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 the thousands of data acquisition frames, the location of each fluorescent fluorophores is then determined with ~20 nm precision by fitting its corresponding point-spread function. Traditionally, the on-off blinking of fluorophores has been controlled through stochastic photoswitching1,2,5 or chemically induced intrinsic blinking6. Other approaches include the induced activation of fluorogens upon transient binding to a fluorogen-activating protein7,8 and the programmable binding-unbinding of labeled DNA oligomers in total internal reflection fluorescence (TIRF) or light sheet excitation9. Recently, we reported a novel and versatile labeling strategy for SMLM10 in which previously reported red-shifted dimeric (DII) states of conventional boron di-pyromethane (BODIPY) conjugates11,12,13 are transiently forming and become specifically excited and detected with red-shifted wavelengths.
BODIPYs are widely used dyes with hundreds of variants that specifically label sub-cellular compartments and biomolecules14,15,16. Because of their ease of use and applicability in living cells, BODIPY variants are commercially available for conventional fluorescence microscopy. Here, we describe a detailed and optimized protocol on how the hundreds of commercially available BODIPY conjugates can be used for live-cell SMLM. By tuning the concentration of BODIPY monomers and by optimizing the excitation laser powers, imaging and data analysis parameters, high-quality super-resolution images and single molecule tracking data is obtained in living cells. When used at low concentration (25-100 nM), BODIPY conjugates can be simultaneously used for SMLM in the red-shifted channel and for correlative conventional fluorescence microscopy in the conventional emission channel. The obtained single molecule data can be analyzed to quantify the spatial organization of immobile structures and to extract the diffusive states of molecules in living cells17. The availability of BODIPY probes in both green and red forms allows for multi-color imaging when used in the right combination with other compatible fluorophores.
In this report, we provide an optimized protocol for acquiring and analyzing live-cell SMLM data using BODIPY-C12, BODIPY (493/503), BODIPY-C12 red and lysotracker-green in multiple colors. We resolve fatty acids and neutral lipids in living yeast and mammalian cells with ~30 nm resolution. We further demonstrate that yeast cells regulate the spatial distribution of externally added fatty acids depending on their metabolic state. We find that added BODIPY-fatty acids (FA) localize to the endoplasmic reticulum (ER) and lipid droplets (LDs) under fed conditions whereas BODIPY-FAs form non-LD clusters in the plasma membrane upon fasting. We further extend the application of this technique to image lysosomes and LDs in living mammalian cells. Our optimized protocol for SMLM using conventional BODIPY conjugates can be a useful resource to study biological processes at the nanoscale with the myriad available BODIPY conjugates.
NOTE: For yeast cloning and endogenous tagging please refer to our recent publication10.
1. Preparation of yeast cell samples for imaging
2. Preparation of mammalian cells for SMLM imaging
3. Equipment preparation
4. Data acquisition
5. Data analysis and single-molecule tracking
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 GFP, mEos2 are shown in Figure 2. We manipulate the metabolic state in yeast by growing them in the same medium for ~48 h and show that BODIPY-C12 forms immobile non-LD clusters in cell periphery upon fasting in contrast to their incorporation into LDs under fed conditions (Figure 3). To further extend the SMLM capability of BODIPY conjugates to mammalian cells, we image BODIPY-C12 and LysoTracker-green in live U2OS cells (Figure 4).
Figure 1: Optimization of SMLM data acquisition and analysis using BODIPY dyes. (A) Workflow for optimizing single molecule fluorescence signals and post-processing of the SMLM data from BODIPY conjugates. (B) LED image (left), conventional fluorescence image (middle, excitation: 488 nm, emission: 525 nm) and anti-Stokes image (right, excitation: 561 nm, emission: 525 nm) of yeast cells stained with the LD marker BODIPY (493/503). (C) Single SMLM frames showing singe BODIPY DII emitters (excitation: 561 nm, emission: 595 nm) at too low density (left), optimal density (middle) and too high density (right). (D) Optimization of SMLM analysis parameters. With a too high photon number threshold, the software misses valid single molecule signals (left), detects all molecules with an optimal photon threshold (middle) and detects false localizations with too low photon thresholds (right). (E) SMLM image of BODIPY (493/503) resolves the size of LDs (left, zoom) with a mean diameter of 125 nm. (F) Single molecule tracking reveals confined diffusion of BODIPY (493/503) inside LDs (left). Traces are used to compute the MSD vs. time curve, which exhibits sub-diffusive behavior inside LDs (right). Scale bar = 1 μm, zoom = 100 nm. Please click here to view a larger version of this figure.
Figure 2: Multi-color SMLM imaging using BODIPY conjugates in living cells. (A) Conventional image of BODIPY-C12 under 488 nm excitation (left). Corresponding SMLM image using DII states of BODIPY-C12 under 561 nm excitation (middle) and zoom (right) revealing BODIPY-C12 in emerging LDs. (B) Conventional fluorescence image of the ER labeled with Sec63-GFP under 488 nm excitation (left). Simultaneously recorded conventional fluorescence image of BODIPY-C12 red with 561 nm excitation (middle) and SMLM image using 640 nm excitation (right). (C) Sequential two-color SMLM imaging of Sec63-mEos2 and BODIPY-C12 green DII states. First, mEos2 is imaged with high 405 nm photo-activation and 561 nm excitation (left) followed by long data acquisition without 405 nm activation (middle). Scale bar = 1 µm. Please click here to view a larger version of this figure.
