Nile red derivative fluorescent imaging probe molecules with excellent labeling ability and high photostability, and reagents for lipid droplet fluorescent imaging.

Abstract

This invention relates to the field of bioimaging technology and discloses fluorescent imaging probe molecules derived from Nile Red, possessing excellent labeling ability and high photostability, as well as reagents for lipid droplet fluorescence imaging. The probes are named 9-diethylamino-6-(2-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one and 9-diethylamino-6-(4-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one, abbreviated as NR-1 and NR-2. Experiments have confirmed that the fluorescent probes NR-1 and NR-2 of this invention not only have photophysical properties very similar to Nile Red, but also exhibit significantly improved lipid droplet labeling ability (5-fold increase in signal-to-noise ratio and 15-fold increase in staining brightness compared to Nile Red staining) and photostability. This invention also discloses the application of these fluorescent probes in cell lipid droplet confocal microscopy, SIM super-resolution, and tissue imaging reagents. NR-1 and NR-2 are expected to replace Nile Red as a new generation of widely used lipid droplet fluorescent probes.

Description

Technical Field

[0001] This invention belongs to the field of bioimaging technology, specifically relating to two Nile Red derivative fluorescent imaging probe molecules with excellent labeling ability and high photostability, and reagents for lipid droplet fluorescent imaging. Background Technology

[0002] Fluorescence imaging is a crucial tool for visualizing biological samples. Its real-time dynamic observation capabilities, high spatiotemporal resolution, and non-invasive nature have led to its widespread use in monitoring and studying lipid droplets. The quality of the imaging is directly related to the performance of the fluorescent probe; parameters such as probe specificity, fluorescence intensity, and photostability directly determine the accuracy and reliability of the observational data. Therefore, researchers have developed a series of targeted fluorescent probes with different molecular structures and functions to support lipid droplet biology research, providing a wealth of tools for this purpose.

[0003] Among the reported lipid droplet fluorescent probes, Nile Red (NR) is currently the most widely used. However, with the increasing demands for imaging and the growing complexity of application scenarios, its limitations are becoming increasingly prominent. This probe suffers from two major drawbacks: first, its lipid droplet labeling ability is generally poor, with insufficient lipid droplet targeting specificity. During staining, it easily generates non-specific binding to other intracellular regions, leading to a high background signal in the imaging, and the fluorescence brightness after labeling is limited; second, its photostability is poor, easily undergoing photobleaching under continuous laser irradiation, making it impossible to achieve long-term continuous observation of lipid droplet dynamic changes. These two major drawbacks severely restrict the application of Nile Red in high-precision, long-term lipid droplet imaging experiments. Therefore, developing novel lipid droplet fluorescent probes that can overcome these shortcomings has significant theoretical research value and practical application value. Summary of the Invention

[0004] To address the shortcomings of commercially available lipid-droplet fluorescent probes like Nile Red, this invention aims to significantly enhance the lipid-droplet labeling ability and photostability of fluorescent probe molecules through rational molecular design. This invention first synthesizes two Nile Red derivative fluorescent imaging probe molecules, NR-1 and NR-2, exhibiting excellent labeling ability and high photostability. Their lipid-droplet labeling ability and photostability were evaluated, and NR-1 was initially used in practical imaging applications.

[0005] The two Nile Red derivative fluorescent imaging probe molecules NR-1 and NR-2 described in this invention have the chemical names 9-diethylamino-6-(2-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one and 9-diethylamino-6-(4-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one, and their chemical structures are shown below:

[0006]

[0007] The preparation reaction formula for this fluorescent imaging probe molecule is as follows:

[0008]

[0009] To address the shortcomings of Nile Red, such as its limited ability to target lipid droplets and poor photostability, this invention uses the Nile Red molecular structure as a core and introduces 2-trifluoromethylphenyl and 4-trifluoromethylphenyl, respectively. Notably, the Nile Red derivatives designed and developed in this invention can be efficiently synthesized in just two mature classical reactions with high product yields, demonstrating excellent potential for scale-up synthesis. Characterization analysis revealed that the prepared fluorescent probe molecules NR-1 and NR-2 retain photophysical properties highly similar to Nile Red while significantly improving lipid droplet labeling ability and photostability. Based on these superior properties, we further successfully applied NR-1 as a lipid droplet fluorescence imaging reagent in structured illumination microscopy (SIM) super-resolution imaging and tissue-level lipid droplet imaging experiments.

