Construction of Novel Pyrrole-Derivative-Based Fluorescent Platforms and Development of Polarity-Sensitive Probes for Imaging Lipid Droplets in Cells and Zebrafish

Construction of Novel Pyrrole-Derivative-Based Fluorescent Platforms and Development of Polarity-Sensitive Probes for Imaging Lipid Droplets in Cells and Zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Construction of Novel Pyrrole-Derivative-Based Fluorescent Platforms and Development of Polarity-Sensitive Probes for Imaging Lipid Droplets in Cells and Zebrafish Jian-Hua Jiang, Yun-Hao Yang, Ying-Kun Liu, Ning Zhang, Jian-Yong Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8235573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted 10 You are reading this latest preprint version Abstract Lipid droplets (LDs), serving as central hubs in lipid metabolism, exhibit dynamic changes closely associated with diseases such as obesity, diabetes, and cancer. Visualizing LDs is crucial for elucidating their role in biological mechanisms and facilitating early disease detection. Donor-acceptor (D-A) type fluorescent probes have been widely designed and employed for LD detection. In this study, we designed and constructed a pyrrole-based molecular scaffold with a symmetrical architecture. Through modulation of para-substituents, a series of novel fluorescent dyes ( 3a-e ) were successfully synthesized. Experimental characterization revealed that the dimethylamino-modified dye 3e , characterized by a typical D-A-D structure, exhibits red emission (λ em ≈ 608 nm in THF), significant solvatochromism, and a large Stokes shift (134 nm). To enhance its stability, the pyrrole N-H group of 3e was further methylated, successfully yielding a fluorescent LD probe, Py-LD . Py-LD not only exhibits a large Stokes shift (142 nm in THF), significantly reducing self-absorption and enhancing imaging signal-to-noise ratio, but also demonstrates excellent polarity sensitivity, enabling polarity detection. Furthermore, Py-LD displays high specificity and stability. It was successfully applied for dynamic tracking of LDs in live cells and lipid imaging within the zebrafish yolk sac, providing a novel tool for LD monitoring. Lipid droplets Polarity-sensitive Fluorescent probe Cell imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Lipid droplets (LDs) are crucial intracellular organelles responsible for lipid storage [ 1 ] . Composed primarily of neutral lipids and cholesteryl esters, their unique structural feature is a hydrophobic core surrounded by a monolayer phospholipid membrane and specific proteins [ 2 – 4 ] . As vital dynamic organelles, LDs participate not only in maintaining energy homeostasis and regulating lipid metabolism but also influence diverse physiological and pathological processes by mediating cellular signaling [ 5 – 8 ] . Studies have demonstrated that aberrant accumulation of LDs is closely associated with metabolic diseases (such as obesity and diabetes) and cardiovascular diseases [ 9 – 11 ] . Furthermore, alterations in the number and function of LDs are correlated with cancer development [ 12 – 14 ] . This is primarily due to the strong affinity cancer cells exhibit for fatty acids and cholesterol, leading to the excessive storage of these lipids within LDs [ 15 ] . Consequently, cancer cells universally display a significant increase in LD number. The substantial accumulation of hydrophobic lipids within LDs in cancer cells results in a reduction in microenvironment polarity [ 16 – 18 ] . Given the high levels of LDs in tumors and their impact on microenvironment polarity, accurately monitoring both the number and polarity of LDs is of significant importance for distinguishing cancer cells from normal cells, as well as for cancer monitoring and treatment. Fluorescent probes are regarded as highly promising and practical detection tools, playing a crucial role in specific detection, especially excelling in cellular imaging within biological systems [ 19 – 21 ] . Compared with other analytical detection methods, fluorescent probe technology exhibits remarkable advantages such as low cost, strong real-time performance, high sensitivity, and high detection accuracy [ 22 – 25 ] . In recent years, various fluorescent probes for specific lipid droplet imaging have been reported, successfully achieving specific labeling and dynamic monitoring of LDs in living cells [ 26 – 30 ] . However, many commonly used probes, such as Nile Red and BODIPY 493/503, still have obvious limitations, including insufficient photostability, short emission wavelengths, and small Stokes shifts [ 31 – 34 ] . These issues restrict their application in high-precision and long-term imaging. Therefore, the development of more novel lipid droplet-specific fluorescent probes with excellent optical properties, such as large Stokes shifts, long emission wavelengths, and high photostability, is of urgent and significant importance for a deeper understanding of the functions of LDs in physiological and pathological processes and for promoting the development of early cancer diagnosis techniques based on lipid droplet imaging. In this work, we designed and investigated a class of pyrrole derivatives. Their ease of modification and unique symmetrical molecular structure render them suitable for constructing donor-acceptor-donor (D-A-D) type fluorescent probes. Studies have shown that probes with strong electron donor-acceptor (D-A) interactions generally exhibit excellent polarity sensitivity. By introducing different electron-donating substituents, we successfully synthesized a series of novel pyrrole derivatives 3a-e . Among them, the compounds containing strong electron-donating groups exhibit typical D-A-D structural features, demonstrating longer emission wavelengths, larger Stokes shifts, and stronger fluorescence intensities. Based on these findings, we successfully constructed a novel polarity probe, Py-LD . Optical tests indicate that the emission wavelength of Py-LD significantly redshifts as the solvent polarity increases, confirming its effectiveness as a polarity-sensitive probe. This probe also possesses good biocompatibility and pH stability. Additionally, Py-LD shows highly specific lipid droplet-targeting ability, with co-localization imaging with the commercial lipid droplet dye (BODIPY ) showing a high degree of overlap (r = 0.92). Py-LD has been successfully applied to lipid droplet imaging studies in cell and zebrafish models. 2. Experimental section 2.1. Reagents and materials Naphthalene-1,4-dione, Iodomethane，KOH, Benzaldehyde, 4-Bromobenzaldehyde, 4-Hydroxybenzaldehyde, p-Tolualdehyde and 4-Dimethylaminobenzaldehyde were bought from Macklin (Shanghai, China). All reagents and drugs involved in the Experimental section were commercially purchased and used directly without secondary purification. All analytical equipment and experimental conditions are described in detail in the Supporting Information, including general procedures for spectroscopic testing, test parameters, and cytotoxicity tests. 2.2. General synthesis procedure of fluorescent dyes 3a–e and Py-LD Scheme 1 outlines the general procedure for the synthesis of pyrrole products 3a−e and Py-LD . Synthesis of compound 3a−e . 1,4-Naphthoquinone (316.4 mg, 2.0 mmol, 1.0 equiv) and benzaldehyde with different substituents ( 2a-e ) (8.0 mmol, 4.0 equiv) and ammonium iodide (869.6 mg, 6.0 mmol, 3.0 equiv) were placed into a 100 mL reaction vial, and 10 mL of toluene was added as solvent. The reaction was stirred at 130 °C for 24 hours. At the end of the reaction, it was cooled to room temperature and volatiles were removed under reduced pressure. The residue was purified by column chromatography on neutral alumina to give the desired products 3a-e (Table 1). Synthesis of compound Py-LD . 3 e (87.3 mg, 0.25 mmol, 1.0 equiv) was added to a suspension of KOH (70.0 mg, 1.25 mmol, 5.0 equiv) in DMF (5.0 mL) at 0 °C. After the solution was stirred for 1 h, CH 3 I (31.0 μL, 0.5 mmol, 2.0 equiv) was added. The reaction was then stirred at 50 °C overnight. At the end of the reaction, the reaction solution was poured into water and the crude product was extracted with dichloromethane. The solution was dried and the solvent was evaporated to obtain the crude product. Finally, the crude product was purified by column chromatography on neutral alumina to give the product Py-LD (72.7 mg, yield: 80%). 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.04 (dd, J = 5.8, 3.3 Hz, 2H), 7.75 (dd, J = 5.8, 3.3 Hz, 2H), 7.51 – 7.42 (m, 4H), 6.87 – 6.79 (m, 4H), 3.34 (s, 3H), 3.01 (s, 12H). 13 C NMR (101 MHz, CDCl 3 ) δ 179.93, 150.84, 140.37, 136.34, 132.55, 131.68, 126.64, 117.84, 111.66, 40.36, 33.80. HRMS (ESI) Found: 450.2174 [M+H] + ; Molecular formula C 29 H 27 N 3 O 2 requires [M+H] + 450.2177. To obtain details regarding the synthesis and characterization of 3a – e and Py-LD , please refer to the Supplementary Information (SI). Detailed characterization is shown in Figs. S7-S23 . 