Single-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells

Nature Communications
Santosh AdhikariElias M Puchner

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.

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Citations

Jan 30, 2020·Methods and Applications in Fluorescence·Angel ManceboElias M Puchner
Sep 25, 2020·The Journal of Membrane Biology·Maria S Ioannou
Sep 29, 2019·Nature Methods·Rita Strack
Apr 30, 2021·ACS Chemical Biology·Aditya IyerBert Poolman
Jun 25, 2021·Current Opinion in Microbiology·Gaurav Kumar, Sharmistha Sinha
Aug 18, 2020·ACS Chemical Biology·Ádám EördöghPablo Rivera-Fuentes
Sep 20, 2021·Journal of Molecular Biology·Nitesh Kumar PodhGunjan Mehta

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Methods Mentioned

BETA
optical diffraction
fluorescence microscopy
fluorescence
fluorescence spectroscopy
super-resolution
conventional
electron microscopy
super-resolution microscopy
PCR
fluorescence imaging

Software Mentioned

INSIGHT
Igor
NL
Pro
Fiji
BODIPY
ImageJ

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