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Protocol
Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging
T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical tr...
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Protocol
Art Ashkin and the Origins of Optical Trap** and Particle Manipulation
A brief history of optical forces, the invention of optical tweezers, and their application to biological problems.
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Protocol
Angular Optical Trap** to Directly Measure DNA Torsional Mechanics
Angular optical trap** (AOT) is a powerful technique that permits direct angular manipulation of a trapped particle with simultaneous measurement of torque and rotation, while also retaining the capabilities...
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Protocol
Probing the Interaction Between Chromatin and Chromatin-Associated Complexes with Optical Tweezers
Single-molecule force spectroscopy is a powerful tool to analyze the architecture and interaction of large macromolecular assemblies that are refractory to high-resolution structural interrogations. Here, we d...
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Protocol
One-Dimensional STED Microscopy in Optical Tweezers
Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that the...
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Protocol
CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy
The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previousl...
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Protocol
High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy
We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in co...
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Protocol
Using Single-Molecule Optical Tweezers to Study the Conformational Cycle of the Hsp90 Molecular Chaperone
The heat shock protein 90 (Hsp90) family of chaperones are well-known, highly important components of the cellular systems which regulate protein homeostasis. Essential in eukaryotes, Hsp90s is also found in p...
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Protocol
Single-Molecule Manipulation Study of Chaperoned SNARE Folding and Assembly with Optical Tweezers
Intracellular membrane fusion is primarily driven by coupled folding and assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE assembly is intrinsically inefficient an...
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Protocol
Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A
KIF1A is a neuron-specific member of the kinesin-3 family of microtubule (MT) plus-end-directed motor proteins. It powers the migration of nuclei in differentiating brain stem cells and the transport of synapt...
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Protocol
High-Speed Optical Traps Address Dynamics of Processive and Non-Processive Molecular Motors
Interactions between biological molecules occur on very different time scales, from the minutes of strong protein–protein bonds, down to below the millisecond duration of rapid biomolecular interactions. Confo...
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Protocol
Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disassembling Microtubule Tips
Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or ...
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Protocol
Quantifying the Interaction Strength Between Biopolymers
The cytoskeleton consists of three types of biopolymers—actin filaments, microtubules, and intermediate filaments—and the interplay between these components is essential for many cellular functions such as cel...
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Protocol
The Invention of the Laser
In this chapter, I describe a scientific rivalry at Columbia University’s physics department in the days of the 1950s before and when the laser invented, and the race to build a laser eventually won by a scien...
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Protocol
Synthesis of Germanium Nanospheres as High-Precision Optical Tweezers Probes
Force spectroscopy on single molecular machines generating piconewton forces is often performed using optical tweezers. Since trap** forces scale with the particle volume, piconewton-force measurements so fa...
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Protocol
Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trap** Assay
Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substr...
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Protocol
Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System
Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the ...
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Protocol
Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding
Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-strand...
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Protocol
Temperature Quantification and Temperature Control in Optical Tweezers
Optical tweezers are widely used to investigate biomolecules and biomolecular interactions. In these investigations, the biomolecules of interest are typically coupled to microscopic beads that can be opticall...
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Protocol
Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation
Ribosomes are at the core of the central dogma of life. They perform the last major step of gene expression by translating the information written in the nucleotide codon sequences into the amino acid sequence...