Method Article

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

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DOI:

10.3791/60950

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June 8th, 2020

 ,  ,  , 
1School of Physics and Astronomy, University of Minnesota, Twin Cities, Physics and Nanotechnology (PAN), 2Middlebury College

Corresponding Authors: Elias M. Puchner <epuchner@umn.edu>

* These authors contributed equally

In This Article

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 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.

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 in a 270 rpm shaking incubator, allowing the cells to grow in exponential phase and to reach an optical density (OD) of ~0.6.
    NOTE: The procedure can vary here depending on which metabolic state is being studied. BODIPY conjugates do not require cells in the exponential growth phase. However, be cognizant of autofluorescence from dead cells during the stationary phase, as it can cause a background signal too strong to analyze single BODIPY-DII emitters.
  3. For studying fasting cells, grow the yeast culture for 2 days without exchange of media.
  4. At ~30 min prior to plating the cells, incubate a chambered coverglass with 80 µL of 0.8 mg/mL sterile Concanavalin A (ConA) in deionized H2O at room temperature. After 30 min, wash the coverglass three times with deionized H2O.
  5. At ~30 min before imaging, pipette the cells on the chambered coverglass, with the correct volume of fresh SCD to achieve an optical density of ~0.12 (typically 60 µL yeast culture at OD ~0.6 in 240 µL SCD). Let the cells settle and adhere to the ConA surface for 30 min.
  6. Add the desired BODIPY conjugate directly to the chambered coverglass at a final concentration of ~100 nM.
    NOTE: A BODIPY concentration optimization experiment may be required depending on BODIPYs local density in a particular cellular compartment.

2. Preparation of mammalian cells for SMLM imaging

  1. Maintain the mammalian U2OS cells in non-fluorescent DMEM with 10% fetal bovine serum, 4 mM glutamine, 1 mM sodium pyruvate and 1% penicillin-streptomycin antibiotics in a T25 flask.
    NOTE: Cells can also be maintained in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, however, the medium needs to be exchanged before imaging with a non-autofluorescent solution.
  2. Split the cells at 70-80% confluency 1:5 in a single well of an 8-well plate. Culture the cells in the 8-well plate for 12 to 24 h before imaging.
  3. Add BODIPY-C12, LysoTracker Green or any other BODIPY conjugate at a final concentration of 100 nM (stock solutions in dimethyl sulfoxide [DMSO]) 10 min prior to imaging. This time can vary based on the desired experiment.
    NOTE: Imaging can be performed at ambient temperature (23 °C) using live cell imaging solution. However, imaging at 37 °C and 5% CO2 with non-fluorescent DMEM mixed with 10% fetal bovine serum, 4 mM glutamine, 1 mM sodium pyruvate and 1% penicillin-streptomycin antibiotics is preferred to keep cells closer to physiological conditions and to make biological conclusions.

3. Equipment preparation

  1. Mount the appropriate filter sets in the emission path based on the emission color of BODIPY being used.
    NOTE: A quad-band dichroic mirror (zt/405/488/561/640rdc) first separates the excitation from the emission light. The green emission (525 nm) and red emission (595 nm) are then split by a dichroic long-pass beam splitter (T562lpxr) followed by band-pass filters ET525/50 in the green channel and ET 595/50 in the red channel. The two channels are then projected to different areas of the same camera chip. Similarly, the red emission (595 nm) and the far-red emission are first split by a dichroic long-pass beam splitter (FF652-Di01) followed by band-pass filters ET 610/75 in the red and FF731/137 in the far-red channel.
  2. Turn on the microscope, microscope stage, lasers (488 nm, 561 nm) and camera. Here an inverted microscope with a perfect focus system and an EMCCD camera cooled to -68 °C are used.
  3. Add a drop of immersion oil on the microscope objective.
  4. Open the Hal4000 software (see the Table of Materials) that controls the LED light for bright field imaging, laser powers, laser shutters and camera settings for imaging. Set the EMCCD gain to 30 and the camera temperature to -68 °C. Prepare the camera and corresponding software to record movies at 20 Hz.
    NOTE: This technique applies to any wide-field microscope capable of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) imaging. Corresponding software may vary.
  5. Turn on the microscope stage heater and set it to a temperature of 37 °C and to a CO2 level of 5%. Adjust the objective correction collar accordingly.
  6. Mount the sample on the microscope stage and focus until the focusing system engages. Move the stage using the stage controller until healthy cells appear in the field of view.
    NOTE: For imaging with yeast cells at room temperature, there is no need to turn on the heater or CO2 control.
  7. Turn on the appropriate lasers for the excitation of monomers as well as dimers. For BODIPY green or LysoTracker green, we use a 561 nm laser to excite DII states for SMLM and a 488 nm laser to excite monomers for conventional fluorescence.
    NOTE: For BODIPY red, use a 640 nm laser to excite DII states for SMLM and a 561 nm laser to excite the monomers for conventional fluorescence. For BODIPY red, adjust the 561 nm and 640 nm laser powers to visualize bulk fluorescence in the red channel and single molecule bursts in the far-red channel. The typical power for 561 nm is ~0.06 W/cm2 and ~5 kW/cm2 for 640 nm. For BODIPY green, expect to also see conventional fluorescence in the green emission channel under 561 nm excitation. For BODIPY red, expect to see conventional fluorescence in the red channel under 640 nm excitation. This signal arises from anti-Stokes emission, which becomes useful for monomer/dimer co-localization images with continuous laser excitation.

