Development of novel red light and dark red light emitting Nile red derivative and application of novel red light and dark red light emitting Nile red derivative in cell lipid droplet fluorescence imaging

Abstract

The invention relates to development of novel red light and dark red light emitting Nile red derivatives and application of the novel red light and dark red light emitting Nile red derivatives in cell lipid droplet fluorescence imaging, and belongs to the technical field of biological imaging. Chemical names of the fluorescent probes are respectively 9-(4-(diethylamino) phenyl)-5H-benzo [a] phenoxazine-5-ketone, (E)-9-(4-(dimethylamino) styryl)-5H-benzo [a] phenoxazine-5-ketone, (E)-9-(4-(-1-pyrrolidinyl) styryl)-5H-benzo [a] phenoxazine-5-ketone and (E)-9-(4-(diethylamino) styryl)-5H-benzo [a] The invention relates to phenoxazine-5-ketone. The series of cell lipid droplet fluorescence imaging probe molecules prepared by the invention are multifunctional fluorescence imaging probe molecules which have ICT emission characteristics, high labeling selectivity, high light stability, environmental sensitivity and excellent biocompatibility and can be used for confocal imaging.

Description

Technical Field

[0001] The invention belongs to the field of biological imaging technology, and specifically relates to the preparation of a new type of red and deep red light emitting Nile red derivatives and their application in cell lipid droplet fluorescence imaging. Background Art

[0002] Fluorescence imaging technology has become one of the most powerful detection tools in the field of biomedical research. It has the advantages of high specificity, high spatiotemporal resolution, and most importantly, non-biodamage observation. The excellent photophysical properties and precise labeling ability of fluorescent imaging probes are important prerequisites for the observation of biological samples. However, on the one hand, for most fluorescent imaging probes, in addition to labeling the target structure, other cells or subcellular structures with adjacent positions or similar structural compositions will also show non-specific coloring, which is very unfavorable for in-depth research on specific structures; on the other hand, a considerable number of existing fluorescent probes emit light in the blue-green visible band. In cell and even tissue imaging, this band has serious autofluorescence, which will also cause artifacts in fluorescent imaging. The existing strategy of changing substituents or targeting groups can improve the selective labeling ability of fluorescent imaging probes, but it requires huge synthesis work and molecular screening, and has strict requirements on the molecular modification sites. It may face the problem of sacrificing luminescence performance, and it is difficult to adjust the luminescence band. Therefore, new design ideas are urgently needed to solve the problem of labeling selectivity.

[0003] Recently, fluorescent molecules with intramolecular charge transfer (ICT) properties have attracted great research interest. Such molecules are usually donor-acceptor structures, and the charge transfer properties give them enhanced solvation effects and polarity sensitivity. Since lipid droplets have significantly lower polarity than other subcellular structures in cells, the emission of enhanced ICT fluorescent molecules targeted to lipid droplets in non-specific coloring areas will be significantly quenched. When such molecules are used for fluorescence imaging, they are expected to emit light only in the target area, thereby improving labeling selectivity. On the other hand, fluorescent molecules designed based on ICT configurations can regulate the intensity of donors and acceptors and the conjugation strength, so it is possible to further adjust the molecular emission band to red or near-infrared light, avoiding the emission band with strong biological autofluorescence background, thereby further improving imaging selectivity and performance. However, this strategy needs further in-depth research. How to accurately adjust the molecular skeleton, donors, acceptors and other related units is a major problem in this research. Summary of the invention

[0004] In view of the shortcomings of the existing lipid droplet imaging fluorescence imaging probe molecular technology, the purpose of the present invention is to provide a new type of red and deep red light emitting Nile red derivatives and their application in cell lipid droplet fluorescence imaging.