3. Results and discussion 3.1 Design and luminescence characterization of dyes 3a-e In recent years, with the rapid development of organic synthesis and fluorescence technology, a large number of new fluorescent organic molecules have been synthesized and studied. Consequently, an increasing number of novel probes have been successfully designed and applied to lipid droplet research. LDs are lipid cores composed of a phospholipid monolayer and fatty acids, with a hydrophobic environment inside. They are surrounded by a polar environment rich in water and inorganic ions. This significant polarity difference between the inside and outside provides ideas for the design of lipid droplet probes [35,36] . Theoretically, if a fluorescent probe can exhibit differential fluorescence properties in these two microenvironments with different polarities, it is expected to become an effective tool for accurately labeling LDs. Based on the above principles, this study developed a novel pyrrole-based fluorescent platform, Py-R . First, the multi-phenyl ring structure of Py-R confers a degree of lipophilicity, which facilitates probe targeting towards LDs. Furthermore, by modifying substituents, the structural features of the Py-R platform can be utilized to construct typical donor-acceptor-donor (D-A-D) conjugated molecules. This design enhances the intramolecular charge transfer (ICT) effect, thereby imparting polarity-sensitive properties [37] . Consequently, we anticipated that it would emit strong fluorescence at short wavelengths in low-polarity solvents. Conversely, in high-polarity solvents, it was expected that significant charge separation might occur, leading to longer emission wavelengths with weaker fluorescence intensity. It was hypothesized that this pronounced solvatochromic effect would enable specific staining of intracellular LDs. Based on the above, we prepared a series of novel fluorescent dyes ( 3a-e ) by a one-step method using benzaldehyde ( 2a-e ) with different para-substituents as raw materials. These dyes based on pyrrole derivatives are typical polar conjugated systems, which motivates us to study their luminescent properties. The UV absorption and fluorescence spectra of these dyes ( 3a-e ) were first tested in different solvents ( Figs. S1-S5 ). It can be seen that the introduction of different substituent modifications can significantly change the luminescent properties of the fluorophores. The fluorescence emission of dyes 3a-e changes significantly over a wide wavelength range of 500-650 nm. As expected, the introduction of a strong electron donor group into the fluorophore leads to the formation of a D-A-D structure, resulting in excellent fluorescence properties ( Fig.1 ). Taking the fluorescent compound 3e as an example, 3e exhibits longer absorption and emission wavelengths due to the better electron-donating effect of dimethylamino groups. Fluorescent probes with longer emission wavelengths have been reported to have the advantages of less background interference, low energy light sources, strong tissue penetration and less tissue damage [38-40] . This prompted us to carry out further studies on compound 3e in order to construct fluorescent probes with excellent photophysical properties. 3.2 Design and photophysical properties of the probe Py-LD As shown in Figure S5, the emission wavelength of fluorescent compound 3e undergoes a significant red-shift with increasing solvent polarity. The compound exhibits markedly higher fluorescence emission intensity in low-polarity/non-protic solvents (toluene) compared to high-polarity/protic solvents (methanol). This behavior indicates the presence of an ICT character: high-polarity solvents stabilize the charge-separated excited state, promoting non-radiative decay and consequent fluorescence quenching. Combined with the lipophilicity imparted by its multi-phenyl ring structure, these properties make it suitable for the design of fluorescent probes targeting hydrophobic microenvironments. Furthermore, the secondary amino group (-NH-) in compound 3e exhibits high reactivity and a propensity for hydrogen bonding, which may induce molecular aggregation and lead to concentration-dependent fluorescence quenching. To enhance chemical stability and suppress aggregation effects, we modified the reactive amino group in 3e through methylation, ultimately affording the fluorescent probe Py-LD ( Scheme 1 ) . Subsequently, we systematically investigated the UV-visible absorption spectra of Py-LD in different solvents and calculated valuable spectral data such as absorption and emission wavelengths, Stokes shifts, fluorescence quantum yields and half-peak widths of the dye Py-LD in different solvents ( Fig. 2a and b, Table 1 ). As expected, Py-LD similarly has a long emission wavelength, large Stokes shift (142 nm) and good polarity response properties. This motivated us to develop and further validate it as a novel lipid droplet-targeted polarity-sensitive probe Py-LD . To further investigate the photophysical-chemical properties of Py-LD , we tested its absorption and fluorescence spectra in different solvents. It can be seen that the maximum absorption wavelength remained relatively constant in different solvents ( Fig. 2a ). Whereas in fluorescence spectra, the maximum emission wavelength of Py-LD undergoes a significant redshift with increasing solvent polarity ( Fig. 2b ). This trend can be observed more clearly from the normalised spectra of Py-LD in different solvents ( Fig. 2c ). Under 365 nm UV irradiation, the solvent colour of Py-LD also changed from yellow to red as the solvent polarity increased, which is consistent with the spectroscopic test results and shows a positive solvatochromic effect ( Fig. 2d ). It is worth noting that Py-LD undergoes a polarity quenching effect in highly polar solvents (Acetone, DMF), resulting in suppression of fluorescence emission and inability to observe fluorescence changes in the solvent. These results all indicate that the probe Py-LD has good polarity response characteristics and has the potential to be a polarity sensitive probe. 3.3 Polarity-sensitivity study of the probe Py-LD To further assess the sensitivity of probe Py-LD to polarity, we quantified the polarity of different solvents using the polarity sensitivity parameter E T (30) and collated photophysical number of probe Py-LD to investigate changes in fluorescence. ( Table 1 ). At the same time, we used these data to draw graphs to intuitively analyze the relationship between Py-LD and polarity change. It is evident from Fig. 3a that the emission intensity of the probe decreases regularly with increasing solvent polarity. In this case, the fluorescence quantum yield also shows a significant decrease with increasing solvent polarity due to the ICT effect of the probe ( Fig. 3b ). We also plotted the fitted curves for the maximum fluorescence emission intensity, Stokes shift, and the solvent polarity parameter E T (30), with correlation coefficients exceeding 0.99 in both cases ( Fig. 3c and d ). These analyses favourably demonstrate that the probe Py-LD is polarity sensitive and has good linear correlation. Table 2 Photophysical properties of compound Py-LD in different solvents. Py-LD Toluene Dioxane THF EtOAc Acetone DMF DMSO MeCN EtOH MeOH PBS λ abs (nm) 458 462 466 462 460 475 482 470 478 475 480 λ em (nm) 584 596 608 608 623 645 653 637 642 647 650 E T (30) a 33.9 36 37.4 38.1 42.2 43.2 45.1 45.6 51.9 55.4 63.1 Stokes shift (nm) 126 134 142 146 163 170 171 167 164 172 170 Φ F b 24.24% 6.17% 5.48% 4.13% 2.67% 1.37% 0.13% 0.09% 0.17% 0.06% 0.43% Log ε max c 3.46 3.90 3.73 3.66 3.40 3.65 5.54 5.59 5.57 3.90 4.03 FWHM d 92.66 95.25 99.63 100.91 99.82 114.12 199.17 233.24 227.81 176.55 94.77 a E T (30) is a solvent polarity parameter and means the molar transition energy in kcal mol −1 [41] . b Fluorescence quantum yield was obtained by the reference method, by applying equation Φ x = Φs(n x /n s ) 2 (A s /A x )(F x /F s ) . c Molar extinction coefficients are calculated in the maximum of the highest peak by the Beer Lambert law. d Full width at half maximum (FWHM) was determined for emission band in solution. 3.4 Theoretical calculation To rationally investigate the electronic properties of Py-LD , we performed theoretical calculations on the energy levels of the compound. Using Density Functional Theory (DFT), we calculated the geometric configuration of probe Py-LD and the electron density distribution of its HOMO and LUMO energy levels ( Fig. 4 ). The dimethylamino group, as an electron-donating moiety, is expected to enhance the intramolecular charge transfer (ICT) efficiency. Therefore, we analyzed the frontier molecular orbitals (HOMO/LUMO) of Py-LD . As shown in Fig. 4, the HOMO and LUMO energies of Py-LD were calculated to be -4.852 eV and -1.971 eV, respectively, under vacuum-level reference conditions. Furthermore, the HOMO orbital is predominantly localized on the dimethylamino donor group, while the LUMO orbital is distributed across the entire isoindole-ketone acceptor skeleton, indicating a strong conjugated system ( Fig. 4 ). Additional calculations of the frontier molecular orbitals and energies in various solvent models further confirmed significant ICT characteristics, providing theoretical evidence for the observed solvatochromic effect and polarity-sensitive behavior of this probe ( Fig. 4 ). 3.5 Lipophilicity study of probe Py-LD Lipophilic analysis is a promising method for verifying the ability of probes to label LDs. LDs are the most hydrophobic organelles, and similar to the principle of similar-phase solubility, the hydrophobicity of the molecular skeleton allows for specific aggregation in the LDs of the cell. The polarity sensitivity of the probe Py-LD gives it a stronger fluorescence emission in less polar solvents, thus significantly brightening the LDs. Because the probe structure itself has multiple alicyclic structures and exhibits specific fluorescence response in lipid solvents, it has been further explored. We characterized the fluorescence emission spectra of the probes in different ratios of 1,4-Dioxane/MeOH mixed solvents, using 1,4-Dioxane as the lipophilic solvent and MeOH as the protonic solvent. The analyses showed that the fluorescence emission intensity of probe Py-LD was significantly enhanced with the increase of 1,4-Dioxane content in the mixed solvent, and the maximum emission wavelength showed an obvious blue shift ( Fig. 5a and b ). This fully demonstrated that the probe Py-LD exhibited excellent sensitivity and good fluorescence emission intensity to the fat-soluble environment. 