Development of novel red and deep red emitting nile red derivatives and their application in cellular lipid droplet fluorescence imaging

Abstract

The preparation of a new type of red and deep red emitting nile red derivative and its application in cell lipid droplet fluorescence imaging belong to the technical field of biological imaging. The chemical names of the fluorescent probes are 9-(4-(diethylamino)phenyl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(dimethylamino)styryl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(1-pyrrolidinyl)styryl)-5H-benzo[a]phenoxazine-5-one and (E)-9-(4-(diethylamino)styryl)-5H-benzo[a]phenoxazine-5-one. The series of cell lipid droplet fluorescence imaging probe molecules prepared by the application are multifunctional fluorescence imaging probe molecules which can be used for confocal imaging and have the characteristics of ICT emission, high labeling selectivity, high light stability, environmental sensitivity and excellent biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of bioimaging technology, specifically relating to the development of a new type of red and deep red light emitting Nile red derivatives and their application in cell lipid droplet fluorescence imaging. Background Technology

[0002] Fluorescence imaging technology has become one of the most powerful detection tools in the field of biomedical research, possessing advantages such as high specificity, high spatiotemporal resolution, and, most importantly, non-biodestructive observation. The excellent photophysical properties and precise labeling capabilities of fluorescence imaging probes are crucial prerequisites for observing biological samples. However, on the one hand, for most fluorescence imaging probes, in addition to labeling the target structure, other nearby or structurally similar cells or subcellular structures also exhibit non-specific staining, which is highly detrimental to in-depth studies of specific structures. On the other hand, a significant portion of existing fluorescence probes emit light in the blue-green visible band, which exhibits severe autofluorescence in cell and tissue imaging, also causing artifacts in fluorescence imaging. Existing strategies of altering substituents or targeting groups can improve the selective labeling capability of fluorescence imaging probes, but these require extensive synthetic work and molecular screening, and have strict requirements on molecular modification sites, potentially sacrificing luminescence performance. Furthermore, it is difficult to adjust the emission band. Therefore, new design approaches are urgently needed to address the labeling selectivity issue.

[0003] Recently, fluorescent molecules with intramolecular charge transfer (ICT) properties have attracted significant research interest. These molecules typically have donor-acceptor structures, and their charge transfer properties endow them with enhanced solvation effects and polar sensitivity. Because lipid droplets possess significantly lower polarity than other subcellular structures within cells, the luminescence of enhanced ICT fluorescent molecules targeting lipid droplets is significantly quenched in non-specifically stained regions. Using these molecules in fluorescence imaging holds promise for achieving luminescence only in the target region, thereby improving labeling selectivity. Furthermore, fluorescent molecules designed based on the ICT configuration can modulate donor-acceptor intensity and conjugation intensity, potentially enabling further tuning of the molecular emission band to red or near-infrared light, avoiding emission bands with strong biofluorescence backgrounds, thus further improving imaging selectivity and performance. However, this strategy requires further in-depth research; precisely tuning the molecular backbone, donor-acceptor units, and other related components remains a major challenge. Summary of the Invention

[0004] To address the shortcomings of existing lipid droplet imaging fluorescent probe molecular technologies, the purpose of this invention is to provide a novel red and deep red emitting Nile Red derivative and its application in cellular lipid droplet fluorescent imaging.

[0005] The present invention describes a novel class of Nile Red derivative fluorescent probes emitting red and deep red light, comprising four structures, with the following chemical names in order: 9-(4-(diethylamino)phenyl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(dimethylamino)styryl)-5H-benzo[a]phenoxazine-5-one, (E)-9-(4-(-1-pyrrolidinyl)styryl)-5H-benzo[a]phenoxazine-5-one, and (E)-9-(4-(diethylamino)styryl)-5H-benzo[a]phenoxazine-5-one, abbreviated as Nile DR-1, Nile DR-2, Nile DR-3, and Nile DR-4. All four structures are Nile Red-based ICT-type cellular lipid droplet fluorescence imaging probe molecules with donor-acceptor structures, and their chemical structures are shown in formula (I):

[0006]

[0007] This invention marks the first time that this fluorescent imaging probe molecule has been prepared and applied to various fluorescent imaging modes. The preparation reaction formula for this fluorescent imaging probe molecule is as follows:

[0008]