[0010] The Nile Red derivative fluorescent imaging probe molecules NR-1 and NR-2 prepared in this invention have excellent lipid droplet labeling ability and high photostability (corresponding to Examples 4-6), and can be used as lipid droplet fluorescent imaging reagents for cell confocal imaging, cell SIM super-resolution imaging and tissue imaging (Examples 7-8).

[0011] The cells described in this invention are HeLa cells, and the tissue is liver tissue from male BALB / c mice.

[0012] Experimental results confirm that the fluorescent imaging probe molecules NR-1 and NR-2 described in this invention not only have similar absorption and emission characteristics to NR, but also exhibit superior lipid droplet labeling ability and higher photostability. This is mainly reflected in their staining signal-to-noise ratio being approximately 5 times that of NR, and their staining brightness being approximately 15 times that of NR. Further practical application verification shows that this probe not only has high compatibility with long-term super-resolution imaging technology, but also demonstrates excellent performance in lipid droplet fluorescence imaging at the tissue level. Specifically, NR-1 achieves a resolution of 129 nm in SIM super-resolution imaging, and after continuous acquisition of more than 11,000 frames, the signal intensity still maintains 52% of its initial value; NR-1 successfully quantitatively monitored changes in the number and volume of lipid droplets in liver tissue of an obese mouse model.

[0013] In summary, the NR-1 and NR-2 probes developed in this invention are not only directly compatible with existing Nile Red-based lipid droplet fluorescence detection platforms, but also exhibit superior overall performance. With these advantages, these probes can achieve more precise, clearer, and more durable visualization and monitoring of lipid droplets within live cells. Therefore, we firmly believe that NR-1 and NR-2 have the potential to replace traditional Nile Red probes and become a new generation of widely used lipid droplet-specific fluorescence imaging tools. Attached Figure Description

[0014] Figure 1 : The proton nuclear magnetic resonance spectrum of the fluorescent imaging probe molecule NR-1 prepared in Example 1 of this invention;

[0015] Figure 2 The proton nuclear magnetic resonance spectrum of the fluorescent imaging probe molecule NR-2 prepared in Example 1 of this invention;

[0016] Figure 3 The fluorescent imaging probe molecules NR-1 and NR-2 prepared in Example 1 of this invention, and the absorption-emission spectra of NR in toluene (corresponding to Example 2);

[0017] The dashed lines represent the absorption spectrum, and the solid lines represent the emission spectrum. The results show that the synthesized fluorescent imaging probe molecule NR-1 (corresponding to...) Figure 3 (b), absorption peak: 533nm, emission peak: 582nm), NR-2 (corresponding to) Figure 3 (c), absorption peak: 533nm, emission peak: 593nm) and NR (corresponding to Figure 3 (a) The absorption peak (522 nm) and emission peak (573 nm) have similar absorption and emission characteristics, which makes it possible to directly match the existing lipid droplet fluorescence detection platform based on Nile Red.

[0018] Figure 4 The fluorescent imaging probe molecules NR-1 and NR-2 prepared in Example 1 of this invention, as well as the confocal images of HeLa cells after NR staining and the quantitative diagram of the signal-to-noise ratio in HeLa cells (corresponding to Example 4).

