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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 (DII) 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-DII 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 limit1,2 and for tracking the dynamics of single biomolecules3,4. 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 .......

Representative 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 DII 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.......

Acknowledgements

The research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R21GM127965.

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Materials

NameCompanyCatalog NumberComments
BODIPY C12ThermoFisherD3822Green fatty acid analog
BODIPY C12 RedThermoFisherD3835Red fatty acid analog
BODIPY(493/503)ThermoFisherD3922Neutral lipid marker
Concanavalin ASigma-AldrichC2010Cell immobilization on glass surface
Drop-out Mix Complete w/o nitrogen baseUS BiologicalD9515Amino acids for SCD
DextroseSigma-AldrichG7021Carbon source for SCD
Eight WellCellvisC8-1.58-NChambered Coverglasses
Eight Well, Lb-Tek IISigma-AldrichChambered Coverglasses
ET525/50ChromaBandpass filter
ET595/50ChromaBandpass filter
ET610/75ChromaBandpass filter
Fetal Bovine Serum (FBS)Gibco26140079Serum
FF652SemrockBeam splitter
FF731/137SemrockBandpass filter
FluoroBrite DMEMThermoFisherA1896701Cell culture medium
Hal4000Zhuang Lab, Harvard UniversityData acquisition software
Ixon89Ultra DU-897UAndorEMCCD camera for photon detection
Laser 405, 488, 561, 640 nmCW-OBISLasers for excitation
Insight3Zhuang Lab, Harvard UniversitySingle molecule localization software
L-GlutamineGibco25030-081Amino acid required for cell culture
live-cell imaging solutionThermoFisherA14291DJImaging buffer
Lysotracker GreenThermoFisherL7526Bodipy based lysosome marker
Mammalian ATCC U2OS cells (Manassas, VA)Dr. Jochen Mueller (University of Minnesota)
Nikon-CFI Apo 100 1.49 N.ANikonOil immersion objective
Penicillin streptomycinGibco15140-122Antibiotics
Sodium PyruvateGibco11360-070Supplement for cell culture
T562lpxrChromaBeam splitter
Trypsin-EDTAGibco15400-054Dissociation of adherent cell
W303 MATa strainHorizon-DharmaconYSC1058Parental yeast strain
Yeast Nitrogen BaseSigma-AldrichY1250Nitrogen base without amino-acids
zt405/488/561/640rdcChromaQuadband dichroic mirror

References

  1. Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods. 3 (10), 793-796 (2006).
  2. Betzig, E., et al. Imaging intracellul....

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BODIPY ConjugatesSuper resolution MicroscopySingle molecule TrackingLive cell ImagingLipid DropletsFatty AcidYeast CellsMammalian CellsEMCCD CameraLaser PowerMicroscope Optimization

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