[0009] To address the shortcomings of existing cellular lipid droplet fluorescence imaging probe molecules, this invention focuses on developing novel lipid droplet imaging fluorescence probe molecules based on the Nile Red backbone, employing donor-acceptor fluorescent molecules. This invention introduces phenyl or vinylphenyl groups to extend the π-bridge backbone on the Nile Red structure; by adjusting the donor units dimethylamine, diethylamine, and pyrrole, the resulting series of fluorescence imaging probe molecules exhibit large Stokes shifts and strong solvation effects, along with environmentally sensitive ICT emission characteristics. The extended π-bridge backbone of phenyl or vinylphenyl groups enhances molecular conjugation, increases the molecular dipole moment, strengthens the ICT effect of the system, and extends the molecular emission band; by adjusting the donor configuration to coordinate the molecular color, the invention ultimately yields 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 of this fluorescent imaging probe molecule enable it to emit fluorescence with high quantum efficiency within low-polarity lipid droplets, while fluorescence is quenched in highly polar environments such as cytoplasm or lipid membranes, thus significantly improving labeling selectivity. Extending the emission wavelength to red or near-infrared also significantly reduces biological autofluorescence background. In fluorescence imaging applications, this series of molecules exhibits ultra-high labeling selectivity, high photostability, and good biocompatibility, enabling high-quality confocal imaging of cellular lipid droplets. Therefore, the development of the Nile DR-1, Nile DR-2, Nile DR-3, and Nile DR-4 series of fluorescent imaging probe molecules provides a powerful tool for cellular lipid droplet fluorescence imaging.

[0010] The cells described in this invention are HeLa cells.

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

[0012] 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 this invention possess environmentally sensitive ICT emission characteristics, which were confirmed by polarity-dependent fluorescence spectroscopy. Taking Nile DR-4 as an example, comparison with the commercial lipid droplet dye BODIPY 493 / 503 revealed that Nile DR-4 can efficiently and selectively label cellular lipid droplets with an ultra-high imaging signal-to-noise ratio. MTT assays confirmed that this probe molecule exhibits excellent biocompatibility and high photostability. Therefore, Nile DR-1, Nile DR-2, Nile DR-3, and Nile DR-4 can serve as lipid droplet fluorescent imaging probe molecules for studying the complex physiological functions of cellular lipid droplets, promoting 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 this invention are a series of novel lipid droplet fluorescence imaging probe molecules. Compared with other lipid droplet fluorescence imaging probe molecules, these probe molecules exhibit ultra-high lipid droplet labeling selectivity due to their environmentally sensitive ICT emission characteristics. Given these characteristics, their application in cellular lipid droplet fluorescence imaging has broad prospects. Attached Figure Description

[0014] Figure 1 The 1H NMR 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 this invention correspond to the following: Figure 1 (a) Figure 1 (b) Figure 1 (c) and Figure 1 (d)

[0015] Figure 2 The absorption 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 this 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 dashed part is the absorption spectrum and the solid part is the emission spectrum.

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

[0017] in, Figure 3 (a) is a co-localization imaging image of Nile DR-1 in the 600-650nm imaging channel under 530nm laser excitation; Figure 3 (b) Figure 3 (c) Figure 3 (d) are colocalization imaging images 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 images of the fluorescent molecular probe prepared in this invention (first column), the fluorescence images of BODIBY 493 / 503 in the 495-525nm imaging channel under 488nm laser excitation (second column), the superposition of the above two fluorescence images and the bright-field image (third column), and the Pearson correlation coefficient of the first two fluorescence channels (fourth column). The colocalization 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 bar: 10μm).

[0018] Figure 4 : Bar graphs showing the cell viability of HeLa cells stained with Nile DR-4 fluorescent imaging probe molecules at different concentrations for 24 hours (corresponding to Example 5).

[0019] Figure 5 Comparison of 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 images of HeLa cells stained with Nile Red and Nile DR-4 imaging probe molecules at frames 1, 12, 25 and 50, respectively (scale bar: 10 μm); Figure 5 (c) is a graph of the luminance statistics function of Nile Red and Nile DR-4 in each of 50 consecutive frames.