Figure 3: Differential fatty acid distribution upon fasting in yeast cells. (A) Schematic describing different metabolic states (fed and fasted condition) based on the duration of growth in the SCD medium. B) Conventional fluorescence images (top) show that BODIPY-C12 red co-localizes with BODIPY (493/503) under fed conditions indicating incorporation into LDs. The SMLM image (lower, left) shows dense BODIPY-C12 puncta in LDs and single molecule traces (lower, right) exhibit diffusion along cellular membranes. (C) Under fasted condition, BODIPY-C12 forms puncta in the cell periphery that do not co-localize with LDs (upper: left, middle, lower left). The SMLM image resolves the puncta and confined traces of BODIPY-C12 red (lower, right). (D) The radial distribution function (left) shows higher clustering of BODIPY-FAs upon fasting. The mean-square displacement vs. time plot of single molecule tracking (right) confirms immobilization of BODIPY-C12 upon fasting. Scale bar = 1 μm. Please click here to view a larger version of this figure.
Figure 4: Imaging of BODIPY dyes in live mammalian U2OS cells. (A) Conventional fluorescence image (left) of BODIPY-C12 at 488 nm excitation. The corresponding SMLM image using DII states (right) at 561 nm excitation shows the nanoscopic distribution of DII states in U2OS cell. The insets show magnifications of lipid droplets (scale bar = 500 nm). (B) Conventional image of lysosomes in U2OS cells using LysoTracker green at 488 nm excitation (left). The corresponding SMLM image of immobile lysosomes (right, scale bar = 5 µm) at 561 nm excitation. Inset: SMLM image of an optically diffraction limited lysosome (scale bar 100 nm). The BODIPY-C12 images were recorded in live cell imaging solution at 23 °C. The images of lysosomes using LysoTracker green were recorded in non-fluorescent DMEM with 10% fetal bovine serum, 4 mM glutamine, 1 mM sodium pyruvate and 1% penicillin-streptomycin antibiotics at 37 °C. Please click here to view a larger version of this figure.
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 with laser parameters, an optimal density of localizations and signal-to-noise can be achieved. We resolved the intracellular distribution and mobility of fatty acid analogs and neutral lipids with ~30 nm resolution (theoretical Thompson’s formula) in living yeast cells under fed and fasted conditions. We also found that ~40% of BODIPY DII states stay on for two or more data acquisition frames at 20 Hz, enabling single-molecule tracking to quantify their mobility under different conditions. Our results show the differential localization and mobility of BODIPY-FAs upon fasting and suggest a protection mechanism against lipotoxicity. Our ability to track single BODIPY molecules and to resolve the size of LDs and BODIPY-FA puncta below the optical diffraction limit under different metabolic states is only possible with the developed SMLM capability of conventional BODIPY conjugates. The exact molecular mechanisms and pathways involved in the spatial regulation of the fatty acid distribution and uptake are the subject of our future studies. Furthermore, we extended the SMLM capability of conventional BODIPY conjugates to living mammalian cells by resolving BODIPY-FAs and lysosomes in U2OS cells.
Using DII states of conventional BODIPY conjugates for SMLM has advantages over other probes since hundreds of different BODIPY variants are commercially available that label specific molecules or cellular compartments in living cells. The sample preparation is as easy as adding the dye at low (~100 nM) concentrations before imaging without any washing. In contrast to other PALM/STORM probes that bleach over time, BODIPY monomers are unaffected by the excitation of their DII states and therefore provide an almost never depleting source for single molecule signals in long term imaging. Since DII states arise due to spontaneous bi-molecular encounters, SMLM using DII states requires no externally added buffer to induce blinking23. Similarly, there is no need for high-energy photo-activation as required for newly synthesized photo-activatable BODIPY versions24 or SMLM of some conventional BODIPY dyes21, which could be detrimental for cell health during long term imaging25,26. Moreover, SMLM with DII states creates an inherent background suppression of non-specifically interacting probes because of the quadratic dependence of DII states on the monomer concentration. Therefore, a higher contrast is achieved in SMLM images compared to traditional probes whose monomeric signal is detected.
BODIPYs exhibit a faint anti-Stokes fluorescence that enables the excitation of monomers and dimers with a single laser at high excitation power. On the one hand, this property can be exploited for simultaneous conventional fluorescence and SMLM imaging to track and resolve moving structures. On the other hand, it makes it harder to combine BODIPY DII states with other probes for multi-color imaging as the BODIPY signal occupies two emission channels. However, multi-color imaging is possible when probes are carefully chosen as shown in Figure 2B with Sec63-GFP and BODIPY-C12 red. Similarly, sequential two-color SMLM is possible with other photo-activatable probes like mEos2 as shown in Figure 2C. Other possible combinations for two-color SMLM include the use of green BODIPY conjugates and a 640 nm excitable dyes such as JF646 bound to the halo tag27.
In summary, we have presented an optimized protocol for SMLM using conventional BODIPY dyes to investigate the spatio-temporal distribution of fatty acids, neutral lipids and lysosomes at the nanoscopic length scale in living yeast and mammalian cells. With minor modifications, this protocol can be equally applicable for SMLM with hundreds of other BODIPY conjugates across different cell types.
The authors have nothing to disclose.
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.
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 |