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.

NOTE: For yeast cloning and endogenous tagging please refer to our recent publication10.
1. Preparation of yeast cell samples for imaging
<ol>
	<li>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&#8211;peptone&#8211;dextrose into a culture tube with ~2 mL of synthetic complete dextrose (SCD) medium. Incubate the tube overnight in a shaking incubator ...

<ol>
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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...

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&#8217;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&#160;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&#160;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1009px;" width="1008">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY C12</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3822</td>
			<td style="width:265px;">Green fatty acid analog</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY C12 Red</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3835</td>
			<td>Red fatty acid analog</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY(493/503)</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3922</td>
			<td>Neutral lipid marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concanavalin A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">C2010</td>
			<td>Cell immobilization on glass surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Drop-out Mix Complete w/o nitrogen base</td>
			<td style="width:196px;">US Biological</td>
			<td style="width:173px;">D9515</td>
			<td>Amino acids for SCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dextrose</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">G7021</td>
			<td>Carbon source for SCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eight Well</td>
			<td style="width:196px;">Cellvis</td>
			<td style="width:173px;">C8-1.58-N</td>
			<td>Chambered Coverglasses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eight Well, Lb-Tek II</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;"></td>
			<td>Chambered Coverglasses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET525/50</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET595/50</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET610/75</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">26140079</td>
			<td>Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">FF652</td>
			<td style="width:196px;">Semrock</td>
			<td style="width:173px;"></td>
			<td>Beam splitter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">FF731/137</td>
			<td style="width:196px;">Semrock</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoroBrite DMEM</td>
			<td style="width:196px;">ThermoFisher</td>
			<td>A1896701</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Hal4000</td>
			<td style="width:196px;">Zhuang Lab, Harvard University</td>
			<td style="width:173px;"></td>
			<td>Data acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ixon89Ultra DU-897U</td>
			<td style="width:196px;">Andor</td>
			<td style="width:173px;"></td>
			<td>EMCCD camera for photon detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Laser 405, 488, 561, 640 nm</td>
			<td style="width:196px;">CW-OBIS</td>
			<td style="width:173px;"></td>
			<td>Lasers for excitation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Insight3</td>
			<td style="width:196px;">Zhuang Lab, Harvard University</td>
			<td style="width:173px;"></td>
			<td>Single molecule localization software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">L-Glutamine</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">25030-081</td>
			<td>Amino acid required for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">live-cell imaging solution</td>
			<td style="width:196px;">ThermoFisher</td>
			<td>A14291DJ</td>
			<td>Imaging buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Lysotracker Green</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">L7526</td>
			<td>Bodipy based lysosome marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mammalian ATCC U2OS cells (Manassas, VA)</td>
			<td colspan="2">Dr. Jochen Mueller (University of Minnesota)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon-CFI Apo 100 1.49 N.A</td>
			<td>Nikon</td>
			<td style="width:173px;"></td>
			<td>Oil immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin</td>
			<td style="width:196px;">Gibco</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Pyruvate</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">11360-070</td>
			<td>Supplement for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">T562lpxr</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Beam splitter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td style="width:196px;">Gibco</td>
			<td>15400-054</td>
			<td>Dissociation of adherent cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">W303 MATa strain</td>
			<td style="width:196px;">Horizon-Dharmacon</td>
			<td style="width:173px;">YSC1058</td>
			<td>Parental yeast strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Yeast Nitrogen Base</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">Y1250</td>
			<td>Nitrogen base without amino-acids</td>
		</tr>
		<tr height="21">
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conventional bodipy conjugates for live-cell super-resolution microscopy and single-molecule tracking

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&#160;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.&#160;

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...

Watch this Scientific Journal Video about Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking at JoVE.com

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

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 ...