3.6 Stability study of the probe Py-LD Photostability is an important characteristic of the probe. Therefore, we investigated the variation of fluorescence intensity over time for the probe Py-LD in toluene, 1,4-Dioxane, and methanol solutions, respectively. As shown in Fig. 6a , the fluorescence intensity of the probe remained stable in different polar environments under prolonged continuous light excitation. To test whether Py-LD adapts to the complex environment inside living cells, we investigated the emission behaviour of Py-LD over a wide pH range in phosphate buffer solution. As shown in Fig. 6b , the fluorescence intensity of the Py-LD probe remains relatively stable across different pH environments, which indicated that the probe was less affected by pH. The results show that the probe exhibits good fluorescence stability in the biological pH range. In addition, a large number of interfering substances exist in the complex cellular environment. In order to study the anti-interference performance of the probe, 1. SO 4 2- 2. PO 4 3- 3. ClO - 4. CO 3 2- 5. Cys 6. HSO 4 - 7. H 2 O 2 8. NO 2- 9. Cl - 10. Na + 11. Fe 3+ 12. Hg 2+ 13. Mg 2+ 14. K + 15. Cu 2+ 16. Ca 2+ were added as the interfering substances. The effect of these analytes on the fluorescence intensity was negligible as shown in Fig. 6c . These results indicate that the probe Py-LD exhibits good anti-interference properties and can be used for polarity detection in complex environments. 3.6 Probe Py-LD for bioimaging applications Based on the above experimental results, we further applied Py-LD to cell imaging experiments. Firstly, MTT assay was performed to detect the biotoxicity of probe Py-LD . After incubating different concentrations of the probe with HeLa cells for 24 h, the cell viability remained above 80% ( Fig. S6) . This indicates that the probe Py-LD has low cytotoxicity and good biocompatibility. Next to validate the specific targeting ability of the probe Py-LD on cellular LDs, co-localisation imaging experiments were performed in a HeLa cell model using the commercial lipid droplet dye BODIPY 493/503 in conjunction with the probe Py-LD ( Fig. 7 ). By confocal fluorescence scanning microscopy ( CLSM ) imaging, we can visualise the red probe Py-LD accumulating in cellular LDs and showing bright fluorescence. In the same cells, Py-LD and BODIPY 493/503 showed the same staining effect, and the fluorescence in the two channels showed significant overlap, and the Pearson correlation coefficient reached 0.92 . This suggests that the probe Py-LD , like BODIPY 493/503 , has a specific targeting effect on LDs. The co-localization curve of the specific region in Fig. 7e also confirms the above viewpoints. The experimental results showed that the probe Py-LD showed good targeting to lipid droplet in living cells and could be used for lipid droplet imaging in living cells. To further investigate the ability of the Py-LD probe to sensitively label LDs in cells, we used oleic acid ( OA ) to stimulate the generation of LDs in cells. Oleic acid is an unsaturated fatty acid that has been proven to be an effective inducer of lipid droplet formation, helping promote the accumulation of a large number of endogenous LDs [42] . Therefore, we treated HeLa cells with the Py-LD probe and incubated them with different concentrations of OA (0 μM, 50 μM, 100 μM) for half an hour before conducting cell imaging. As shown in Fig. 8a , as the concentration of OA increased, the size and number of LDs in the cells significantly increased. The accumulation of more LDs resulted in a stronger fluorescent signal from Py-LD , and the fluorescence intensity was positively correlated with the OA concentration ( Fig. 8b ). Furthermore, after staining with Py-LD , the fluorescence images clearly showed the presence of small LDs in the cells. These results indicate that the Py-LD probe can sensitively label LDs in cells and visually display the size and quantity of LDs. Therefore, Py-LD is an excellent tool for monitoring LDs. Finally, we assessed the performance of the Py-LD probe in biological imaging, using zebrafish as a model organism to evaluate its imaging capabilities. During zebrafish development, the yolk sac is an important structure that provides nutritional support, composed of approximately 70% neutral lipids. As shown in Fig. 9 , after incubating with the Py-LD probe for 30 minutes, the yolk sac in the zebrafish was clearly stained red in the red channel. This indicates that the Py-LD probe successfully marked the lipid structures within the yolk sac of the zebrafish. Therefore, the Py-LD probe shows great potential for applications in biological imaging. 4. Conclusions In summary, we designed and developed a novel symmetrical molecular scaffold, Py-R , based on pyrrole derivatives. Through the introduction of diverse substituents, a series of fluorescent dyes ( 3a-e ) were successfully synthesized. Based on systematic investigations into the photoluminescent properties of these dyes, we further engineered a polarity-sensitive probe, Py-LD , for targeted lipid droplet imaging. Remarkably, Py-LD exhibits not only excellent polarity-sensitive characteristics but also high fluorescence stability, low cytotoxicity, and a large Stokes shift (142 nm). Additionally, Py-LD efficiently labels intracellular LDs, enabling real-time monitoring of lipid droplet levels and successful application in zebrafish bioimaging. This study provides new insights for the development of similar probes and offers a powerful tool for cellular lipid droplet research. Declarations Declaration of Competing Interest The authors declare that they have no known competingfinan-cial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Jin-Hua Jiang and Jian-Yong Wang: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project Administration. Yun-Hao Yang and Ying-Kun Liu: Formal Analysis, Resources, Project Administration, Writing original draft preparation. Ning Zhang: Formal Analysis, Data Curation, Software. Acknowledgement This work was financially supported by Natural Science Foundation of China (21801145) and Major Scientific Research Project for the Construction of State Key Lab (No. 2025ZDGZ02). References Zehmer JK, Huang Y, Peng G, et al. A role for lipid droplets in inter-membrane lipid traffic. Proteomics 2009;9(4):914-921. https://doi.org/10.1002/pmic.200800584. Yang J, Guo Y, Pistolozzi M, et al. Research Progress of Multi-Functional Fluorescent Probes for Alzheimer’s Disease Monitoring. Dyes Pigm 2021;193:109466. https://doi.org/10.1016/j.dyepig.2021.109466. Yin J, Ma Y, Li G, et al. 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Punna Rao AML, Sridhar Rao A, Saratchandra Babu M, et al. Triphenylphosphine (PPh3) Catalyzed Erlenmeyer Reaction for Azlactones under Solvent-free Conditions. J Heterocyclic Chem 2017;54: 429–435. https://doi.org/10.1002/jhet.2600. Malhi H, Bronk SF, Werneburg NW, et al. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J Biol Chem 2006;281(17):12093–101. https://doi.org/10.1074/jbc.m510660200. Table Table 1 is available in the Supplementary Files section. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files PyLDSI.doc Table1Structureofstartingcompounds2.docx scheme1.png Scheme 1Synthesis route of 3a−e and Py-LD. Cite Share Download PDF Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted Editorial decision: Revision requested 08 Jan, 2026 Reviews received at journal 31 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 07 Dec, 2025 Reviewers invited by journal 07 Dec, 2025 Editor assigned by journal 03 Dec, 2025 Submission checks completed at journal 02 Dec, 2025 First submitted to journal 29 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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10:51:08","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3216,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/73809c2a1a516d4c1fa04e76.png"},{"id":97900241,"identity":"9d23110f-aaa1-4d0d-965c-90a0f2f0dcb5","added_by":"auto","created_at":"2025-12-10 15:45:19","extension":"xml","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124606,"visible":true,"origin":"","legend":"","description":"","filename":"6e60ba9af9fc4a7e8d09d495dc2318ce1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/bdbb2e11bb481e6cfdf4f428.xml"},{"id":97874247,"identity":"2afa0528-d7a4-4e9e-93df-76e6d4a26c42","added_by":"auto","created_at":"2025-12-10 10:51:08","extension":"html","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137719,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/42ae918e67b311b339c279c5.html"},{"id":97899313,"identity":"764ef2d3-9c96-48de-a321-dba4db096e05","added_by":"auto","created_at":"2025-12-10 15:42:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127004,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Normalized absorption spectra of compounds \u003cstrong\u003e3a–e\u003c/strong\u003e (10 µM) in \u003cstrong\u003eTHF\u003c/strong\u003e solution. (b) Normalized emission spectra of compounds \u003cstrong\u003e3a–e\u003c/strong\u003e (10 µM) in \u003cstrong\u003eTHF\u003c/strong\u003e solution.