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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.
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.
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....
NOTE: For yeast cloning and endogenous tagging please refer to our recent publication10.
1. Preparation of yeast cell samples for imaging
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.......
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.......
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.
....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 |
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