Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Summary

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Abstract

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (D_II) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-D_II states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work. 

Introduction

Single-molecule localization microscopy (SMLM) techniques such as stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM) have emerged as methods for generating super-resolution images with information beyond Abbe’s optical diffraction limit^1,2 and for tracking the dynamics of single biomolecules^3,4. One of the requirements for probes compatible with SMLM is the ability to control the number of active fluorophores at any time to avoid spatial overlap of their point spread functions (PSF). In each of....

Protocol

NOTE: For yeast cloning and endogenous tagging please refer to our recent publication¹⁰.

1. Preparation of yeast cell samples for imaging

1. Prepare a liquid overnight culture of a w303 yeast strain. Using a sterile wooden stick, spot a small amount of yeast cells from an agar plate containing yeast extract–peptone–dextrose into a culture tube with ~2 mL of synthetic complete dextrose (SCD) medium. Incubate the tube overnight in a shaking incubator at 270 rpm and 30 °C.
2. Perform a 1:50 morning dilution of the cells in SCD. Continue to culture the diluted cells for 4 h at 30 °C ....

Results

Here, we present an optimized sample preparation, data acquisition and analysis procedure for SMLM using BODIPY conjugates based on the protocol above (Figure 1A). To demonstrate an example of the workflow for acquiring and analyzing SMLM data, we employ BODIPY (493/503) in yeast to resolve LDs below the optical diffraction limit (Figure 1B-F). Examples of the different multi-color imaging modes of BODIPY in conjunction with other probes such as.......

Discussion

In this protocol, we demonstrated how conventional BODIPY conjugates can be used to obtain SMLM images with an order of magnitude improvement in spatial resolution. This method is based on exploiting previously reported, red-shifted D_II states of conventional BODIPY dyes, which transiently form through bi-molecular encounters. These states can be specifically excited and detected with red-shifted wavelengths and are sparse and short-lived enough for SMLM. By tuning the concentration of BODIPY monomers along wi.......

Materials

Name	Company	Catalog Number	Comments
BODIPY C12	ThermoFisher	D3822	Green fatty acid analog
BODIPY C12 Red	ThermoFisher	D3835	Red fatty acid analog
BODIPY(493/503)	ThermoFisher	D3922	Neutral lipid marker
Concanavalin A	Sigma-Aldrich	C2010	Cell immobilization on glass surface
Drop-out Mix Complete w/o nitrogen base	US Biological	D9515	Amino acids for SCD
Dextrose	Sigma-Aldrich	G7021	Carbon source for SCD
Eight Well	Cellvis	C8-1.58-N	Chambered Coverglasses
Eight Well, Lb-Tek II	Sigma-Aldrich		Chambered Coverglasses
ET525/50	Chroma		Bandpass filter
ET595/50	Chroma		Bandpass filter
ET610/75	Chroma		Bandpass filter
Fetal Bovine Serum (FBS)	Gibco	26140079	Serum
FF652	Semrock		Beam splitter
FF731/137	Semrock		Bandpass filter
FluoroBrite DMEM	ThermoFisher	A1896701	Cell culture medium
Hal4000	Zhuang Lab, Harvard University		Data acquisition software
Ixon89Ultra DU-897U	Andor		EMCCD camera for photon detection
Laser 405, 488, 561, 640 nm	CW-OBIS		Lasers for excitation
Insight3	Zhuang Lab, Harvard University		Single molecule localization software
L-Glutamine	Gibco	25030-081	Amino acid required for cell culture
live-cell imaging solution	ThermoFisher	A14291DJ	Imaging buffer
Lysotracker Green	ThermoFisher	L7526	Bodipy based lysosome marker
Mammalian ATCC U2OS cells (Manassas, VA)	Dr. Jochen Mueller (University of Minnesota)
Nikon-CFI Apo 100 1.49 N.A	Nikon		Oil immersion objective
Penicillin streptomycin	Gibco	15140-122	Antibiotics
Sodium Pyruvate	Gibco	11360-070	Supplement for cell culture
T562lpxr	Chroma		Beam splitter
Trypsin-EDTA	Gibco	15400-054	Dissociation of adherent cell
W303 MATa strain	Horizon-Dharmacon	YSC1058	Parental yeast strain
Yeast Nitrogen Base	Sigma-Aldrich	Y1250	Nitrogen base without amino-acids
zt405/488/561/640rdc	Chroma		Quadband dichroic mirror