[0019] Figures (a) to (c) show confocal images of HeLa cells stained with different fluorescent imaging probe molecules (scale bar: 10 μm); Figure (d) shows the signal-to-noise ratio (S / N ratio) (n=5) quantification of HeLa cells stained with different fluorescent imaging probe molecules. Compared to NR, the newly synthesized series of derivatives showed significantly reduced fluorescence signals in the cytoplasm, allowing for more precise and clear labeling of lipid droplets. We used the signal-to-noise ratio (S / N ratio, i.e., the ratio of fluorescence intensity between a lipid droplet and its surrounding cytoplasmic region) to quantify the specificity of the fluorescent probes in labeling lipid droplets. The S / N ratios of NR-1 (20.25±2.34, n=5) and NR-2 (19.05±4.02, n=5) were approximately 5 times higher than that of NR (3.87±0.49, n=5), demonstrating their ultra-high lipid droplet labeling selectivity.

[0020] Figure 5 The fluorescent imaging probe molecules NR-1 and NR-2 prepared in Example 1 of this invention, as well as confocal images of HeLa cells after NR staining and a quantitative diagram of imaging brightness in HeLa cells (corresponding to Example 5).

[0021] Figures (a) to (c) show confocal images of HeLa cells stained with different fluorescent imaging probe molecules, scale bar: 10 μm; Figure (d) is a quantitative graph (n=5) showing the ratio of the staining brightness of HeLa cells stained with different fluorescent imaging probe molecules to that NR staining brightness. Under the same experimental conditions (power excitation, staining time, concentration, etc.), the lipid droplet staining brightness of NR-1 and NR-2 is approximately 15 times that of NR, demonstrating their excellent lipid droplet imaging capabilities.

[0022] Figure 6 : The fluorescence imaging probe molecules NR-1, NR-2 and NR photostability prepared in Example 1 of this invention (corresponding to Example 6).

[0023] Figure (a) shows the first, 25th, 75th, and 100th frames of continuous confocal images after HeLa cells were stained with different fluorescent imaging probe molecules under high-intensity confocal illumination of 488 nm (50 times the intensity of ordinary imaging laser). Figure (b) shows the curves of relative fluorescence intensity of different fluorescent imaging probe molecules as a function of the number of image frames. Under high-intensity confocal illumination of 488 nm, NR-1 and NR-2 maintained stable fluorescence signals after 100 consecutive scans; while NR was significantly photobleached after 100 images taken with the same excitation power. Further quantitative statistics showed that NR-1 and NR-2 could still maintain 84.6% and 73.7% of their initial fluorescence signal intensity values, respectively, while NR only maintained 28.6%. This data directly confirms the excellent photostability of NR-1 and NR-2.

[0024] Figure 7 The result of using SIM super-resolution imaging after staining HeLa cells with the fluorescent imaging probe NR-1 prepared in Example 1 of this invention (corresponding to Example 7).

[0025] Among them, Figure (a) left image shows a comparison of the imaging effects of HeLa cells stained with NR-1 under wide-field microscopy and SIM microscopy, scale bar: 10 μm; Figure (a) right image (the upper image is a magnified view under SIM microscopy, and the lower image is a magnified view under wide-field microscopy) shows a magnified view of the area marked by the box, scale bar: 1 μm; Figure (b) shows the signal intensity distribution map through the lipid droplet under wide-field and SIM microscopy (indicated by lines), and its full width at half maximum (FWHM) resolution was calculated by Gaussian fitting; Figure (c) shows the images of HeLa cells stained with NR-1 captured by SIM super-resolution imaging at different frame numbers. Figure (d) shows the decay curves of relative fluorescence intensity of different fluorescent imaging probe molecules as a function of imaging frame number; Figure (e) shows the image of HeLa cells stained with NR-1 captured by SIM super-resolution imaging at frame 11,000, scale bar: 1 μm; Figure (f) shows the signal intensity distribution of lipid droplets at frame 11,000 under SIM (indicated by lines), its full width at half maximum (FWHM) resolution was calculated by Gaussian fitting; Figure (g) shows the dynamic process of lipid droplets observed by SIM imaging when imaging HeLa cells stained with NR-1, scale bar: 1 μm. It can be seen that in wide-field imaging mode, due to the limitation of optical diffraction limit, it is difficult to accurately distinguish the individual boundaries of multiple neighboring lipid droplets. In contrast, in the same cell sample, SIM images show extremely clear subcellular structural details. Lipid droplets that originally overlapped under wide field were successfully separated into independent, sharply defined individuals, and their true number, size distribution, and spatial arrangement were accurately restored. Figure 7 As shown in (b), through statistical analysis of the fluorescence intensity profiles at lipid droplet positions, the imaging resolution was improved to 129 nm, significantly breaking the diffraction limit. We then applied this to long-term SIM imaging to observe the long-term dynamic movement of lipid droplets in cells under starvation conditions. The results showed that after continuously acquiring more than 11,000 frames using SIM super-resolution microscopy, the signal intensity still maintained 52% of its initial value, while the resolution reached 136 nm. Furthermore, we clearly observed the movement, fusion, and division of lipid droplets during this process. This not only demonstrates the excellent lipid droplet staining ability and photostability of NR-1 but also verifies its high compatibility with super-resolution imaging.

