scholarly journals High-throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

2009 ◽  
Vol 18 (5) ◽  
pp. 1115-1120 ◽  
Author(s):  
Toshihiko Sugiki ◽  
Chie Yoshiura ◽  
Yutaka Kofuku ◽  
Takumi Ueda ◽  
Ichio Shimada ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Correlation Spectroscopy ◽  
High Throughput Screening ◽  
Optimal Solution ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Biological Studies

scholarly journals Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy

1999 ◽  
Vol 4 (6) ◽  
pp. 335-353 ◽  
Author(s):  
Keith J. Moore ◽  
Sandra Turconi ◽  
Stephen Ashman ◽  
Martin Ruediger ◽  
Ulrich Haupts ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
High Throughput Screening ◽  
Equilibrium Constants ◽  
Correlation Spectroscopy ◽  
Single Molecule Detection ◽  
General Applicability ◽  
Detection Techniques ◽  
Fluorescence Correlation

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of -1 fl (10-15 L) such that the signal is insensitive to miniaturization of HTS assays to 1 A1 or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Two-photon fluorescence excitation and related techniques in biological microscopy

2005 ◽  
Vol 38 (2) ◽  
pp. 97-166 ◽  
Author(s):  
Alberto Diaspro ◽  
Giuseppe Chirico ◽  
Maddalena Collini
Keyword(s):  
Optical Microscopy ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Basic Principles ◽  
Fluorescence Correlation ◽  
Two Photon ◽  
Photon Excitation ◽  
Biological Studies ◽  
Laser Sources ◽  
Saturation Effects

1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy

2013 ◽  
Vol 455 (3) ◽  
pp. 339-345 ◽  
Author(s):  
Takayasu Mori ◽  
Eriko Kikuchi ◽  
Yuko Watanabe ◽  
Shinya Fujii ◽  
Mari Ishigami-Yuasa ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Library Screening ◽  
Chemical Library ◽  
Fluorescence Correlation ◽  
Fluorescent Correlation Spectroscopy

To discover WNK–OSR1/SPAK signalling inhibitors, we generated a new high-throughput system using fluorescent correlation spectroscopy capable of screening compounds that disrupt the binding of two molecules. We finally identified two novel and promising compounds for WNK–OSR1/SPAK signalling inhibition.

scholarly journals High-throughput fluorescence correlation spectroscopy enables analysis of surface components of cell-derived vesicles

2020 ◽  
Vol 412 (11) ◽  
pp. 2589-2597 ◽  
Author(s):  
Xu Fu ◽  
Yongwook Song ◽  
Abdullah Masud ◽  
Kanthi Nuti ◽  
Jason E. DeRouchey ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation
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