Estimation of protein dynamic states with single molecule fluorescence data analysis at microsecond scale

2016 ◽  
Author(s):  
Kan Liu ◽  
Zhu Liu ◽  
Yi Peng ◽  
Yiming Ding ◽  
Chun Tang
Keyword(s):  
Data Analysis ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Protein Dynamic ◽  
Dynamic States

scholarly journals Improved methods for protein and single-molecule RNA detection in C. elegans embryos

2021 ◽  
Author(s):  
Dylan M Parker ◽  
Lindsay P Winkenbach ◽  
Annemarie Parker ◽  
Sam Boyson ◽  
Erin Osborne Nishimura
Keyword(s):  
Data Analysis ◽  
Single Molecule ◽  
Gene Products ◽  
Biological Functions ◽  
Single Molecule Fluorescence ◽  
Rna Detection ◽  
C Elegans ◽  
Improved Methods ◽  
Data Analysis Methods

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule Fluorescence In Situ Hybridization (smFISH) and Immunofluorescence (IF) visualize the abundance and localization of mRNAs and proteins, respectively, allowing researchers to elucidate the localization, dynamics, and functions of many genes. Here, we describe several improvements and optimizations to existing IF and smFISH approaches specifically for use in C. elegans embryos. We present 1) optimized fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility, 2) a streamlined, in-tube approach that negates freeze-cracking, 3) the smiFISH (single molecule inexpensive FISH) adaptation that reduces cost, 4) an assessment of optimal anti-fade products, and 5) straightforward quantification and data analysis methods. Most importantly, published IF and smFISH protocols have predominantly been mutually exclusive, preventing exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we present methods to combine IF and smFISH protocols in C. elegans embryos including an efficient method harnessing nanobodies. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol.

scholarly journals Single Molecule Fluorescence Imaging and Blinking

2006 ◽  
Vol 46 (3) ◽  
pp. 164-168
Author(s):  
Hiroaki YOKOTA ◽  
Tetsuichi WAZAWA ◽  
Yoshiharu ISHII
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence

scholarly journals Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

2021 ◽  
Author(s):  
Sujay Ray ◽  
Nibedita Pal ◽  
Nils G Walter
Keyword(s):  
Cluster Analysis ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Holliday Junction ◽  
Energetic Cost ◽  
Genomic Integrity ◽  
Dna Molecules ◽  
Single Molecule Fluorescence ◽  
And Cluster Analysis ◽  
Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

scholarly journals Low-Light Photodetectors for Fluorescence Microscopy

2021 ◽  
Vol 11 (6) ◽  
pp. 2773
Author(s):  
Hiroaki Yokota ◽  
Atsuhito Fukasawa ◽  
Minako Hirano ◽  
Toru Ide
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Science Studies ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Oxide Semiconductor ◽  
Wide Field ◽  
Single Molecule Fluorescence ◽  
Low Light

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

Evidence of an Intermediate and Parallel Pathways in Protein Unfolding from Single-Molecule Fluorescence

2008 ◽  
Vol 130 (25) ◽  
pp. 7898-7907 ◽  
Author(s):  
Angel Orte ◽  
Timothy D. Craggs ◽  
Samuel S. White ◽  
Sophie E. Jackson ◽  
David Klenerman
Keyword(s):  
Single Molecule ◽  
Protein Unfolding ◽  
Single Molecule Fluorescence ◽  
Parallel Pathways
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