[0005] The novel red and deep red light emitting Nile red derivative fluorescent probes described in the present invention have a total of four structures, whose chemical names are 9-(4-(diethylamino)phenyl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(dimethylamino)phenylvinyl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(-1-pyrrolidinyl)phenylvinyl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(-1-pyrrolidinyl)phenylvinyl)-5H-benzo[a]phenoxazine-5-one, and (E)-9-(4-(diethylamino)phenylvinyl)-5H-benzo[a]phenoxazine-5-one, respectively abbreviated as Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4. The four structures of Nile red-based ICT type cell lipid droplet fluorescent imaging probe molecules are all donor-acceptor structures, and their chemical structures are shown in formula (I):

[0006]

[0007] The present invention prepares the fluorescent imaging probe molecule for the first time and applies it to a variety of fluorescent imaging modes. The preparation reaction formula of the fluorescent imaging probe molecule is as follows:

[0008]

[0009] In view of the shortcomings of existing cell lipid droplet fluorescence imaging probe molecules, the present invention selects donor-acceptor type fluorescent molecules based on the Nile Red skeleton in the development of new lipid droplet imaging fluorescence imaging probe molecules. Based on the structure of Nile Red, the present invention introduces phenyl or vinylphenyl to expand the π bridge skeleton respectively; adjusts the donor units of dimethylamine, diethylamine and pyrrole, and the prepared series of fluorescent imaging probe molecules show large Stokes shift and strong solvation effect, and have environmentally sensitive ICT emission characteristics. The extended π bridge skeleton of phenyl or vinylphenyl enhances molecular conjugation, increases the molecular dipole moment, enhances the ICT effect of the system and extends the molecular luminescence band; adjusts the donor configuration to coordinate the molecular light color, and finally obtains the Nile DR-1 molecule in the red light band and the Nile DR-2, Nile DR-3 and Nile DR-4 series molecules in the near-infrared band. The environmentally sensitive ICT emission characteristics enable the fluorescent imaging probe molecule to emit light with high quantum efficiency in low-polarity lipid droplets, while the fluorescence will be quenched in environments such as high-polarity cytoplasm or lipid membranes, thereby significantly improving the labeling selectivity; the emission band extended to red or near-infrared also significantly reduces the biological autofluorescence background. In terms of fluorescence imaging applications, the series of molecules have ultra-high labeling selectivity, high photostability and good biocompatibility, achieving high-quality confocal imaging of cell lipid droplets. Therefore, the development of the series of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, NileDR-3 and Nile DR-4 provides a powerful tool for cell lipid droplet fluorescence imaging.

[0010] The cells described in the present invention are HeLa cells.

[0011] The series of cell lipid droplet fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 prepared by the present invention are multifunctional fluorescent imaging probe molecules that can be used for confocal imaging and have ICT emission characteristics, high labeling selectivity, high photostability, environmental sensitivity and excellent biocompatibility (Examples 3 to 6).

[0012] The experimental results confirm that the series of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 described in the present invention have environmentally sensitive ICT emission characteristics, and their environmentally sensitive characteristics are confirmed by polarity-dependent fluorescence spectrum testing. Taking Nile DR-4 as a representative, by comparing with the commercial lipid droplet dye BODIPY 493 / 503, it is found that NileDR-4 can efficiently and selectively label cell lipid droplets and has an ultra-high imaging signal-to-noise ratio. The MTT test confirms that the probe molecule has excellent biocompatibility. High photostability. Therefore, Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 can be used as lipid droplet fluorescent imaging probe molecules to study the complex physiological functions of cell lipid droplets and promote the development of lipid droplet cell biology. The design strategy of this molecule has important guiding significance for the development of fluorescent imaging probe molecules targeting different organelles.

[0013] In summary, Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 described in the present invention are a series of new lipid droplet fluorescence imaging probe molecules. Compared with other lipid droplet fluorescence imaging probe molecules, this probe molecule has ultra-high lipid droplet labeling selectivity due to its environmentally sensitive ICT emission characteristics. In view of the above characteristics, its application in cell lipid droplet fluorescence imaging has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : The nuclear magnetic resonance hydrogen spectra of the series of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 prepared in Example 1 of the present invention correspond to Figure 1 (a) Figure 1 (b) Figure 1 (c) and Figure 1 (d).

[0015] Figure 2 : Absorption spectra and emission spectra of the series of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 prepared in Example 1 of the present invention in toluene solution (corresponding to Example 2); wherein, Figure 2 (a)~ Figure 2 (d) Corresponds to Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 respectively. The dotted line part is the absorption spectrum and the solid line part is the emission spectrum.