4. Data acquisition

  1. Load laser shutter sequences for the excitation of monomers as well as dimers.
    NOTE: We typically use nine single molecule excitation frames at 561 nm followed by one conventional excitation frame at 488 nm. This offers a brighter conventional fluorescence signal and avoids leaking of another 488 nm excitable fluorophore such as green fluorescent protein (GFP) into the red single-molecule detection channel in multi-color imaging applications. Alternatively, turn on the 561 nm laser continuously and rely on the anti-Stokes emission for conventional images in the shorter wavelength channel.
  2. Tune the laser powers such that single molecule fluorescence bursts are detected in the red-shifted emission channel under 561 nm excitation, and conventional fluorescence appears in the green emission channel with 488 nm excitation. Typical laser powers of the 561 nm laser will be around 0.8-1 kW/cm2 for SMLM, and 0.035-0.07 W/cm2 for the 488 nm laser in the conventional fluorescence imaging mode.
  3. Choose a destination folder for movies and record 5,000-20,000 acquisition frames to collect enough localizations for reconstructing super-resolution images.
  4. Move to different fields of view and repeat the steps above to collect data from more cells.

5. Data analysis and single-molecule tracking

  1. Load the movie into a SMLM analysis software.
    NOTE: Any software18 can be used. We use INSIGHT (see the Table of Materials) and cross-validate the results using the ThunderSTORM19 plugin for imageJ (Fiji).
  2. Visually screen the movie and adjust contrast settings such that single-molecule fluorescence blinking is visible. If needed restrict the region or the frame range for SMLM data analysis if parts of the sample are continuously fluorescing.
  3. Set single molecule identification parameters for fitting with 2D Gaussian PSFs (ROI: 7 x 7 pixels with pixel size 160 nm, width 260-650 nm, height > 50 photons). Visually screen through some example frames to check the identification parameters and to reliably detect the distinct single molecule fluorescence bursts (see Figure 1C).
    NOTE: Certain identification parameters such as height and width can be slightly adjusted to optimize the recognition of visually perceived single molecule fluorescence signals.
  4. Perform SMLM image analysis with the optimized identification parameters and then render each single molecule as a 2D Gaussian whose width is weighted by the inverse square root of the detected number of photons.
  5. Assess the quality of the data. Use restricted frame ranges to observe single molecule distributions at more specific instances in time. This can account for organelle movement during data acquisition.
  6. To further analyze the spatial distribution and dynamics of the molecule distributions, export the obtained molecule list containing the coordinates, frames of appearance, photons, widths and heights of the localizations. Import the molecule list in custom written analysis procedures.
  7. For obtaining spatial information of the single molecule distribution, calculate the radial distribution function ρ(r), which represents the density of localizations as a function of the radial distance20. To obtain ρ(r), calculate unique pair-wise distances of all localizations, construct the histogram with bins centered at ri with height H(ri) and with a width dr and divide by 2πri*dr; (ρ(ri) = H(ri)/π(ri+dr)2-ri2). The radial distribution function can then be used to quantify and compare the degree of clustering as well as the characteristic size of clusters.
  8. To obtain dynamic information about the diffusion of molecules, link localizations that appear for example within 3 pixels (0.48 µm) in consecutive data acquisition frames to create single molecule traces.
    NOTE: The linking distance will depend on the diffusion of molecules and the density of localizations. The maximum linking distance can be estimated by analyzing the density of localizations in each frame21,22. The average density was determined to be 0.043 localizations per µm2; thus a 0.48 µm radius was within a low enough density to ensure that different molecules were not linked together.
  9. Average the displacements for different lag times Δt from multiple traces lasting at least three lag times to create a mean squared displacement (MSD) vs. Δt plot. Fit the MSD vs. Δt curve with the equation MSD = 4DΔt + σ2 to calculate the average diffusion coefficient D3.

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 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).

SMLM diagram with BODIPY staining, LED fluorescence, and diffusion analysis for cell density optimization.
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.

Simultaneous fluorescence, SMLM imaging of BODIPY, Sec63-GFP, mEos2; microscopy analysis.
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.

Flowchart and microscopy images of BODIPY-stained cells; fed vs. fasted lipid analysis.
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.

Fluorescence microscopy, BODIPY-C12, Lysotracker signals; cell lipid, lysosome localization.
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.

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 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.

Disclosures

The authors declare no competing interests.

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.

Materials

List of materials used in this article
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

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Single Molecule Localization MicroscopyLipid Droplet ImagingFatty Acid TrackingLive Cell ImagingFluorescence MicroscopyLaser Power OptimizationBODIPY-C12
Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking
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