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/9ff4f061cb4da3eff07ba41f.png"},{"id":97874193,"identity":"ac4642ce-a5d3-44f5-b287-9ce8efa561f4","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":361335,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Absorption spectra of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different solvents. (b) Emission spectra of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different solvents. (c) Normalized fluorescence spectra of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different solvents. (d) Photographs of probe\u003cstrong\u003e Py-LD\u003c/strong\u003e in different polar solvents under UV light.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/d7d443b3340256bdfabe243e.png"},{"id":97900725,"identity":"cbc376ea-0965-4662-a268-37318e2a6481","added_by":"auto","created_at":"2025-12-10 15:45:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108408,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Maximum emission fluorescence intensity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different solvents. (b) The quantum yields of probe\u003cstrong\u003e Py-LD\u003c/strong\u003e in different solvents. (c) Linearity relationship of the fluorescence emission intensity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) with respect to the polarity-dependent parameter E\u003csub\u003eT\u003c/sub\u003e(30). (d) Linearity relationship for Stokes shift of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e with polarity dependent parameter E\u003csub\u003eT\u003c/sub\u003e(30).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/a01f19b878fbef99193d8375.png"},{"id":97899840,"identity":"47a71b5b-07af-41e4-886f-23ba2fa586e6","added_by":"auto","created_at":"2025-12-10 15:44:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":291063,"visible":true,"origin":"","legend":"\u003cp\u003eCalculation of the frontier molecular orbitals of the probe \u003cstrong\u003ePy-LD \u003c/strong\u003eindifferent solvent models, calculated with Gaussian’09 at the B3LYP/6-31+G(d,p) level.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/5c0dd4971d5181ab7a896f41.png"},{"id":97874198,"identity":"3a802fb3-cdbf-42b1-a89f-f2bfc4e7f582","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104707,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The fluorescence spectra of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different ratios of 1,4-Dioxane/MeOH. (b) Dotted line diagram of the relationship between the fluorescence emission intensity of the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 µM) in different ratios of 1,4-Dioxane /MeOH.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/14ec887bb1585a24cdc1c023.png"},{"id":97874204,"identity":"5956f36b-9a25-4927-8b35-8dbd3d3601ef","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103537,"visible":true,"origin":"","legend":"\u003cp\u003ea) Fluorescence stability of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 μM) in toluene, 1,4-Dioxane, and methanol solutions. (b) Fluorescence emission intensity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 μ M) in PBS buffers (1,4-Dioxane/PBS =1/1) of different pH (Slit: 20 nm). (c) Selectivity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 μM) at different ions. Analytes：1. SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e 2. PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e 3. ClO\u003csup\u003e-\u003c/sup\u003e 4. CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e 5. Cys 6. HSO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e 7. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 8. NO\u003csup\u003e2-\u003c/sup\u003e 9. Cl\u003csup\u003e-\u003c/sup\u003e 10. Na\u003csup\u003e+\u003c/sup\u003e 11. Fe\u003csup\u003e3+\u003c/sup\u003e 12. Hg\u003csup\u003e2+\u003c/sup\u003e 13. Mg\u003csup\u003e2+\u003c/sup\u003e 14. K\u003csup\u003e+\u003c/sup\u003e 15. Cu\u003csup\u003e2+\u003c/sup\u003e 16. Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/ffbdcd130e526282e2ebbc52.png"},{"id":97874209,"identity":"27a37552-29be-4b6f-8302-b8e0f269236f","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":332577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCo-localized\u003c/em\u003e cellular images of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (10µM) in live HeLa cells. (a) \u003cstrong\u003ePy-LD\u003c/strong\u003e (10µM) staining. (b) \u003cstrong\u003eBODIPY\u003c/strong\u003e (10µM) staining. (d) Merged image of (a) and (b). (d) Brightfield of HeLa cells; (e) Fluorescence intensity distribution along the \u003cstrong\u003eROI\u003c/strong\u003e line . The red channel of \u003cstrong\u003ePy-LD\u003c/strong\u003e: λ\u003csub\u003eex\u003c/sub\u003e = 405 nm, collected 550-700 nm. The green channel of \u003cstrong\u003eBODIPY\u003c/strong\u003e: λ\u003csub\u003eex\u003c/sub\u003e = 493 nm, collected 480-530 nm. scale bar:10 μm\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/a16019fade9d0b5405985ea0.png"},{"id":97899596,"identity":"74bf5faf-a313-4a1e-902b-ddc16c117ece","added_by":"auto","created_at":"2025-12-10 15:44:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":362036,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence images of \u003cstrong\u003ePy-LD\u003c/strong\u003e (10µM) in HeLa cells pretreated with oleic acid (0 μM, 50 μM, 100 μM) of different concentrations. (b) Histogram of the fluorescence intensities of the red channel in (a). λ\u003csub\u003eex\u003c/sub\u003e = 405 nm, collected 550-700 nm. Scale bar:10 μm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/49ca20850818eb20d90e9362.png"},{"id":97874220,"identity":"99805c7a-0777-487b-919a-04b874382e99","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":194033,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent images of living zebrafish treated with \u003cstrong\u003ePy-LD\u003c/strong\u003e (10 μM). (a) Bright field. (b) Red channel, λ\u003csub\u003eex\u003c/sub\u003e= 405 nm, collected 550-700 nm. (c) Merge images. Scale bar: 250μm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/ebef1d6ab9176402c44df286.png"},{"id":104740469,"identity":"64447e82-ed9a-4567-9432-83ebc93ce5e5","added_by":"auto","created_at":"2026-03-16 16:18:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2983136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/d1dea39b-8c33-42c1-b07c-5d6d1fe94404.pdf"},{"id":97900929,"identity":"1e861130-cebb-4506-8205-ae5bd70daac6","added_by":"auto","created_at":"2025-12-10 15:46:08","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1645423,"visible":true,"origin":"","legend":"","description":"","filename":"PyLDSI.doc","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/02a2b7b6bfa80e462367dc4b.doc"},{"id":97874194,"identity":"9c5d3713-cefc-49c1-b825-3a89aab49e79","added_by":"auto","created_at":"2025-12-10 10:51:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":117893,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Structureofstartingcompounds2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/264f51b53bc298b485e50c22.docx"},{"id":97899994,"identity":"e8b38df0-e63a-4216-bc84-81c4a54a08b3","added_by":"auto","created_at":"2025-12-10 15:45:09","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":36117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003eSynthesis route of \u003cstrong\u003e3a−e\u003c/strong\u003e and\u003cstrong\u003e Py-LD\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-8235573/v1/d7afc4cb8e3a7da4abcae325.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Construction of Novel Pyrrole-Derivative-Based Fluorescent Platforms and Development of Polarity-Sensitive Probes for Imaging Lipid Droplets in Cells and Zebrafish","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLipid droplets (LDs) are crucial intracellular organelles responsible for lipid storage \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Composed primarily of neutral lipids and cholesteryl esters, their unique structural feature is a hydrophobic core surrounded by a monolayer phospholipid membrane and specific proteins \u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. As vital dynamic organelles, LDs participate not only in maintaining energy homeostasis and regulating lipid metabolism but also influence diverse physiological and pathological processes by mediating cellular signaling \u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that aberrant accumulation of LDs is closely associated with metabolic diseases (such as obesity and diabetes) and cardiovascular diseases \u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Furthermore, alterations in the number and function of LDs are correlated with cancer development \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. This is primarily due to the strong affinity cancer cells exhibit for fatty acids and cholesterol, leading to the excessive storage of these lipids within LDs \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Consequently, cancer cells universally display a significant increase in LD number. The substantial accumulation of hydrophobic lipids within LDs in cancer cells results in a reduction in microenvironment polarity \u003csup\u003e[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Given the high levels of LDs in tumors and their impact on microenvironment polarity, accurately monitoring both the number and polarity of LDs is of significant importance for distinguishing cancer cells from normal cells, as well as for cancer monitoring and treatment.