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 at 270 rpm and 30 &#176;C.</li>
	<li>Perform a 1:50 morning dilution of the cells in SCD. Continue to culture the diluted cells for 4 h at 30 &#176;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.</li>
	<li>For studying fasting cells, grow the yeast culture for 2 days without exchange of media.</li>
	<li>At ~30 min prior to plating the cells, incubate a chambered coverglass with 80 &#181;L 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.</li>
	<li>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 &#181;L yeast culture at OD ~0.6 in 240 &#181;L SCD). Let the cells settle and adhere to the ConA surface for 30 min.</li>
	<li>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.</li>
</ol>
2. Preparation of mammalian cells for SMLM imaging
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Add BODIPY-C12, LysoTracker Green or any other BODIPY conjugate at a final concentration of 100&#8201;nM (stock solutions in dimethyl sulfoxide [DMSO]) 10&#8201;min prior to imaging. This time can vary based on the desired experiment. 
	NOTE: Imaging can be performed at ambient temperature (23 &#176;C) using live cell imaging solution. However, imaging at 37 &#176;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.</li>
</ol>
3. Equipment preparation
<ol>
	<li>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.</li>
	<li>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 &#176;C are used.</li>
	<li>Add a drop of immersion oil on the microscope objective.</li>
	<li>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 &#176;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.</li>
	<li>Turn on the microscope stage heater and set it to a temperature of 37 &#176;C and to a CO2 level of 5%. Adjust the objective correction collar accordingly.</li>
	<li>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&#160;CO2 control.</li>
	<li>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.</li>
</ol>
4. Data acquisition
<ol>
	<li>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&#160;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.</li>
	<li>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.</li>
	<li>Choose a destination folder for movies and record 5,000-20,000 acquisition frames to collect enough localizations for reconstructing super-resolution images.</li>
	<li>Move to different fields of view and repeat the steps above to collect data from more cells.</li>
</ol>
5. Data analysis and single-molecule tracking
<ol>
	<li>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).</li>
	<li>Visually screen the movie and adjust contrast settings such that single-molecule fluorescence&#160;blinking is visible. If needed restrict the region or the frame range for SMLM data analysis if parts of the sample are continuously fluorescing.</li>
	<li>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 &#62; 50 photons). Visually screen through some example frames to check the identification parameters and to reliably detect the distinct single molecule fluorescence&#160;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;localizations. Import the molecule list in custom written analysis procedures.</li>
	<li>For obtaining spatial information of the single molecule distribution, calculate the radial distribution function &#961;(r), which represents the density of localizations as a function of the radial distance20. To obtain &#961;(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 2&#960;ri*dr; (&#961;(ri) = H(ri)/&#960;(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.</li>
	<li>To obtain dynamic information about the diffusion of molecules, link localizations that appear for example within 3 pixels (0.48 &#181;m) 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 &#181;m2; thus a 0.48 &#181;m radius was within a low enough density to ensure that different molecules were not linked together.</li>
	<li>Average the displacements for different lag times &#916;t from multiple traces lasting at least three lag times to create a mean squared displacement (MSD) vs. &#916;t plot. Fit the MSD vs. &#916;t curve with the equation MSD = 4D&#916;t + &#963;2 to calculate the average diffusion coefficient D3.</li>
</ol>

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The authors declare no competing interests.

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">
			<td height="21" style="height:21px;width:375px;">zt405/488/561/640rdc</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Quadband dichroic mirror</td>
		</tr>
	</tbody>
</table>

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 ...

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

Research

JoVE Journal

Biochemistry

8.1K Views.  University of Minnesota, Twin Cities, Physics and Nanotechnology (PAN). 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.

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...