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

[0022] in, Figure 6 (a) Figure 6 (b) Confocal images of HeLa cells stained with Nile Red and Nile DR-4 imaging probe molecules, respectively (scale bar: 10 μm); Figure 6 (c)~ Figure 6 (e) is Figure 6 (a) Figure 6 (b) Enlarged view of the area marked by the box and the corresponding signal-to-noise ratio statistical bar chart (scale: 1μm). Detailed Implementation

[0023] Example 1:

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

[0025] 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), 1,8-diazabicyclo[5.4.0]undec-7-ene (5.96 mL, 39.9 mmol) was added, and the mixture was refluxed for 12 hours. The precipitated solid was washed three times with methanol and dried to give 8.09 g (24.8 mmol, 93%) of a 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)styryl)-5H-benzo[a]phenoxazine-5-one (Nile DR-1)

[0028] To NR-Br (0.25 g, 0.77 mmol), N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)aniline (1.05 g, 3.83 mmol), tetra(triphenylphosphine)palladium (88.6 mg, 0.077 mmol), and potassium carbonate (1.06 g, 7.70 mmol), a degassed solvent mixture of toluene, ethanol, and water (V / V / V = 8:1:1, 25 mL) was added, 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 layers were washed with brine, dried over anhydrous magnesium sulfate, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 0.176 g (0.45 mmol, 58%) of Nile DR-1, a purple solid.

[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)styryl)-5H-benzo[a]phenoxazine-5-one (Nile DR-2)

[0031] Degassed triethylamine (10 mL) was added 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 tris(o-tolyl)phosphine (37.3 mg, 123 mmol). 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, dried over anhydrous magnesium sulfate, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 137 mg (0.392 mmol, 64%) 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)styryl)-5H-benzo[a]phenoxazine-5-one (Nile DR-3)

[0034] Degassed triethylamine (10 mL) was added 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 tris(o-tolyl)phosphine (37.3 mg, 123 mmol). 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, dried over anhydrous magnesium sulfate, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 137 mg (0.392 mmol, 64%) 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)styryl)-5H-benzo[a]phenoxazine-5-one (Nile DR-4)

[0037] Degassed triethylamine (10 mL) was added 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 tris(o-tolyl)phosphine (37.3 mg, 123 mmol). 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, dried over anhydrous magnesium sulfate, and filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to give 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 1H NMR spectra of the fluorescent imaging probe molecules Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 synthesized in Example 1 show that the target products Nile DR-1, Nile DR-2, Nile DR-3 and Nile DR-4 were successfully prepared.

[0040] Example 2: Spectroscopic determination 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. Absorption spectra were collected using a UV absorption spectrometer in the wavelength range of 300–800 nm, and emission spectra were collected using a marine optical fiber fluorescence spectrometer under excitation light at a wavelength of 470 nm. Figure 2 As shown, the dashed lines represent the absorption spectrum, and the solid lines represent the emission spectrum, illustrating the absorption-emission peak positions and large Stokes shifts 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] In this embodiment, all percentages are volume fractions.

[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% penicillin-streptomycin mixture. The fetal bovine serum, penicillin-streptomycin mixture, and high-glucose DMEM were purchased directly from a biological reagent company.

[0045] Once the cells reached the logarithmic growth phase, they were passaged: The original 5 mL of culture medium in the T25 cell culture flask was aspirated, and the cell surface was washed with 2 mL of high-glucose DMEM medium (containing 1% penicillin and antibiotics) without fetal bovine serum (FBS). This medium was then aspirated, and the cells were digested with 1 mL of trypsin for 2 minutes. After most cells detached from the cell wall, 2 mL of high-glucose DMEM medium containing 10% FBS and 1% penicillin and antibiotics was added and the mixture was thoroughly mixed. The entire cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and 1 mL of high-glucose DMEM medium containing 10% FBS and 1% penicillin and antibiotics was added and the mixture was thoroughly mixed again. Cell suspensions were then transferred at ratios of 1:3 and 1:10 to new cell culture flasks and culture dishes, respectively, and placed in a CO2 cell culture incubator. Cells in the culture dishes were ready for subsequent passage or confocal imaging experiments once the cell concentration was suitable.

