Name: investigating receptor-ligand systems of the cellulosome with afm-based single-molecule force spectroscopy
Uploaded: 2013-12-20T13:45:00
Description: Watch this Scientific Journal Video about Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy at JoVE.com

The authors acknowledge funding from a European Research Council advanced grant to Hermann Gaub. Michael A. Nash gratefully acknowledges funding from Society in Science - The Branco Weiss Fellowship program. The authors thank Edward A. Bayer, Yoav Barak, and Daniel B. Fried at the Weizmann Institute of Science for generously providing the proteins used in this study. The authors thank Hermann E. Gaub, Elias M. Puchner, and Stefan W. Stahl for helpful discussions.

A schematic of the pulling geometry used in this work to probe the cohesin-dockerin interaction is shown in Figure 1A. The protein immobilization protocol reported here for cantilever and cover glass functionalization is a modified version of the procedure published previously27. The proteins were expressed from plasmid vectors in E. coli using conventional methods. The proteins were designed with a solvent-accessible thiol group, which was used in combination with maleimide chemistry...

<ol>
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To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.
The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to ...

Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g.&#160;C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides1. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein (&#39;scaffoldin&#39;), which comprises a polyprotein of cohesin domains that are specific for the type I dockerin module. At higher levels, cellulosome architecture can become very complex, incorporating alternative cohesin and dockerin pairs (e.g.&#160;type II, type III) that anchor the structures to the cell surface and allow for the assembly of branched structures containing multiple scaffoldins2. The various cohesin-dockerin types, despite having related structures, exhibit differential binding specificities suppressing cross reactivity with unintended scaffoldins or components from other cellulosome-producing bacterial species. While bioinformatic approaches have successfully identified thousands of unique cellulosomal components at the genetic level, comparatively few protein structures are known, and the mechanisms at work in cohesin-dockerin specificity determination remains an active area of structural biology research.
Since the invention of the atomic force microscope (AFM) by Binnig et al.3, various AFM operational modes have been developed and continuously improved, including noncontact imaging, oscillation mode imaging4, and single molecule force spectroscopy (SMFS)5,6. SMFS has evolved into a widely used technique to directly probe individual proteins7-11, nucleic acids12-15, and synthetic polymers16-19. In a typical SMFS experiment to investigate receptor-ligand binding20,21, an AFM cantilever tip is modified with one of the binding partners, while a flat glass surface is modified with the complementary binding partner. The modified cantilever is brought into contact with the surface allowing the partners to bind. The base of the cantilever is then withdrawn at constant speed and the force is measured using the optical lever deflection method. The resultant force-distance data traces exhibit sawtooth-like peaks if binding was established. In cases where the binding partners are fused to multiple protein domains, each peak in the force-distance trace can be correlated to the unfolding of a single protein domain or folded subdomain, while the last peak corresponds to rupture of the protein binding interface. The specific positions of the force-resistant elements can be used as a fingerprint to identify the various protein domains of interest. This method can be used to interrogate important amino acids involved in protein folding and stabilization. Many models have been reported in the literature to treat the characteristic force extension behavior observed in SMFS experiments. The most commonly used models include the freely jointed chain (FJC) model22, the worm-like chain (WLC) model18,23-25, and the freely rotating chain (FRC) model25,26.
In our prior work11, we used single-molecule force spectroscopy to investigate the interaction of cohesin and dockerin modules. Here, we present an experimental protocol for glass surface and cantilever functionalization with enzyme-dockerin and CBM-cohesin protein constructs. We also present an AFM-based SMFS protocol including data acquisition and analysis procedures. The described protocol can easily be generalized to other molecular systems, and should prove particularly useful to researchers interested in high-affinity receptor ligand pairs.

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			<td height="42" style="height:42px;width:216px;">3-Aminopropyl dimethyl ethoxysilane</td>
			<td style="width:139px;">ABCR GmbH</td>
			<td style="width:119px;">AB110423</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5 kDa NHS-PEG-maleimide</td>
			<td style="width:139px;">Rapp Polymer</td>
			<td style="width:119px;">13 5000-65-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TCEP Disulfide reducing gel</td>
			<td style="width:139px;">Thermo Scientific, Pierce</td>
			<td align="right" style="width:119px;">77712</td>
			<td><a href="http://www.thermoscientific.com/pierce">www.thermoscientific.com/pierce</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BioLever mini silicon nitride cantilevers</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">BL-AC40TS-C2</td>
			<td>Soft batches</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">XYZ Piezoelectric actuators</td>
			<td style="width:139px;">Physik Instrumente GmbH</td>
			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:216px;">Infrared &#8220;broad spectrum&#8221; IR laser</td>
			<td style="width:139px;">Superlum</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MFP-3D AFM Controller</td>
			<td style="width:139px;">Asylum Research</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Igor Pro 6.31</td>
			<td style="width:139px;">Wavemetrics</td>
			<td style="width:119px;"></td>
			<td>Data acquisition and analysis</td>
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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
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			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
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			<td height="21" style="height:21px;width:216px;">pH Meter</td>
			<td style="width:139px;"></td>
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			<td height="21" style="height:21px;width:216px;">Sodium borate</td>
			<td style="width:139px;"></td>
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			<td height="21" style="height:21px;width:216px;">Tweezers</td>
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			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:216px;">Cover glasses</td>
			<td style="width:139px;">Thermo Scientific, Menzel-Gl&#228;ser</td>
			<td style="width:119px;"></td>
			<td>24 mm diameter, 0.5 mm thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PTFE sample holder</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>custom made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator bath</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric acid (concentrated)</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide (30%)</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Orbital shaker</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toluene</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter paper</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slides</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtubes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipettes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>suitable for microtubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotator</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beakers</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optical microscope</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating receptor-ligand systems of the cellulosome with afm-based single-molecule force spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

We used the described procedure to investigate a type I cohesin-dockerin pair from C. thermocellum. Upon successful binding of the cohesin-dockerin pair, the recorded force distance traces showed characteristic peak patterns. A typical trace is shown in Figure 4a. Every peak in the trace represents the unfolding of one protein subdomain with the last peak corresponding to the dissociation of the receptor-ligand complex.
For the CBM-cohesin-dockerin-xylanase complex in...

Watch this Scientific Journal Video about Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy at JoVE.com

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

investigating-receptor-ligand-systems-cellulosome-with-afm-based

Research

JoVE Journal

Bioengineering

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...