[0026] Figure 8 The image shows the results of tissue imaging using the fluorescent imaging probe molecule NR-1 prepared in Example 1 of this invention (corresponding to Example 8);

[0027] Figure (a) shows confocal microscopy images of tissue blocks and sections stained with NR and NR-1 (20 μM, 2 h), along with magnified views of the boxed areas; Figure (b) shows the statistical analysis results of their signal-to-noise ratio (n=5), scale bar: 10 μm; Figure (c) shows representative confocal fluorescence images of liver tissue corresponding to the obesity model established by feeding BALB / c mice with a high-cholesterol, high-fat diet for different weeks, along with magnified views of the boxed areas, scale bar: 20 μm (Overview), 10 μm (Enlarged); Figures (d) and (e) show the quantitative analysis of the number and volume of lipid droplets in liver tissue sections of mice fed a high-cholesterol, high-fat diet for one, two, and three weeks. First, we compared the imaging of tissue sections and tissue blocks using NR and NR-1. We can clearly see that NR has a very strong background signal when staining tissue, which will seriously affect the accurate observation of lipid droplet distribution, size, and number. In contrast, NR-1 can observe lipid droplets more clearly and accurately at both the tissue section and tissue block levels. Furthermore, we statistically analyzed the signal-to-noise ratio (SNR) of NR and NR-1 in tissue imaging. NR-1 (Block: 16.58±3.98, n=5; Section: 7.09±0.67, n=5) showed significantly higher SNR than NR (Block: 4.47±0.67, n=5; Section: 2.41±0.42, n=5) in both tissue blocks and tissue sections, indicating that NR-1 has greater potential in tissue imaging. Next, we simulated different stages of obesity in BALB / c mice by feeding them a high-cholesterol diet (HCD) for 1, 2, and 3 weeks. Subsequently, liver sections from mice at different stages were prepared, stained with NR-1, and imaged under a confocal microscope. Statistical analysis of the number and volume of lipid droplets was successfully achieved. It is evident that, thanks to the excellent lipid droplet specificity and staining brightness of NR-1, it can perform excellent lipid droplet fluorescence imaging at the tissue level. Detailed Implementation

[0028] Example 1:

[0029] Synthesis of 9-diethylamino-6-(2-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one and 9-diethylamino-6-(4-trifluoromethylphenyl)-5H-benzo[a]phenoxazine-5-one

[0030] NBS (0.61 g, 3.5 mmol) was slowly added to a solution of Nile Red (1.00 g, 3.14 mmol) in dichloromethane (50 mL), and the mixture was stirred at room temperature for 1 hour. The solution was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography to give 1.09 g (2.51 mmol, 87%) of the monobrominated solid product. ¹H NMR (400 MHz, CDCl₃): δ 8.61 (d, J = 8.1 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.73–7.55 (m, 3H), 6.81–6.43 (m, 2H), 3.46 (q, J = 7.2 Hz, 4H), 1.27 (t, J = 7.4 Hz, 6H).