[0046] Example 4: Confocal Imaging

[0047] Confocal microscopy was performed on cell culture dishes, with sample preparation as described in Example 3. HeLa cells were cultured in 20 mm diameter glass-bottomed dishes and multiplied in a CO2 incubator for 1 day. After removal from the incubator, the original DMEM culture medium was removed from the dishes. For standard confocal imaging, 1 mL of DMEM culture medium containing one target dye molecule (Nile DR-1 / Nile DR-2 / Nile DR-3 / Nile DR-4) at a molecular concentration of 2 μM was added, and the cells were incubated for another 2 hours. After removal, the cells were washed three times with HBSS solution, followed by confocal fluorescence imaging in HBSS solution. The confocal imaging system used was a Leica DMI8 imaging system. The specific operation was as follows: the processed cell culture dish was placed on the stage, and a suitable amount of immersion oil was applied to the bottom of the dish. Then, simultaneous observation and focusing were performed under a wide-field fluorescence microscope until the cells were clearly visible in the field of view. After focusing, focal length correction was performed under the confocal field of view, setting the excitation intensity, detector width, and detector gain to 530nm; 550nm-795nm; and 800%, respectively. Once set, the imaging operation could be performed to acquire the confocal fluorescence image.

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

[0049] We used 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to perform a cytotoxicity test on the fluorescent imaging probe molecule Nile DR-4 (HeLa cells). HeLa cells were seeded in 96-well plates (approximately 1*10⁻⁶ cells per well). 4 The cells were cultured in a CO2 cell incubator for 24 hours. Then, the culture medium in the 6×10 wells of the central array was replaced with medium containing different concentrations (0, 0.5, 1.0, 2.0, 5.0, and 10.0 μM) of the fluorescent imaging probe molecule Nile DR-4 and 1% (v / v) DMSO (10 parallel experiments per concentration). After another 24 hours of incubation, 10 μL of MTT reagent was added to each well, and the cells were returned to the cell incubator for another 4 hours. The original culture medium was then removed from the wells, and 100 μL of DMSO was added to dissolve the generated formazan crystals. The cells were then incubated at room temperature on a shaker for 30 minutes, and the absorbance of each well was measured at 490 nm using a microplate reader. Since only living cells can react with MTT reagent to form formazan crystals, the average absorbance values ​​of the wells with different concentrations in each group can be compared with the average absorbance values ​​of the control group (10 wells with a probe molecule concentration of 0) to calculate cell viability. The results are as follows: Figure 4As shown, the fluorescent imaging probe molecule Nile DR-4 has virtually no cytotoxicity, and a concentration of 10.0 μM of this probe molecule did 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 20mm diameter glass-bottomed culture dishes and multiplied them in a CO2 incubator for one day. After removing them from the incubator, we removed the original DMEM culture medium and added 1 mL of DMEM culture medium containing one target dye molecule: NileDR-1 / Nile DR-2 / Nile DR-3 / Nile DR-4 (2μM); commercial probes BODIPY 493 / 503 (2μM) and 1% (v / v) DMSO. We then placed the cells back into the cell culture incubator and continued culturing for 2 hours. After removal, the cells were washed three times with HBSS solution, and then fluorescence imaging was performed 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 be well co-localized 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 cellular lipid droplet labeling ability.

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

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

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

[0054] After removing the two culture dishes containing HeLa cells from the cell culture incubator in Example 3, we removed the original DMEM culture medium and added DMEM culture medium containing 2 μM Nile DR-4 and 2 μM Nile Red (containing 1% DMSO), respectively. We then returned them to the incubator and continued culturing for 2 hours. Afterward, we removed the two culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. Figure 6 (a)~ Figure 6 As shown in (d). We selected regions in two petri dishes, magnified them, and compared the signal-to-noise ratio of the selected regions within the magnified areas, as shown. Figure 6 As shown in (e), the fluorescent imaging probe molecule Nile DR-4 exhibited a much higher imaging signal-to-noise ratio than Nile Red, indicating its ultra-high lipid droplet labeling selectivity and confirming the effectiveness of environmentally sensitive ICT emission characteristics in improving the labeling selectivity of fluorescent imaging probes.

Claims

1. A class of Nile Red derivative fluorescent probes emitting red and deep red light, characterized in that: The structure of the fluorescent probe is shown below. 。 2. The application of the Nile Red derivative fluorescent probe emitting red and deep red light as described in claim 1 in the preparation of cell lipid droplet fluorescence imaging reagents.

3. The application of the Nile Red derivative fluorescent probe emitting red and deep red light as described in claim 2 in the preparation of cell lipid droplet fluorescence imaging reagents, characterized in that: The cells in question are HeLa cells.

Images