Abstract
Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.
Highlights
Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale
We demonstrate that these BODIPY probes are applicable for SMLM in living mammalian cells by resolving and tracking single fatty acid analogs, as well as lysosomes, using a BODIPY-Lysotracker
BODIPY-C12 localizes to the endoplasmic reticulum (ER) and lipid droplets (LDs) and has been widely used to follow the uptake and distribution of fatty acid molecules with conventional fluorescence microscopy[26,28,32]
Summary
Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. We employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. The concentration of the monomeric BODIPY can be used to tune the density of singlemolecule localizations to the typical range of SMLM experiments Due to their favorable spectroscopic properties[19], hundreds of BODIPY conjugates labeling different cellular compartments and biomolecules have been developed for conventional fluorescence microscopy applications[20,21,22,23]. We develop the multi-color SMLM capability of various conventional BODIPY conjugates to quantify the nanoscopic spatial distribution and the dynamics of single fatty acid analogs and lipid droplet (LD) probes in living yeast cells with
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