\u003c/p\u003e\u003cp\u003eFluorescent probes are regarded as highly promising and practical detection tools, playing a crucial role in specific detection, especially excelling in cellular imaging within biological systems \u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Compared with other analytical detection methods, fluorescent probe technology exhibits remarkable advantages such as low cost, strong real-time performance, high sensitivity, and high detection accuracy \u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In recent years, various fluorescent probes for specific lipid droplet imaging have been reported, successfully achieving specific labeling and dynamic monitoring of LDs in living cells \u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. However, many commonly used probes, such as Nile Red and BODIPY 493/503, still have obvious limitations, including insufficient photostability, short emission wavelengths, and small Stokes shifts \u003csup\u003e[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. These issues restrict their application in high-precision and long-term imaging. Therefore, the development of more novel lipid droplet-specific fluorescent probes with excellent optical properties, such as large Stokes shifts, long emission wavelengths, and high photostability, is of urgent and significant importance for a deeper understanding of the functions of LDs in physiological and pathological processes and for promoting the development of early cancer diagnosis techniques based on lipid droplet imaging.\u003c/p\u003e\u003cp\u003eIn this work, we designed and investigated a class of pyrrole derivatives. Their ease of modification and unique symmetrical molecular structure render them suitable for constructing donor-acceptor-donor (D-A-D) type fluorescent probes. Studies have shown that probes with strong electron donor-acceptor (D-A) interactions generally exhibit excellent polarity sensitivity. By introducing different electron-donating substituents, we successfully synthesized a series of novel pyrrole derivatives \u003cb\u003e3a-e\u003c/b\u003e. Among them, the compounds containing strong electron-donating groups exhibit typical D-A-D structural features, demonstrating longer emission wavelengths, larger Stokes shifts, and stronger fluorescence intensities. Based on these findings, we successfully constructed a novel polarity probe, \u003cb\u003ePy-LD\u003c/b\u003e. Optical tests indicate that the emission wavelength of \u003cb\u003ePy-LD\u003c/b\u003e significantly redshifts as the solvent polarity increases, confirming its effectiveness as a polarity-sensitive probe. This probe also possesses good biocompatibility and pH stability. Additionally, \u003cb\u003ePy-LD\u003c/b\u003e shows highly specific lipid droplet-targeting ability, with co-localization imaging with the commercial lipid droplet dye \u003cb\u003e(BODIPY\u003c/b\u003e) showing a high degree of overlap (r\u0026thinsp;=\u0026thinsp;0.92). \u003cb\u003ePy-LD\u003c/b\u003e has been successfully applied to lipid droplet imaging studies in cell and zebrafish models.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cp\u003e\u003cstrong\u003e2.1. Reagents and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Naphthalene-1,4-dione, Iodomethane，KOH, Benzaldehyde, 4-Bromobenzaldehyde, 4-Hydroxybenzaldehyde, p-Tolualdehyde and 4-Dimethylaminobenzaldehyde were bought from Macklin (Shanghai, China). All reagents and drugs involved in the Experimental section were commercially purchased and used directly without secondary purification. All analytical equipment and experimental conditions are described in detail in the Supporting Information, including general procedures for spectroscopic testing, test parameters, and cytotoxicity tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. General synthesis procedure of fluorescent dyes 3a\u0026ndash;e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScheme 1 outlines the general procedure for the synthesis of pyrrole products \u003cstrong\u003e3a\u0026minus;e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003ePy-LD\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eSynthesis of compound \u003cstrong\u003e3a\u0026minus;e\u003c/strong\u003e. 1,4-Naphthoquinone (316.4 mg, 2.0 mmol, 1.0 equiv) and benzaldehyde with different substituents (\u003cstrong\u003e2a-e\u003c/strong\u003e) (8.0 mmol, 4.0 equiv) and ammonium iodide (869.6 mg, 6.0 mmol, 3.0 equiv) were placed into a 100 mL reaction vial, and 10 mL of toluene was added as solvent. The reaction was stirred at 130 \u0026deg;C for 24 hours. At the end of the reaction, it was cooled to room temperature and volatiles were removed under reduced pressure. The residue was purified by column chromatography on neutral alumina to give the desired products \u003cstrong\u003e3a-e\u003c/strong\u003e (Table 1).\u003c/p\u003e\n\u003cp\u003eSynthesis of compound \u003cstrong\u003ePy-LD\u003c/strong\u003e. \u003cstrong\u003e3\u003c/strong\u003ee (87.3 mg, 0.25 mmol, 1.0 equiv) was added to a suspension of KOH (70.0 mg, 1.25 mmol, 5.0 equiv) in DMF (5.0 mL) at 0 \u0026deg;C. After the solution was stirred for 1 h, CH\u003csub\u003e3\u003c/sub\u003eI (31.0 \u0026mu;L, 0.5 mmol, 2.0 equiv) was added. The reaction was then stirred at 50 \u0026deg;C overnight. At the end of the reaction, the reaction solution was poured into water and the crude product was extracted with dichloromethane. The solution was dried and the solvent was evaporated to obtain the crude product. Finally, the crude product was purified by column chromatography on neutral alumina to give the product \u003cstrong\u003ePy-LD\u003c/strong\u003e (72.7 mg, yield: 80%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta; 8.04 (dd, J = 5.8, 3.3 Hz, 2H), 7.75 (dd, J = 5.8, 3.3 Hz, 2H), 7.51 \u0026ndash; 7.42 (m, 4H), 6.87 \u0026ndash; 6.79 (m, 4H), 3.34 (s, 3H), 3.01 (s, 12H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; 179.93, 150.84, 140.37, 136.34, 132.55, 131.68, 126.64, 117.84, 111.66, 40.36, 33.80. HRMS (ESI) Found: 450.2174 [M+H]\u003csup\u003e+\u003c/sup\u003e; Molecular formula C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e requires [M+H]\u003csup\u003e+\u003c/sup\u003e 450.2177.\u003c/p\u003e\n\u003cp\u003eTo obtain details regarding the synthesis and characterization of \u003cstrong\u003e3a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ee\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;Py-LD\u003c/strong\u003e, please refer to the Supplementary Information (SI). Detailed characterization is shown in \u003cstrong\u003eFigs. S7-S23\u003c/strong\u003e.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Design and luminescence characterization of dyes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3a-e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn recent years, with the rapid development of organic synthesis and fluorescence technology, a large number of new fluorescent organic molecules have been synthesized and studied. Consequently, an increasing number of novel probes have been successfully designed and applied to lipid droplet research. LDs are lipid cores composed of a phospholipid monolayer and fatty acids, with a hydrophobic environment inside. They are surrounded by a polar environment rich in water and inorganic ions. This significant polarity difference between the inside and outside provides ideas for the design of lipid droplet probes \u003csup\u003e[35,36]\u003c/sup\u003e. Theoretically, if a fluorescent probe can exhibit differential fluorescence properties in these two microenvironments with different polarities, it is expected to become an effective tool for accurately labeling LDs. Based on the above principles, this study developed a novel pyrrole-based fluorescent platform, \u003cstrong\u003ePy-R\u003c/strong\u003e. First, the multi-phenyl ring structure of \u003cstrong\u003ePy-R\u003c/strong\u003e confers a degree of lipophilicity, which facilitates probe targeting towards LDs. Furthermore, by modifying substituents, the structural features of the \u003cstrong\u003ePy-R\u003c/strong\u003e platform can be utilized to construct typical donor-acceptor-donor (D-A-D) conjugated molecules. This design enhances the intramolecular charge transfer (ICT) effect, thereby imparting polarity-sensitive properties \u003csup\u003e[37]\u003c/sup\u003e. Consequently, we anticipated that it would emit strong fluorescence at short wavelengths in low-polarity solvents. Conversely, in high-polarity solvents, it was expected that significant charge separation might occur, leading to longer emission wavelengths with weaker fluorescence intensity. It was hypothesized that this pronounced solvatochromic effect would enable specific staining of intracellular LDs.\u003c/p\u003e\n\u003cp\u003eBased on the above, we prepared a series of novel fluorescent dyes (\u003cstrong\u003e3a-e\u003c/strong\u003e) by a one-step method using benzaldehyde (\u003cstrong\u003e2a-e\u003c/strong\u003e) with different para-substituents as raw materials.\u0026nbsp;These dyes based on pyrrole derivatives are typical polar conjugated systems, which motivates us to study their luminescent properties.\u0026nbsp;The UV absorption and fluorescence spectra of these dyes (\u003cstrong\u003e3a-e\u003c/strong\u003e) were first tested in different solvents (\u003cstrong\u003eFigs. S1-S5\u003c/strong\u003e).