[0016] Figure 3: Photographs of co-localization of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 with commercial lipid droplet dye BODIPY 493 / 503 in HeLa cells (corresponding to Example 6);

[0017] in, Figure 3 (a) is a co-localization imaging photograph of Nile DR-1 in the 600-650nm imaging channel under 530nm laser excitation; Figure 3 (b) Figure 3 (c) Figure 3 (d) are co-localization imaging photos of Nile DR-2, Nile DR-3 and Nile DR-4 in the 650-700nm imaging channel under 530nm laser excitation; in each imaging result, from left to right are the fluorescence photo of the fluorescent molecular probe prepared by the present invention (first column), the fluorescence photo of BODIBY 493 / 503 in the 495-525nm imaging channel under 488nm laser excitation (second column), the superposition of the above two fluorescence photos and the bright field photo (third column), and the Pearson correlation coefficient of the first two fluorescence channels (fourth column); the co-localization coefficients of Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 are 0.83, 0.94, 0.94 and 0.86, respectively (scale: 10μm).

[0018] Figure 4 : A bar graph showing the cell survival rate after HeLa cells were stained with different concentrations of the fluorescent imaging probe molecule Nile DR-4 for 24 hours (corresponding to Example 5).

[0019] Figure 5 : Comparison of the photostability of the fluorescent imaging probe molecule Nile DR-4 and the commercial lipid droplet probe Nile Red in HeLa cells (corresponding to Example 7);

[0020] in, Figure 5 (a) Figure 5 (b) Confocal photographs of HeLa cells stained with Nile Red and Nile DR-4 imaging probe molecules at the 1st, 12th, 25th and 50th frames, respectively (scale bar: 10 μm); Figure 5 (c) is the brightness statistical function diagram of Nile Red and Nile DR-4 in each frame of 50 consecutive frame samples.

[0021] Figure 6 : Quantification of the signal-to-noise ratio of the fluorescent imaging probe molecule Nile DR-4 and the commercial lipid droplet dye Nile Red in HeLa cells (corresponding to Example 8);

[0022] in, Figure 6 (a) Figure 6 (b) Confocal photographs of HeLa cells stained with Nile Red and Nile DR-4 imaging probe molecules (scale bar: 10 μm); Figure 6 (c)~ Figure 6 (e) Figure 6 (a) Figure 6 (b) Enlarged image of the boxed area and the corresponding signal-to-noise ratio statistical bar graph (scale bar: 1 μm). DETAILED DESCRIPTION

[0023] Embodiment 1:

[0024] Synthesis of 9-bromo-5H-benzo[a]phenoxazine-5-one (NR-Br)

[0025] 1,8-diazabicyclo[5.4.0]undec-7-ene (5.96 mL, 39.9 mmol) was added to a solution of 2-amino-5-bromophenol (5.00 g, 26.6 mmol) and 2-chloro-1,4-naphthoquinone (10.24 g, 53.2 mmol) in dichloromethane (100 mL), and the mixture was refluxed for 12 hours. The precipitated solid was washed three times with methanol and dried to obtain 8.09 g (24.8 mmol, 93%) of pale yellow solid NR-Br.

[0026] 1 H NMR (400MHz, DMSO-d6): δ8.64(d,J=7.4Hz,1H),8.15(d,J=7.7Hz,1H),7.96–7. 84(m,2H),7.81(d,J=8.4Hz,2H),7.63(dd,J=8.6Hz,J=1.5Hz,1H),6.44(s,1H).

[0027] Synthesis of (E)-9-(4-(dimethylamino)phenylvinyl)-5H-benzo[a]phenoxazine-5-one (Nile DR-1)

[0028] A degassed solvent mixture of toluene, ethanol and water (V / V / V=8:1:1, 25 mL) was added to NR-Br (0.25 g, 0.77 mmol), N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.05 g, 3.83 mmol), tetrakis(triphenylphosphine)palladium (88.6 mg, 0.077 mmol) and potassium carbonate (1.06 g, 7.70 mmol), and the reaction mixture was refluxed for 12 hours. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with dichloromethane. The combined organic layer was washed with brine, then dried over anhydrous magnesium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 0.176 g (0.45 mmol, 58%) of purple solid Nile DR-1.

[0029] 1 H NMR (400MHz, CDCl3): δ8.72(d,J=7.7Hz,1H),8.30(d,J=7.4Hz,1H),7.82–7.66(m,3H),7.61–7.53 (m,3H),7.47(s,1H),6.75(d,J=8.6Hz,2H),6.44(s,1H),3.50–3.35(m,4H),1.22(t,J=7.0Hz,6H).