[0031] A mixture of the monobrominated product (0.10 mg, 0.25 mmol), (2-(trifluoromethyl)phenyl)boronic acid (95 mg, 0.50 mmol), Pd2(dba)3 (29 mg, 0.025 mmol), K2CO3 (0.11 g, 0.75 mmol), and Dicyclohexyl(2′,6′-dimethoxy[1,1′-biphenyl]-2-yl)phosphane (42 mg, 0.10 mmol) was added to a mixture of degassed toluene, ethanol, and water (8:1:1, 30 mL, v / v). The reaction mixture was then 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. The filtrate was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography to give 85 mg (0.18 mmol, 73%) of NR-1 solid. 1 H NMR (500 MHz, CDCl3) δ 8.72 (dd, J = 7.9, 1.3Hz, 1H), 8.35 (dd, J = 7.8, 1.4 Hz, 1H), 7.85 (dd, J = 7.9, 1.2 Hz, 1H), 7.75(td, J = 7.6, 1.4 Hz, 1H), 7.70–7.62 (m, 3H), 7.56 (t, J = 7.7 Hz, 1H), 7.38(d, J = 7.6 Hz, 1H), 6.68 (dd, J = 9.1, 2.7 Hz, 1H), 6.22 (d, J = 2.7 Hz, 1H), 3.40 (qd, J = 7.3, 2.0 Hz, 4H), 1.19 (t, J = 7.1 Hz, 6H).

[0032] A mixture of the monobrominated product (0.10 mg, 0.25 mmol), (4-(trifluoromethyl)phenyl)boronic acid (95 mg, 0.50 mmol), Pd2(dba)3 (29 mg, 0.025 mmol), K2CO3 (0.11 g, 0.75 mmol), and Dicyclohexyl(2′,6′-dimethoxy[1,1′-biphenyl]-2-yl)phosphane (42 mg, 0.10 mmol) was added to a mixture of degassed toluene, ethanol, and water (8:1:1, 30 mL, v / v). The reaction mixture was then 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. The filtrate was concentrated under reduced pressure, and the resulting mixture was purified by silica gel column chromatography to give 97 mg (0.21 mmol, 83%) of NR-2 solid. 1 H NMR (500 MHz, CDCl3) δ 8.70 (dd, J = 8.1, 1.3Hz, 1H), 8.36 (dd, J = 7.9, 1.4 Hz, 1H), 7.78–7.72 (m, 3H), 7.71–7.61 (m, 4H), 6.69 (dd, J = 9.1, 2.7 Hz, 1H), 6.30 (d, J = 2.7 Hz, 1H), 3.44 (q, J = 7.1Hz, 4H), 1.23 (t, J = 7.1 Hz, 6H).

[0033] Figure 1 and Figure 2 The 1H NMR spectra of the fluorescent imaging probe molecules NR-1 and NR-2 synthesized in Example 1 show that the target products NR-1 and NR-2 were successfully prepared.

[0034] Example 2: Determination of the absorption-emission spectra of the fluorescent imaging probe molecules NR-1, NR-2, and NR prepared in Example 1 in toluene.

[0035] The fluorescent imaging probe molecules NR-1, NR-2, and NR 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 350–700 nm, and emission spectra were collected using a marine optical fiber fluorescence spectrometer under excitation light at a wavelength of 470 nm. Figure 3 As shown, the dashed line represents the absorption spectrum, and the solid line represents the emission spectrum.

[0036] Example 3: Cell Culture

[0037] In this embodiment, all percentages are volume fractions.

[0038] 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.