\u0026nbsp;It can be seen that the introduction of different substituent modifications can significantly change the luminescent properties of the fluorophores. The fluorescence emission of dyes\u003cstrong\u003e\u0026nbsp;3a-e\u003c/strong\u003e changes significantly over a wide wavelength range of 500-650 nm. As expected, the introduction of a strong electron donor group into the fluorophore leads to the formation of a D-A-D structure, resulting in excellent fluorescence properties (\u003cstrong\u003eFig.1\u003c/strong\u003e). Taking the fluorescent compound \u003cstrong\u003e3e\u003c/strong\u003e as an example, \u003cstrong\u003e3e\u003c/strong\u003e exhibits longer absorption and emission wavelengths due to the better electron-donating effect of dimethylamino groups. Fluorescent probes with longer emission wavelengths have been reported to have the advantages of less background interference, low energy light sources, strong tissue penetration and less tissue damage \u003csup\u003e[38-40]\u003c/sup\u003e. This prompted us to carry out further studies on compound \u003cstrong\u003e3e\u003c/strong\u003e in order to construct fluorescent probes with excellent photophysical properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Design and photophysical properties of the probe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure S5, the emission wavelength of fluorescent compound\u003cstrong\u003e\u0026nbsp;3e\u003c/strong\u003e undergoes a significant red-shift with increasing solvent polarity. The compound exhibits markedly higher fluorescence emission intensity in low-polarity/non-protic solvents (toluene) compared to high-polarity/protic solvents (methanol). This behavior indicates the presence of an ICT character: high-polarity solvents stabilize the charge-separated excited state, promoting non-radiative decay and consequent fluorescence quenching. Combined with the lipophilicity imparted by its multi-phenyl ring structure, these properties make it suitable for the design of fluorescent probes targeting hydrophobic microenvironments. Furthermore, the secondary amino group (-NH-) in compound\u003cstrong\u003e\u0026nbsp;3e\u0026nbsp;\u003c/strong\u003eexhibits high reactivity and a propensity for hydrogen bonding, which may induce molecular aggregation and lead to concentration-dependent fluorescence quenching. To enhance chemical stability and suppress aggregation effects, we modified the reactive amino group in \u003cstrong\u003e3e\u003c/strong\u003e through methylation, ultimately affording the fluorescent probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (\u003cstrong\u003eScheme\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e Subsequently, we systematically investigated the UV-visible absorption spectra of \u003cstrong\u003ePy-LD\u003c/strong\u003e in different solvents and calculated valuable spectral data such as absorption and emission wavelengths, Stokes shifts, fluorescence quantum yields and half-peak widths of the dye \u003cstrong\u003ePy-LD\u003c/strong\u003e in different solvents (\u003cstrong\u003eFig. 2a and b, Table 1\u003c/strong\u003e). As expected, \u003cstrong\u003ePy-LD\u003c/strong\u003e similarly has a long emission wavelength, large Stokes shift (142 nm) and good polarity response properties. This motivated us to develop and further validate it as a novel lipid droplet-targeted polarity-sensitive probe \u003cstrong\u003ePy-LD\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo further investigate the photophysical-chemical properties of \u003cstrong\u003ePy-LD\u003c/strong\u003e, we tested its absorption and fluorescence spectra in different solvents.\u0026nbsp;It can be seen that the maximum absorption wavelength remained relatively constant in different solvents (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Whereas in fluorescence spectra, the maximum emission wavelength of \u003cstrong\u003ePy-LD\u003c/strong\u003e undergoes a significant redshift with increasing solvent polarity (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). This trend can be observed more clearly from the normalised spectra of \u003cstrong\u003ePy-LD\u003c/strong\u003e in different solvents (\u003cstrong\u003eFig. 2c\u003c/strong\u003e). Under 365 nm UV irradiation, the solvent colour of \u003cstrong\u003ePy-LD\u003c/strong\u003e also changed from yellow to red as the solvent polarity increased, which is consistent with the spectroscopic test results and shows a positive solvatochromic effect (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). It is worth noting that \u003cstrong\u003ePy-LD\u003c/strong\u003e undergoes a polarity quenching effect in highly polar solvents (Acetone, DMF), resulting in suppression of fluorescence emission and inability to observe fluorescence changes in the solvent. These results all indicate that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e has good polarity response characteristics and has the potential to be a polarity sensitive probe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Polarity-sensitivity study of the probe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further assess the sensitivity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e to polarity, we quantified the polarity of different solvents using the polarity sensitivity parameter E\u003csub\u003eT\u003c/sub\u003e(30) and collated photophysical number of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e to investigate changes in fluorescence. (\u003cstrong\u003eTable 1\u003c/strong\u003e). At the same time, we used these data to draw graphs to intuitively analyze the relationship between \u003cstrong\u003ePy-LD\u003c/strong\u003e and polarity change. It is evident from \u003cstrong\u003eFig. 3a\u003c/strong\u003e that the emission intensity of the probe decreases regularly with increasing solvent polarity. In this case, the fluorescence quantum yield also shows a significant decrease with increasing solvent polarity due to the ICT effect of the probe (\u003cstrong\u003eFig. 3b\u003c/strong\u003e).\u0026nbsp;We also plotted the fitted curves for the maximum fluorescence emission intensity, Stokes shift, and the solvent polarity parameter E\u003csub\u003eT\u003c/sub\u003e(30), with correlation coefficients exceeding \u003cstrong\u003e0.99\u003c/strong\u003e in both cases (\u003cstrong\u003eFig. 3c and d\u003c/strong\u003e). These analyses favourably demonstrate that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e is polarity sensitive and has good linear correlation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Photophysical properties of compound \u003cstrong\u003ePy-LD\u003c/strong\u003e in different solvents.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"756\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eToluene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDioxane\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTHF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEtOAc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMSO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeCN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEtOH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeOH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u0026lambda;\u003csub\u003eabs\u003c/sub\u003e (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e482\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e480\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u0026lambda;\u003csub\u003eem\u003c/sub\u003e (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e584\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e596\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e608\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e608\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e653\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e650\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e(30)\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e33.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e38.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e42.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e43.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e45.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e45.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e51.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e55.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e63.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eStokes shift (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e126\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e134\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 52px;\"\u003e\n \u003cp\u003e142\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 58px;\"\u003e\n \u003cp\u003e146\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e163\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e170\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026Phi;\u003c/em\u003e\u003csub\u003eF\u0026nbsp;\u003c/sub\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e24.24%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.17%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e5.48%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e4.13%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e1.37%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.13%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e0.09%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e0.17%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e0.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.43%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eLog\u003cem\u003e\u0026epsilon;\u003c/em\u003e\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.46\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.90\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e3.73\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e3.