Stable Super‐Resolution Imaging of Lipid Droplet Dynamics through a Buffer Strategy with a Hydrogen‐Bond Sensitive Fluorogenic Probe

Abstract

Although super‐resolution imaging offers an opportunity to visualize cellular structures and organelles at the nanoscale level, cellular heterogeneity and unpredictability still pose a significant challenge in the dynamic imaging of live cells. It is thus vital to develop better‐performing and more photostable probes for long‐term super‐resolution imaging. Herein, we report a probe, LD‐FG, for imaging lipid droplet (LD) dynamics using structured illumination microscopy (SIM). LD‐FG allows wash‐free imaging of LDs, owing to a hydrogen‐bond sensitive fluorogenic response. The replacement of photobleached LD‐FG by intact probe molecules outside the LDs ensures the long‐time stability of the fluorescence imaging. With this buffering fluorogenic probe, fast and unpredictable dynamic processes of LDs can be visualized. Using this probe, two LD coalescence modes were discovered. The dynamic imaging also allowed us to propose a new model of LD maturation during adipocyte differentiation, i.e., a fast LD coalescence followed by a slow ripening step. The excellent performance of LD‐FG makes the buffer strategy an effective method for designing fluorescent probes for cell dynamic imaging.

A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets

“…Under the STED laser intensity of 5 MW cm −2 , the STED imaging with gate delay times (tg) of 1 ns, 3 ns or 6 ns provided FWHM resolutions of 93 ± 9 nm, 60 ± 7 nm, or even up to 37 ± 4 nm (Figure 3D and Figure S17B). The resolution of 37 ± 4 nm is substantially broken the diffraction limit of light (~250 nm) and represents the highest resolution of fluorescence imaging of LDs up to date (Table S3) [11][12][13][14][15][16]. This is also a state-of-the-art resolution of living cell STED super-resolution imaging (Table S4) [33][34][35][36][37][38][39][40][41][42][43][44][45].…”

“…To visualize LDs and reveal their versatile functions, the confocal and wide-field fluorescence microscopies have been widely employed with sub-micrometer resolution (~250 nm) [6][7][8][9][10]. The emerging super-resolution fluorescence microscopies, such as stimulated emission depletion microscopy (STED) and photoactivated localization microscopy (PALM), successfully broke the resolution limit of light diffraction and make it possible to further visualize the small/nascent LDs with nanoscale resolution [11][12][13][14][15][16]. For example, our group developed a super-photostable LDs fluorescent probe Lipi-DSB for STED super-resolution imaging, successfully visualizing the nanoscale fusion process of nascent LDs [12].…”

“…Xiao and coworkers designed a series of visible-light-activated fluorescence switch probes for PALM super-resolution imaging of LDs, achieving the localization precision of 13 nm in the LDs images 13 . Xu and coworkers reported a hydrogen-bond-sensitive fluorogenic LDs probe LD-FG for structured illumination microscopy (SIM) super-resolution imaging, impressively tracking various dynamic process of cellular LDs 14 . Niu and coworkers developed a series of rigidly structured solvatochromic fluorescent probes for SIM super-resolution imaging of cytosolic and nuclear LDs, acquiring the unprecedented spatial resolution for nuclear LDs of 142 nm 15 .…”

pH-Triggered Charge Reversible Fluorescent Probe for Simultaneous Imaging of Lipid Droplets and Nucleoli in Living Cells

“…10 shrinkage and heterogeneity and composition variations. 32 Although various dynamics of LDs and their cytoplasmic functions have been thoroughly explored, the functions and changes of LDs in the nucleus are ambiguous. In 2019, Fujimoto et al reported that nuclear LDs in hepatocytes were derived from apolipoprotein B (ApoB)-free lumenal LDs by immunofluorescence microscopy costained with BODIPY 558 (commercial LD probe) and Hoechst 33342 (commercial nucleus probe).…”

“…In recent years, the fluorescent probe has been extensively employed in biological detection and analysis due to its advantages of nondestructive in real time and in situ detection. − Yu and co-workers reported a fluorescent probe for the simultaneous visualization of LDs and ER successfully by two different colors . In addition, Xu et al tracked various types of LD dynamics, such as growth and shrinkage and heterogeneity and composition variations . Although various dynamics of LDs and their cytoplasmic functions have been thoroughly explored, the functions and changes of LDs in the nucleus are ambiguous.…”