[0030] Synthesis of (E)-9-(4-(-1-pyrrolidinyl)phenylvinyl)-5H-benzo[a]phenoxazine-5-one (Nile DR-2)

[0031] To a mixture of NR-Br (200 mg, 0.613 mmol), N-dimethyl-4-vinylaniline (127.7 mg, 1.23 mmol), palladium acetate (13.8 mg, 61.3 mmol) and tri(o-tolyl)phosphine (37.3 mg, 123 mmol) was added degassed triethylamine (10 mL). After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with dichloromethane. The combined organic layers were washed with brine, then dried over anhydrous magnesium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 137 mg (0.392 mmol, 64%) of Nile DR-2 as a red solid.

[0032] 1H NMR (400MHz, CDCl3): δ8.72(d,J=9.2Hz,1H),8.31(d,J=6.4Hz,1H),7.82–7.70(m,3H),7.53–7.35(m, 4H),7.21(d,J=17.5Hz,1H),6.93(d,J=16.5Hz,1H),6.73(d,J=7.3Hz,2H),6.45(s,1H),3.02(s,6H).

[0033] Synthesis of (E)-9-(4-(-1-pyrrolidinyl)phenylvinyl)-5H-benzo[a]phenoxazine-5-one (Nile DR-3)

[0034] To a mixture of NR-Br (200 mg, 0.613 mmol), NR-Br and 1-(4-vinylphenyl)pyrrolidine (127.7 mg, 1.23 mmol), palladium acetate (13.8 mg, 61.3 mmol) and tri(o-tolyl)phosphine (37.3 mg, 123 mmol) was added degassed triethylamine (10 mL). After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with dichloromethane. The combined organic layers were washed with brine, then dried over anhydrous magnesium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 137 mg (0.392 mmol, 64%) of Nile DR-2 as a red solid.

[0035] 1 H NMR (400MHz, CDCl3): δ8.74(d,J=7.1Hz,1H),8.34(d,J=6.5Hz,1H),7.85–7.71(m,3H),7.52–7.42(m,3H),7.39(s, 1H),7.22(d,J=16.2Hz,1H),6.92(d,J=16.3Hz,1H),6.61(d,J=7.5Hz,2H),6.47(s,1H),3.37(s,4H),2.06(s,4H).

[0036] Synthesis of (E)-9-(4-(diethylamino)phenylvinyl)-5H-benzo[a]phenoxazine-5-one (Nile DR-4)

[0037] To a mixture of NR-Br (200 mg, 0.613 mmol), 4-vinyl-N,N-diethylaniline (127.7 mg, 1.23 mmol), palladium acetate (13.8 mg, 61.3 mmol) and tri(o-tolyl)phosphine (37.3 mg, 123 mmol) was added degassed triethylamine (10 mL). After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with dichloromethane. The combined organic layers were washed with brine, then dried over anhydrous magnesium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 137 mg (0.392 mmol, 64%) of Nile DR-2 as a red solid.

[0038] 1 H NMR (500MHz, CD2Cl2): δ8.74 (dd, J=7.7Hz, J=0.7Hz, 1H), 8.27 (dd, J=7.7Hz, J=1.0Hz, 1 H),7.82(td,J=7.6Hz,J=1.4Hz,1H),7.80–7.74(m,2H),7.51(dd,J=8.3Hz,J=1.6Hz,1H) ,7.46(d,J=8.8Hz,2H),7.41(d,J=1.6Hz,1H),7.24(d,J=16.2Hz,1H),6.95(d,J=16.2H z, 1H), 6.71 (d, J = 8.8Hz, 2H), 6.42 (s, 1H), 3.44 (q, J = 7.1Hz, 4H), 1.23 (t, J = 7.1Hz, 6H).

[0039] Figure 1 The hydrogen nuclear magnetic spectra of the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 synthesized in Example 1 indicate that the target products Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 are prepared.