[0039] Once the cells reached the logarithmic growth phase, we passaged them: after removing the original 5 mL of culture medium from the cell culture flask, we washed the cell surface with 2 mL of high-glucose DMEM culture medium (containing 1% penicillin and antibiotics) without fetal bovine serum. After removing this culture medium, we digested the cells with 0.5 mL of trypsin for 2 minutes. After most of the cells detached from the cell wall, we added 2 mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% penicillin and antibiotics and mixed it thoroughly. We then transferred an appropriate amount of this cell dispersion to new cell culture flasks and culture dishes, and placed them in a CO2 cell culture incubator for culture. Once the cell concentration in the culture dishes was suitable, we used them for confocal or super-resolution imaging experiments.

[0040] Example 4: Quantification of the signal-to-noise ratio of fluorescent imaging probe molecules NR-1, NR-2, and NR prepared in Example 1 of this invention in HeLa cells.

[0041] After removing the three culture dishes containing HeLa cells from the cell culture incubator as in Example 3, we removed the original DMEM culture medium and added DMEM culture medium containing 2 μM NR-1, 2 μM NR-2, and 2 μM NR (containing 1% DMSO), respectively. We then returned them to the incubator and continued culturing for 2 hours. Afterward, we removed the three culture dishes, washed each dish three times with HBSS solution, and then performed fluorescence imaging. We selected a region within each of the three culture dishes, magnified it, and measured and compared the signal-to-noise ratio of the selected region within the magnified area. Figure 4 As shown, in addition to staining spherical lipid droplet structures, NR also labels other cellular structures, and a clear fluorescent signal can be observed in the cytoplasm. Compared to NR, NR-1 and NR-2 show significantly reduced fluorescence signals in the cytoplasm, and the signal-to-noise ratio of stained lipid droplets is significantly improved, allowing for more precise and clear labeling of lipid droplets.

[0042] Example 5: Quantification of imaging brightness of fluorescent imaging probe molecules NR-1, NR-2, and NR prepared in Example 1 of this invention in HeLa cells.

[0043] After removing the three culture dishes filled with 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 NR-1, 2 μM NR-2, and 2 μM NR (containing 1% DMSO), respectively. We then returned them to the incubator and continued culturing for 2 hours. Afterward, we removed the three culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. Figure 5 As shown, we selected regions in three culture dishes and magnified them, then measured the fluorescence intensity of the images of those regions using ImageJ software. We found that the fluorescence brightness of lipid droplets stained with the fluorescent imaging probe molecules NR-1 and NR-2 was significantly higher than that of NR.

[0044] Example 6: Quantification of the photostability of the fluorescent imaging probe molecules NR-1, NR-2, and NR prepared in Example 1 of this invention in HeLa cells.

[0045] After removing the three 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 1% DMSO) containing 2 μM NR-1, 2 μM NR-2, and 2 μM NR, respectively. We then returned them to the incubator and continued culturing for 2 hours. Afterward, we removed the three culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. Confocal images were recorded within the selected area using a Nikon A1RMP microscope under the same excitation light intensity (488 nm). The fluorescence intensity of each image was then measured using ImageJ software, normalized to the intensity value of the first image, and plotted as a function of the number of recorded confocal images. Figure 6 As shown, under high-intensity confocal illumination of 488nm (50 times the intensity of ordinary imaging laser), NR-1 and NR-2 can still maintain stable fluorescence signals after 100 consecutive scans; while NR is significantly photobleached after 100 scans with the same excitation power.

[0046] Example 7: The fluorescent imaging probe molecule NR-1 prepared in Example 1 of this invention was used to stain HeLa cells for SIM super-resolution imaging.