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e3.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e3.65\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e5.54\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e5.59\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e5.57\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e3.90\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e4.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eFWHM\u003cem\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e92.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e95.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e99.63\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e100.91\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e99.82\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e114.12\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e199.17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e233.24\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e227.81\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e176.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e94.77\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e E\u003csub\u003eT\u003c/sub\u003e(30) is a solvent polarity parameter and means the molar transition energy in kcal mol\u003csup\u003e\u0026minus;1\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[41]\u003c/sup\u003e. \u003csup\u003eb\u003c/sup\u003e Fluorescence quantum yield was obtained by the reference method, by applying equation \u003cem\u003e\u0026Phi;\u003csub\u003ex\u003c/sub\u003e = \u0026Phi;s(n\u003csub\u003ex\u003c/sub\u003e/n\u003csub\u003es\u003c/sub\u003e)\u003csup\u003e2\u003c/sup\u003e(A\u003csub\u003es\u003c/sub\u003e/A\u003csub\u003ex\u003c/sub\u003e)(F\u003csub\u003ex\u003c/sub\u003e/F\u003csub\u003es\u003c/sub\u003e)\u003c/em\u003e. \u003csup\u003ec\u0026nbsp;\u003c/sup\u003eMolar extinction coefficients are calculated in the maximum of the highest peak by the Beer Lambert law. \u003csup\u003ed\u003c/sup\u003e Full width at half maximum (FWHM) was determined for emission band in solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Theoretical calculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo rationally investigate the electronic properties of \u003cstrong\u003ePy-LD\u003c/strong\u003e, we performed theoretical calculations on the energy levels of the compound. Using Density Functional Theory (DFT), we calculated the geometric configuration of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e and the electron density distribution of its HOMO and LUMO energy levels (\u003cstrong\u003eFig. 4\u003c/strong\u003e). The dimethylamino group, as an electron-donating moiety, is expected to enhance the intramolecular charge transfer (ICT) efficiency. Therefore, we analyzed the frontier molecular orbitals (HOMO/LUMO) of \u003cstrong\u003ePy-LD\u003c/strong\u003e. As shown in Fig. 4, the HOMO and LUMO energies of \u003cstrong\u003ePy-LD\u003c/strong\u003e were calculated to be -4.852 eV and -1.971 eV, respectively, under vacuum-level reference conditions. Furthermore, the HOMO orbital is predominantly localized on the dimethylamino donor group, while the LUMO orbital is distributed across the entire isoindole-ketone acceptor skeleton, indicating a strong conjugated system (\u003cstrong\u003eFig. 4\u003c/strong\u003e). Additional calculations of the frontier molecular orbitals and energies in various solvent models further confirmed significant ICT characteristics, providing theoretical evidence for the observed solvatochromic effect and polarity-sensitive behavior of this probe (\u003cstrong\u003eFig. 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Lipophilicity study of probe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipophilic analysis is a promising method for verifying the ability of probes to label LDs. LDs are the most hydrophobic organelles, and similar to the principle of similar-phase solubility, the hydrophobicity of the molecular skeleton allows for specific aggregation in the LDs of the cell. The polarity sensitivity of the probe \u003cstrong\u003ePy-LD\u0026nbsp;\u003c/strong\u003egives it a stronger fluorescence emission in less polar solvents, thus significantly brightening the LDs. Because the probe structure itself has multiple alicyclic structures and exhibits specific fluorescence response in lipid solvents, it has been further explored. We characterized the fluorescence emission spectra of the probes in different ratios of 1,4-Dioxane/MeOH mixed solvents, using 1,4-Dioxane as the lipophilic solvent and MeOH as the protonic solvent. The analyses showed that the fluorescence emission intensity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e was significantly enhanced with the increase of 1,4-Dioxane content in the mixed solvent, and the maximum emission wavelength showed an obvious blue shift (\u003cstrong\u003eFig. 5a and b\u003c/strong\u003e). This fully demonstrated that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e exhibited excellent sensitivity and good fluorescence emission intensity to the fat-soluble environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Stability study of the probe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotostability is an important characteristic of the probe. Therefore, we investigated the variation of fluorescence intensity over time for the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e in toluene, 1,4-Dioxane, and methanol solutions, respectively. As shown in \u003cstrong\u003eFig. 6a\u003c/strong\u003e, the fluorescence intensity of the probe remained stable in different polar environments under prolonged continuous light excitation. To test whether \u003cstrong\u003ePy-LD\u003c/strong\u003e adapts to the complex environment inside living cells, we investigated the emission behaviour of \u003cstrong\u003ePy-LD\u003c/strong\u003e over a wide \u003cstrong\u003epH\u003c/strong\u003e range in phosphate buffer solution. As shown in \u003cstrong\u003eFig. 6b\u003c/strong\u003e, the fluorescence intensity of the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe remains relatively stable across different pH environments, which indicated that the probe was less affected by pH. The results show that the probe exhibits good fluorescence stability in the biological \u003cstrong\u003epH\u003c/strong\u003e range. In addition, a large number of interfering substances exist in the complex cellular environment. In order to study the anti-interference performance of the probe, 1. SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e 2. PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e 3. ClO\u003csup\u003e-\u003c/sup\u003e 4. CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e 5. Cys 6. HSO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e 7. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 8. NO\u003csup\u003e2-\u003c/sup\u003e 9. Cl\u003csup\u003e-\u003c/sup\u003e 10. Na\u003csup\u003e+\u003c/sup\u003e 11. Fe\u003csup\u003e3+\u003c/sup\u003e 12. Hg\u003csup\u003e2+\u003c/sup\u003e 13. Mg\u003csup\u003e2+\u003c/sup\u003e 14. K\u003csup\u003e+\u003c/sup\u003e 15. Cu\u003csup\u003e2+\u003c/sup\u003e 16. Ca\u003csup\u003e2+\u003c/sup\u003e were added as the interfering substances. The effect of these analytes on the fluorescence intensity was negligible as shown in \u003cstrong\u003eFig. 6c\u003c/strong\u003e. These results indicate that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e exhibits good anti-interference properties and can be used for polarity detection in complex environments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Probe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePy-LD\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;for bioimaging applications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the above experimental results, we further applied \u003cstrong\u003ePy-LD\u003c/strong\u003e to cell imaging experiments. Firstly, MTT assay was performed to detect the biotoxicity of probe \u003cstrong\u003ePy-LD\u003c/strong\u003e. After incubating different concentrations of the probe with \u003cstrong\u003eHeLa\u003c/strong\u003e cells for 24 h, the cell viability remained above 80% (\u003cstrong\u003eFig. S6)\u003c/strong\u003e. This indicates that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e has low cytotoxicity and good biocompatibility. Next to validate the specific targeting ability of the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e on cellular LDs, co-localisation imaging experiments were performed in a \u003cstrong\u003eHeLa\u003c/strong\u003e cell model using the commercial lipid droplet dye\u003cstrong\u003e\u0026nbsp;BODIPY 493/503\u003c/strong\u003e in conjunction with the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e (\u003cstrong\u003eFig. 7\u003c/strong\u003e).\u0026nbsp;By confocal fluorescence scanning microscopy (\u003cstrong\u003eCLSM\u003c/strong\u003e) imaging, we can visualise the red probe \u003cstrong\u003ePy-LD\u003c/strong\u003e accumulating in cellular LDs and showing bright fluorescence. In the same cells,\u003cstrong\u003e\u0026nbsp;Py-LD\u003c/strong\u003e and \u003cstrong\u003eBODIPY 493/503\u003c/strong\u003e showed the same staining effect, and the fluorescence in the two channels showed significant overlap, and the Pearson correlation coefficient reached \u003cstrong\u003e0.92\u003c/strong\u003e. This suggests that the probe \u003cstrong\u003ePy-LD\u003c/strong\u003e, like \u003cstrong\u003eBODIPY 493/503\u003c/strong\u003e, has a specific targeting effect on LDs. The co-localization curve of the specific region in \u003cstrong\u003eFig. 7e\u003c/strong\u003e also confirms the above viewpoints. The experimental results showed that the probe \u003cstrong\u003ePy-LD\u0026nbsp;\u003c/strong\u003eshowed good targeting to lipid droplet in living cells and could be used for lipid droplet imaging in living cells.