[0040] Example 2: Determination of the spectra of the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 prepared in Example 1 in toluene solution

[0041] Absorption-emission spectra in toluene: The fluorescent imaging probe molecules NileDR-1, NileDR-2, NileDR-3 and NileDR-4 synthesized in Example 1 were prepared into a 10 μM solution using toluene solvent. The absorption spectrum was collected by ultraviolet absorption spectrometer in the wavelength range of 300-800 nm, and the emission spectrum was collected by Ocean Optics fiber optic fluorescence spectrometer under the excitation of 470 nm excitation light, as shown in FIG. Figure 2 As shown, the dotted line part is the absorption spectrum, and the solid line part is the emission spectrum, which illustrates the absorption-emission peak position and its large Stokes shift of the series of fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4.

[0042] Example 3: Cell culture

[0043] All percentages in this embodiment are volume percentages.

[0044] HeLa cells were cultured in an incubator at 37°C and 5% CO2 concentration in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture). Among them, fetal bovine serum, double antibody and high-glucose DMEM were purchased directly from a biological reagent company.

[0045] When the cells grow to the logarithmic phase, the cells are passaged: after the original 5mL culture medium in the T25 cell culture bottle is sucked away, the cell surface is washed with 2mL of high-glucose DMEM culture medium (containing 1% double antibody) without fetal bovine serum, the culture medium is sucked away, and the cells are digested with 1mL of trypsin for 2 minutes. After most of the cells are detached from the wall, 2mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody is added and blown evenly. The treated cell suspension is transferred to a 15mL centrifuge tube, centrifuged at 1500r / min for 5 minutes, the supernatant is removed after centrifugation, 1mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody is added and blown evenly again, and the cell suspension is taken out at a ratio of 1:3 and 1:10 and transferred to a new cell culture bottle and culture dish respectively, and placed in a CO2 cell culture incubator for culture. The cells in the culture dish can be used for subsequent passage or confocal imaging experiments when the concentration is appropriate.

[0046] Example 4: Confocal imaging

[0047] The confocal experiment was performed on a cell culture dish, and the sample preparation method was as described in Example 3. HeLa cells were cultured in a 20 mm diameter glass-bottomed culture dish and propagated in a CO2 incubator for 1 day. After taking out from the incubator, the original DMEM culture medium in the culture dish was removed. For standard confocal imaging, 1 mL of DMEM culture medium containing a target dye molecule (Nile DR-1 / NileDR-2 / Nile DR-3 / Nile DR-4) was added; wherein the molecule concentration was 2 μM, and placed in a cell culture incubator for further culturing for 2 hours. After removal, wash with HBSS solution 3 times, and then perform confocal fluorescence imaging in HBSS solution. The confocal imaging system is a Leica DMI8 imaging system. The specific operation is as follows: place the treated cell culture dish on the stage, apply an appropriate amount of immersion oil to the bottom of the dish, then observe and focus under a wide-field fluorescence microscope. The focus adjustment is completed when the cells in the field of view are clear. Then, perform focus correction under the confocal field of view, set the excitation intensity and detector width, and the detector gain to 530nm; 550nm-795nm; 800% respectively. After the settings are completed, you can perform shooting operations to obtain confocal fluorescence images.

[0048] Example 5: Test of cytotoxicity of the fluorescent imaging probe molecule Nile DR-4 prepared in Example 1

[0049] We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to test the cytotoxicity of the fluorescent imaging probe Nile DR-4 (HeLa cells). HeLa cells were seeded on a 96-well plate (the number of cells in each well was about 1*10 4 ), and cultured in a CO2 cell culture incubator for 24 hours. After that, the culture medium in the culture wells of the 6×10 array area in the central area was replaced with a culture medium containing different concentrations (0, 0.5, 1.0, 2.0, 5.0 and 10.0 μM) of fluorescent imaging probe molecules Nile DR-4 and 1% (volume fraction) DMSO (10 groups of parallel experiments were set for each concentration). After another 24 hours of culture, MTT reagent (10 μL per well) was added to these wells, and they were returned to the cell culture incubator for further culture for 4 hours. After removing the original culture medium in these wells, DMSO (100 μL per well) was added to dissolve the generated formazan crystals, and after being placed on a shaker at room temperature for 30 minutes, the absorbance value of each well was measured at 490 nm using an enzyme marker. Since only living cells can react with MTT reagent to generate formazan crystals, the average absorbance value of each group of wells with different concentrations can be compared with the average absorbance value of the control group (10 wells with probe molecule concentration of 0) to calculate the cell survival rate. The results are as follows: Figure 4As shown, it is shown that the fluorescent imaging probe molecule Nile DR-4 has basically no cytotoxicity, and the probe molecule at a concentration of 10.0 μM will not affect the normal growth of HeLa cells within 24 hours. Example 6: Co-staining experiment of the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 prepared in Example 1 with the commercial lipid droplet dye BODIPY 493 / 503 in HeLa cells

