ER-Cor-P To verify the feasibility of cell experiments, this invention selected HepG2 cells and used the MTT assay to investigate the cytotoxicity of the probe ER-Cor-P. Figure 6 As shown, the probe ER-Cor-P was added to HepG2 cells at concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM and incubated for 24 h. It was found that when the concentration of probe ER-Cor-P was as high as 50 μM, the cell viability was still over 70%, indicating that the endoplasmic reticulum probe ER-Cor-P of the present invention has low cytotoxicity and good biocompatibility, and can be used for further research.

[0039] Example 7: Cellular uptake time and intracellular distribution assay of ER-Cor-P The cellular uptake time of ER-Cor-P can be determined by flow cytometry. Figure 7AFirst, add HepG2 cell suspension (1×10⁻⁶) to the 12-well plate. 5 Cells per well were incubated in a CO2 cell culture incubator for 24 h. When the cell density reached 80%, the old culture medium was discarded, and fresh medium containing ER-Cor-P (10 μM) was added. Incubation was continued for 4 h, 3 h, 2 h, 1 h, 0.5 h, and 0 h, respectively. Subsequently, the old culture medium was discarded, cells were washed three times with PBS, and cells were digested with trypsin. After digestion was terminated, cells were collected in centrifuge tubes and centrifuged (300 g, 5 min). Cells were resuspended in PBS (500 μL) for flow cytometry. Cell uptake of the ER-Cor-P probe was measured using the VL3 (405 ex, 603 em) channel. Data were analyzed using FlowJo V10 software.

[0040] The distribution of ER-Cor-P in cells can be determined by laser confocal fluorescence imaging. HepG2 cell suspension (1×10⁻⁶) was used. 5 Cells (per well) were added to laser confocal microscopy dishes and incubated overnight in a CO2 cell culture incubator. When the cells reached 50% confluence, the old culture medium was discarded, and medium containing ER-Cor-P (5 μM) was added. Incubation continued for 30 min. The cell culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 8 min, the paraformaldehyde was discarded, and the cells were washed three times with PBS. DAPI staining solution was then added, and staining was performed for 8 min. The DAPI dye was discarded, and the cells were washed three times with PBS. The cells were then observed and photographed using a laser confocal microscope. Figure 7B (Laser confocal microscope parameter settings: ER-Cor-P excitation wavelength is 561 nm, emission wavelength is 570-720 nm; DAPI excitation wavelength is 405 nm, emission wavelength is 410-500 nm) The results in Figure 7 indicate that the probe ER-Cor-P can be rapidly taken up by HepG2 cells within 30 min and selectively distributed in the cytoplasm after take-up.

[0041] Example 8: Subcellular localization assay of ER-Cor-P To further investigate the specific localization of the ER-Cor-P probe, 5 μM of the ER-Cor-P probe was co-stained with commercial subcellular dyes Lyso-Tracker Green (1 μM), Mito-Tracker Green (250 nM), or ER-Tracker Green (2 μM) simultaneously in HepG2 cells, and the results were observed using laser confocal fluorescence imaging. Figure 8As shown, (a) is the fluorescence signal of commercial Tracker Green in the green channel; (b) is the fluorescence signal of probe ER-Cor-P in the red channel; (c) is the bright field image; (d) is the superposition of the fluorescence signal of probe ER-Cor-P and the fluorescence signal of commercial Tracker Green; (e) is a magnified view; and (f) represents the correlation between the fluorescence signal of probe ER-Cor-P and the fluorescence signal of commercial Tracker Green. Figure 8 The results showed that the red channel fluorescence signal of the probe ER-Cor-P highly overlapped with the green channel fluorescence signal of the commercial endoplasmic reticulum probe ER-Tracker Green, with a Pearson correlation coefficient as high as 0.92. Therefore, ER-Cor-P can be used as a red fluorescent probe targeting the endoplasmic reticulum in biological systems.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent probe targeting the endoplasmic reticulum, characterized in that, The fluorescent probe targeting the endoplasmic reticulum is an azide-based phosphorus carbole complex with the following structure: 。 2. A method for preparing the fluorescent probe according to claim 1, characterized in that, It is prepared through the following steps: Step I: 4-Nitrobenzaldehyde reacts with sodium azide in the first solvent in a nucleophilic substitution reaction to produce 4-azidobenzaldehyde; Step II: 4-Azidebenzaldehyde and pentafluorodipyrrolidine undergo a condensation reaction in a second solvent with hydrochloric acid as a catalyst, followed by cyclic closure by 2,3-dichloro-5,6-dicyanobenzoquinone in a third solvent to generate carboxylic compounds. Step III: The carbole compound and phosphorus trichloride undergo a coordination reaction in a fourth solvent under nitrogen protection to obtain an azide phosphorus carbole complex, which is the fluorescent probe targeting the endoplasmic reticulum.

3. The preparation method according to claim 2, characterized in that, In step I, the molar ratio of reactants is 4-nitrobenzaldehyde:sodium azide = 1:2; the first solvent is... N -Methylpyrrolidone; the nucleophilic substitution reaction was carried out at 80±10℃ for 8±2h.

4. The preparation method according to claim 2, characterized in that, In step II, the molar ratio of reactants is 4-azidobenzaldehyde: pentafluorodipyrrolidine: 2,3-dichloro-5,6-dicyanobenzoquinone = 1: 2: 2; the second solvent is methanol, and the third solvent is dichloromethane; the condensation reaction and oxidative cyclization are carried out at 25-30°C for 2-3 hours.

5. The preparation method according to claim 2, characterized in that, In step III, the molar ratio of reactants is carboxylic acid compound: phosphorus trichloride = 1:300~400; the fourth solvent is pyridine; the coordination reaction is carried out at 125±5℃ for 3±1h.

6. The preparation method according to claim 2, characterized in that, In step I, the ratio of 4-nitrobenzaldehyde to the first solvent is 1 mmol: 1~2 mL; In step II, the addition ratio of 4-azidobenzaldehyde to the second solvent and the third solvent is 1 mmol: 40 ± 10 mL: 60 ± 10 mL; In step III, the ratio of carboxylic acid compound to the fourth solvent is 1 mmol: 200~250 mL.

7. The use of the fluorescent probe of claim 1 for non-diagnostic and / or therapeutic purposes in the detection of endoplasmic reticulum in live cells.

8. The application according to claim 7, characterized in that, The specific detection method is as follows: cells are incubated with a culture medium containing the fluorescent probe described in claim 1, and then observed and photographed using a laser confocal microscope to detect the localization and morphological changes of the endoplasmic reticulum in real time.

9. The application according to claim 8, characterized in that, The concentration of the fluorescent probe in the culture medium is 5~10μM; the incubation time is 30±5min; the parameters of the laser confocal microscope are set as follows: excitation wavelength Ex = 561nm, emission wavelength Em = 570-720nm.

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