Name: conventional bodipy conjugates for live-cell super-resolution microscopy and single-molecule tracking
Uploaded: 2020-06-08T13:00:00
Description: Watch this Scientific Journal Video about Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking at JoVE.com

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">
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		<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>
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			<td>Nitrogen base without amino-acids</td>
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			<td height="21" style="height:21px;width:375px;">zt405/488/561/640rdc</td>
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			<td>Quadband dichroic mirror</td>
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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

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 ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

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 ...

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 ...