[0050] We cultured HeLa cells in a 20 mm diameter glass-bottomed culture dish and propagated them in a CO2 incubator for 1 day. After taking it out of the incubator, remove the original DMEM culture medium in the culture dish, add 1 mL of DMEM culture medium containing a target dye molecule: NileDR-1 / Nile DR-2 / Nile DR-3 / Nile DR-4 (2 μM); and commercial probe BODIPY 493 / 503 (2 μM) and 1% (volume fraction) DMSO, and continue to culture in a cell culture incubator for 2 hours. After taking it out, wash it 3 times with HBSS solution, and then perform fluorescence imaging in HBSS solution. Figure 3 As shown, we can clearly observe that all the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3, and Nile DR-4 prepared in Example 1 can achieve good co-localization with the commercial lipid droplet dye BODIPY 493 / 503 in HeLa cells, indicating that the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3, and Nile DR-4 prepared in Example 1 have excellent cell lipid droplet labeling capabilities.

[0051] Example 7: Comparison of photostability of the fluorescent imaging probe molecule Nile DR-4 prepared in Example 1 and the commercial lipid droplet dye NileRed in HeLa cells

[0052] After we took out the two culture dishes filled with HeLa cells in Example 3 from the cell culture incubator, removed the original DMEM culture solution, added 2μM Nile DR-4 and 2μM Nile Red DMEM culture solution (containing 1% DMSO) respectively, put them back into the incubator and continued to culture for 2 hours, then took out the two culture dishes, washed them three times with HBSS solution respectively, and then performed fluorescence imaging. The confocal images in the 500-700nm channel were recorded using a Nikon A1RMP microscope, and the excitation laser power was the same and 50 times higher than the conventional excitation intensity (488nm), as shown in Figure 2. Figure 5 (a)~ Figure 5(b) The total signal intensity of each image was measured using ImageJ, normalized to the value of the first image, and then plotted as a function of the number of confocal images recorded, as shown in Figure 5 As shown in (c), the results indicate that Nile DR-4 has a higher photostability than Nile Red.

[0053] Example 8: Comparison of the imaging signal-to-noise ratio of the fluorescent imaging probe molecule Nile DR-4 prepared in Example 1 and the commercial lipid droplet dye NileRed in HeLa cells

[0054] After taking out the two culture dishes filled with HeLa cells in Example 3 from the cell culture incubator, the original DMEM culture medium was removed, and DMEM culture medium containing 2 μM Nile DR-4 and 2 μM Nile Red (containing 1% DMSO) was added respectively. After returning to the incubator and continuing to culture for 2 hours, the two culture dishes were taken out, washed three times with HBSS solution respectively, and then fluorescence imaging was performed, as shown in FIG. Figure 6 (a)~ Figure 6 (d) shows. We selected areas in two culture dishes and zoomed in, and compared the signal-to-noise ratio of the selected area in the zoomed area, as shown in Figure 6 As shown in (e), it was found that the fluorescent imaging probe molecule Nile DR-4 exhibited an imaging signal-to-noise ratio much higher than that of Nile Red, indicating its ultra-high lipid droplet labeling selectivity and confirming the effectiveness of the environmentally sensitive ICT emission characteristics in improving the labeling selectivity of fluorescent imaging probes.

Claims

1. A type of Nile red derivative fluorescent probe emitting red and deep red light, characterized in that: The structural formula of the fluorescent probe is shown as one of the following:

2. Use of a class of red and deep red emitting Nile red derivative fluorescent probes as described in claim 1 in the preparation of cell lipid droplet fluorescent imaging agents.

3. Use of a class of red and deep red emitting Nile red derivative fluorescent probes as claimed in claim 2 in the preparation of cell lipid droplet fluorescent imaging agents, characterized in that: The cells are HeLa cells.

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