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the ability of the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe to sensitively label LDs in cells, we used oleic acid (\u003cstrong\u003eOA\u003c/strong\u003e) to stimulate the generation of LDs in cells. Oleic acid is an unsaturated fatty acid that has been proven to be an effective inducer of lipid droplet formation, helping promote the accumulation of a large number of endogenous LDs \u003csup\u003e[42]\u003c/sup\u003e. Therefore, we treated \u003cstrong\u003eHeLa\u003c/strong\u003e cells with the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe and incubated them with different concentrations of \u003cstrong\u003eOA\u003c/strong\u003e (0 \u0026mu;M, 50 \u0026mu;M, 100 \u0026mu;M) for half an hour before conducting cell imaging. As shown in \u003cstrong\u003eFig. 8a\u003c/strong\u003e, as the concentration of \u003cstrong\u003eOA\u003c/strong\u003e increased, the size and number of LDs in the cells significantly increased. The accumulation of more LDs resulted in a stronger fluorescent signal from \u003cstrong\u003ePy-LD\u003c/strong\u003e, and the fluorescence intensity was positively correlated with the \u003cstrong\u003eOA\u003c/strong\u003e concentration (\u003cstrong\u003eFig. 8b\u003c/strong\u003e). Furthermore, after staining with \u003cstrong\u003ePy-LD\u003c/strong\u003e, the fluorescence images clearly showed the presence of small LDs in the cells. These results indicate that the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe can sensitively label LDs in cells and visually display the size and quantity of LDs. Therefore, \u003cstrong\u003ePy-LD\u003c/strong\u003e is an excellent tool for monitoring LDs.\u003c/p\u003e\n\u003cp\u003eFinally, we assessed the performance of the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe in biological imaging, using zebrafish as a model organism to evaluate its imaging capabilities. During zebrafish development, the yolk sac is an important structure that provides nutritional support, composed of approximately 70% neutral lipids. As shown in \u003cstrong\u003eFig. 9\u003c/strong\u003e, after incubating with the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe for 30 minutes, the yolk sac in the zebrafish was clearly stained red in the red channel. This indicates that the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe successfully marked the lipid structures within the yolk sac of the zebrafish. Therefore, the \u003cstrong\u003ePy-LD\u003c/strong\u003e probe shows great potential for applications in biological imaging.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, we designed and developed a novel symmetrical molecular scaffold, \u003cb\u003ePy-R\u003c/b\u003e, based on pyrrole derivatives. Through the introduction of diverse substituents, a series of fluorescent dyes (\u003cb\u003e3a-e\u003c/b\u003e) were successfully synthesized. Based on systematic investigations into the photoluminescent properties of these dyes, we further engineered a polarity-sensitive probe, \u003cb\u003ePy-LD\u003c/b\u003e, for targeted lipid droplet imaging. Remarkably, \u003cb\u003ePy-LD\u003c/b\u003e exhibits not only excellent polarity-sensitive characteristics but also high fluorescence stability, low cytotoxicity, and a large Stokes shift (142 nm). Additionally, \u003cb\u003ePy-LD\u003c/b\u003e efficiently labels intracellular LDs, enabling real-time monitoring of lipid droplet levels and successful application in zebrafish bioimaging. This study provides new insights for the development of similar probes and offers a powerful tool for cellular lipid droplet research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competingfinan-cial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJin-Hua Jiang and Jian-Yong Wang: Conceptualization, Resources, Writing - Review \u0026amp; Editing, Supervision, Project Administration. Yun-Hao Yang and Ying-Kun Liu: Formal Analysis, Resources, Project Administration, Writing original draft preparation. Ning Zhang: Formal Analysis, Data Curation, Software.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was financially supported by Natural Science Foundation of China (21801145) and Major Scientific Research Project for the Construction of State Key Lab (No. 2025ZDGZ02).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZehmer JK, Huang Y, Peng G, et al. A role for lipid droplets in inter-membrane lipid traffic. Proteomics 2009;9(4):914-921. https://doi.org/10.1002/pmic.200800584.\u003c/li\u003e\n\u003cli\u003eYang J, Guo Y, Pistolozzi M, et al. Research Progress of Multi-Functional Fluorescent Probes for Alzheimer\u0026rsquo;s Disease Monitoring. Dyes Pigm 2021;193:109466. https://doi.org/10.1016/j.dyepig.2021.109466.\u003c/li\u003e\n\u003cli\u003eYin J, Ma Y, Li G, et al. A Versatile Small-Molecule Fluorescence Scaffold: Carbazole Derivatives for Bioimaging. Coord. Chem. 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J Heterocyclic Chem 2017;54: 429\u0026ndash;435. https://doi.org/10.1002/jhet.2600.\u003c/li\u003e\n\u003cli\u003eMalhi H, Bronk SF, Werneburg NW, et al. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J Biol Chem 2006;281(17):12093\u0026ndash;101. https://doi.org/10.1074/jbc.m510660200.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lipid droplets, Polarity-sensitive, Fluorescent probe, Cell imaging","lastPublishedDoi":"10.21203/rs.3.rs-8235573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8235573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipid droplets (LDs), serving as central hubs in lipid metabolism, exhibit dynamic changes closely associated with diseases such as obesity, diabetes, and cancer. Visualizing LDs is crucial for elucidating their role in biological mechanisms and facilitating early disease detection. Donor-acceptor (D-A) type fluorescent probes have been widely designed and employed for LD detection. In this study, we designed and constructed a pyrrole-based molecular scaffold with a symmetrical architecture. Through modulation of para-substituents, a series of novel fluorescent dyes (\u003cb\u003e3a-e\u003c/b\u003e) were successfully synthesized. Experimental characterization revealed that the dimethylamino-modified dye \u003cb\u003e3e\u003c/b\u003e, characterized by a typical D-A-D structure, exhibits red emission (λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;608 nm in THF), significant solvatochromism, and a large Stokes shift (134 nm). To enhance its stability, the pyrrole N-H group of \u003cb\u003e3e\u003c/b\u003e was further methylated, successfully yielding a fluorescent LD probe, \u003cb\u003ePy-LD\u003c/b\u003e. \u003cb\u003ePy-LD\u003c/b\u003e not only exhibits a large Stokes shift (142 nm in THF), significantly reducing self-absorption and enhancing imaging signal-to-noise ratio, but also demonstrates excellent polarity sensitivity, enabling polarity detection. Furthermore, \u003cb\u003ePy-LD\u003c/b\u003e displays high specificity and stability. It was successfully applied for dynamic tracking of LDs in live cells and lipid imaging within the zebrafish yolk sac, providing a novel tool for LD monitoring.\u003c/p\u003e","manuscriptTitle":"Construction of Novel Pyrrole-Derivative-Based Fluorescent Platforms and Development of Polarity-Sensitive Probes for Imaging Lipid Droplets in Cells and Zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 10:50:57","doi":"10.21203/rs.3.rs-8235573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-08T12:07:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T08:45:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-14T14:57:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119529733133975764276683233442400365669","date":"2025-12-08T21:10:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308086622999014667163794543952981815536","date":"2025-12-08T14:20:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207117127443787612683371207890325193197","date":"2025-12-07T17:35:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-07T17:25:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T08:50:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-02T15:58:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photochemical \u0026 Photobiological Sciences","date":"2025-11-29T08:23:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f2590379-c991-45da-8100-b5d83cfe4887","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:14:56+00:00","versionOfRecord":{"articleIdentity":"rs-8235573","link":"https://doi.org/10.1007/s43630-026-00876-2","journal":{"identity":"photochemical-and-photobiological-sciences","isVorOnly":false,"title":"Photochemical \u0026 Photobiological Sciences"},"publishedOn":"2026-03-12 15:57:48","publishedOnDateReadable":"March 12th, 2026"},"versionCreatedAt":"2025-12-10 10:50:57","video":"","vorDoi":"10.1007/s43630-026-00876-2","vorDoiUrl":"https://doi.org/10.1007/s43630-026-00876-2","workflowStages":[]},"version":"v1","identity":"rs-8235573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8235573","identity":"rs-8235573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}