[0047] After removing the culture dish containing HeLa cells from the incubator in Example 4, we removed the original DMEM culture medium and added DMEM culture medium (containing 1% DMSO) containing 2 μM NR-1, 2 μM NR-2, and 2 μM NR. After incubating for 2 hours, we removed the dish, washed it three times with HBSS solution, and then performed imaging in HBSS solution. In the selected area, we excited the fluorescent imaging probe molecule with 488 nm excitation light, and collected fluorescence signals in wide-field imaging mode and SIM super-resolution imaging mode in the detection range of 600–700 nm. We then used the software provided with the Hessen SIM super-resolution microscope to deconvolve the fluorescence images. By statistically analyzing the fluorescence intensity profiles at lipid droplet positions, we calculated the full width at half maximum (FWHM) to obtain the imaging resolution. This resolution was then used for long-term SIM imaging to observe the long-term dynamic movement of lipid droplets in starved cells. Simultaneously, we statistically analyzed the fluorescence intensity of each frame, normalized it to the intensity value of the first frame, and plotted it as a function of the number of recorded SIM super-resolution imaging frames. Figure 7 As shown in (a) and 7(b), compared to wide-field imaging mode, the true number, size distribution, and spatial arrangement of lipid droplets in the SIM images were accurately reproduced. Through statistical analysis of the fluorescence intensity profiles at lipid droplet locations, the imaging resolution was improved to 129 nm, significantly exceeding the diffraction limit. We then applied this to long-term SIM imaging to observe the long-term dynamic movement of lipid droplets in cells under starvation conditions. Figure 7 As shown in (c), 7(d), and 7(e), after acquiring more than 11,000 frames using SIM super-resolution microscopy, the lipid droplets still exhibited considerable fluorescence brightness. During this process, as... Figure 7 As shown in (g), we also clearly observed the movement, fusion and splitting of lipid droplets, fully demonstrating the stability and biological applicability of NR-1 in long-term super-resolution observations.

[0048] Example 8: The fluorescent imaging probe molecule NR-1 prepared in Example 1 of this invention is used for tissue imaging.

[0049] All procedures involving animals were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" of Jilin University, and the experiments were approved by the Animal Ethics Committee of Jilin University (Agreement No.: SY202503044).

[0050] Six-week-old male BALB / c mice were randomly divided into four groups. One group served as the control group, fed a standard diet. The other three groups were fed a high-cholesterol diet (HCD) for 1, 2, or 3 weeks, respectively. After the intervention, the mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg / kg), and their circulating blood was cleared by cardiac perfusion with ice-cold PBS. Liver tissue was then collected. The tissue was stained overnight at 4°C with NR and NR-1 (20 μM) to label lipid droplets (LDs). After washing, the samples were either mounted with mounting media or directly imaged on a Leica TCS SP8 laser scanning confocal microscope. The staining signal-to-noise ratio or the number and volume of lipid droplets within the selected field of view were statistically analyzed. The imaging conditions were as follows: excitation wavelength λ ex =488nm, emission wavelength λ em =600~700nm. For example... Figure 8 (a) and Figure 8 As shown in (b), compared to NR, NR-1 can observe lipid droplets more clearly and accurately at both the tissue section and tissue block levels, exhibiting a higher signal-to-noise ratio in tissue imaging. Furthermore, as... Figure 8 As shown in (c), 8(d) and 8(e), thanks to the excellent lipid droplet specificity and staining brightness of NR-1, it can be used to quantitatively monitor the changes in the number and volume of lipid droplets in the liver tissue of an obese mouse model.

Claims

1. A Nile red derivative fluorescent imaging probe molecule with excellent labeling ability and high photostability, the structure of which is shown in one of the following formulas: 。 2. The Nile Red derivative fluorescent imaging probe molecule with excellent labeling ability and high photostability as described in claim 1 is used as a lipid droplet fluorescent imaging reagent.

3. The Nile Red derivative fluorescent imaging probe molecule with excellent labeling ability and high photostability as described in claim 2, used as a lipid droplet fluorescent imaging reagent, is characterized in that: Fluorescence imaging includes cell confocal fluorescence imaging, micro-super-resolution imaging of cell structures under illumination, or tissue imaging.

4. The Nile Red derivative fluorescent imaging probe molecule with excellent labeling ability and high photostability as described in claim 2, used as a lipid droplet fluorescent imaging reagent, is characterized in that: The cells were HeLa cells, and the tissue was liver tissue from male BALB / c mice.

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