Our protocol describes the use of conventional BODIPY dyes for super-resolution microscopy using their sparse, red-shifted states. This enables us to study organelles and biomolecules in living cells with 30-nanometer resolution. The advantage of this method is its simplicity and the versatility of different available BODIPY conjugates that provide a long-lasting source of single molecule signals.Our demonstrated application could become important to gain insights in diseases such as fatty liver disease or type 2 diabetes by resolving lipid droplets and fatty acid below the diffraction limit. We gain insight in fatty acid and lipid droplet biology in yeast and mammalian cells. However, this technique is applicable to all other transparent cell types with low background fluorescence.When using the technic for the first time, make sure to optimize the di concentrations and laser powers, to observe bright single molecule signals. A visual demonstration of this technic is important to see how the di concentration and optimization of laser power results in bright single molecule signals. Maintain the mammalian U2OS cells in non-fluorescent DMBM with 10%fetal bovine serum four millimolar glutamine, one millimolar sodium pyruvate, and one percent penicillin streptomycin antibiotics in a T-25 flask.Split the cells at 70 to 80%confluency to one to five, and pipette in a single well of an eight well plate. Alter the cells in the eight well plate for 12 to 24 hours. Ten minutes prior to imaging it, add a BODIPY-C12 Lysotracker green or and the other BODIPY conjugate, add a final concentration of 100 nanomolar.Mount the appropriate filter sets in the emission path based on the emission color of BODIPY being used. Turn on the microscope. Microscope stage, 488 nanometer and 561-nanometer lasers, as well as the camera.Add a drop of emerging oil on the microscope objective. Open the HAL4000 software 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 minus 68 degrees Celsius.Prepare the camera and corresponding software to record movies at 20 Hertz. Turn on the microscope stage heater and set it to temperature of 37 degrees celsius and to a carbon dioxide level of five percent. Adjust the objective correction color accordingly.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. Load laser shutter sequences for the excitation of monumus as well as dimmers, to whom laser power of the 561-nanometer laser to between 821 kilowatts per square centimeters for a single molecule localization microscopy, such that, single molecule fluorescence pores are detected in the red-shifted emission channel.Adjust the laser power between point 035 and point 07 watts per square centimeter for the 488-nanometer laser, so that conventional fluorescence appears in the green emission channel. Choose a destination folder for movies and record 5000 to 20000 acquisition frames to collect enough localizations for reconstructing super resolution images. Move to different fields of view and repeat to collect data for more cells.Load the movie into a single molecule localization microscopy analysis software. Visually screen the movie and adjust contrast settings, such that single molecule fluorescent blinking is visible. To set single molecule identification parameters for fitting the 2D Gaussian, PSFs.Visually screen through some example frames to check the identification parameters, and reliably detect the distinct single molecule fluorescent bursts. Press analysis to perform single molecule localization microscopy imagine 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.Assess the quality of the data. Use restricted frame ranges to observe single molecule distributions at more specific instances and time. This accounts for organelle movement during data acquisition.In this study, we presented an optimized sample preparation, data acquisition and analysis procedure with single molecule localization microscopy using BODIPY conjugates. To demonstrate an example of the workflow for acquiring and analyzing single molecule localization microscopy data, BODIPY in yeast was employed to resolve lipid droplets below the optical diffraction limit. Examples of the different multicolor imaging modes of BODIPY in conjugation with other probes such as GFP and mEos2 are shown here.BODIPY-C12 formed in mobile non-lipid droplet clusters in cell periphery upon fasting, in contrast to their incorporation into lipid droplets under fed conditions. To further extend the single molecule localization microscopy capability of BODIPY conjugates to mammalian cells, BODIPY-C12 and Lysotracker green in live U2OS cells were imaged. Optimizing BODIPY concentration as well as exciting the laser powers are critical steps in order to visualize bright single molecule signals, and to reconstruct super resolution images.This method can be used with any other BODIPY conjugate to gain high resolution insight, in the special temporal distribution of specific bio-molecules inside living cells. Our technic paved the way, to further interrogate lipid droplet and fatty acid biology on the nanoscopic lens scale. However, this application goes far beyond the specific philopectetes due to the availability of various functional BODIPY conjugates.Please be sure to follow the standard operating procedures for handling the biological samples and dyes, and also be our obliged hedgers and follow the laser safety procedures.

conventional-bodipy-conjugates-for-live-cell-super-resolution

Research

JoVE Journal

Biochemistry

8.1K visualizações.  University of Minnesota, Twin Cities, Physics and Nanotechnology (PAN). Nosso protocolo descreve o uso de corantes BODIPY convencionais para microscopia de super-resolução usando seus estados esparsos e vermelhos. Isso nos permite estudar organelas e biomoléculas em células vivas com resolução de 30 nanômetros. A vantagem deste método é sua simplicidade e a versatilidade de diferentes conjugados BODIPY disponíveis que fornecem uma fonte duradoura de sinais de molécula única.Nossa aplicação demonstrada pode se tornar importante para obter insi...