Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

Rigler, Rudolf; Mets, Ülo; Widengren, Jerker; Kask, Peet

doi:10.1007/bf00185777

Cited by 953 publications

(932 citation statements)

References 11 publications

Supporting

Mentioning

909

Contrasting

Unclassified

Order By: Relevance

“…25 The light of an argon-ion laser (488 nm; Lasos, Jena, Germany) or an argon-krypton ion laser (488 nm, 568 nm; Melles Griot, Carlsbad, CA, USA) was filtered (488/10 nm, TRAST measurements. The instrumentation was based on a previously described set-up for total internal reflection fluorescence microscopy, here used in a confocal setting.…”

Section: Fcsmentioning

confidence: 99%

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

et al. 2011

View full text Add to dashboard Cite

Inter-or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleedthrough, thereby tolerating a high excess (up to 100 fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50 % in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in-vitro and in-vivo FRET-based spectroscopy and imaging.

show abstract

Section: Fcsmentioning

confidence: 99%

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Stick Theory- (6) with Tirado and Garcia de la Torre- (7) with Broersma's Relations- (8) with and δ = ln(2L/d) In all of the expressions above L is the rod length, d is the rod diameter, k B is the Boltzmann constant, T is the temperature, η and is the solvent viscosity. Broersma's relations are appropriate for long cylinders of aspect ratios > 3.5 (our shortest rods -5×13 nm -can not be properly described by this model).…”

Section: Translational Diffusion Coefficientsmentioning

confidence: 99%

“…Both rotational and translational diffusion constants were found to be inversely proportional to viscosity, as expected from theory (eqns. [6][7][8][9][10][11][12]. Viscosity values for glycerol/water mixtures were taken from the literature.…”

Section: Viscosity Effectsmentioning

confidence: 99%

Rotational and Translational Diffusion of Peptide-Coated CdSe/CdS/ZnS Nanorods Studied by Fluorescence Correlation Spectroscopy

2006

View full text Add to dashboard Cite

CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D trans and D rot ) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories, when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence D rot ~ L −3 versus D trans ~ L −1 . By titrating peptide-coated NRs with Bovine Serum Albumin (BSA) we monitored (nonspecific) binding through rotational diffusion and showed that D rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be a useful tool for observing binding and conformational dynamics in biological systems.

show abstract

“…One advantage of FCS is the extreme sensitivity down to the single molecule level (Rigler et al, 1993). This requires high signal to noise ratio of the sample and minimal background interference.…”

Section: Autofluorescence Studymentioning

confidence: 99%

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy

Shi

Teo

Pan

et al. 2009

Developmental Dynamics

View full text Add to dashboard Cite

Zebrafish and Drosophila are animal models widely used in developmental biology. High-resolution microscopy and live imaging techniques have allowed the investigation of biological processes down to the cellular level in these models. Here, using fluorescence correlation spectroscopy (FCS), we show that even processes on a molecular level can be studied in these embryos. The two animal models provide different advantages and challenges. We first characterize their autofluorescence pattern and determine usable penetration depth for FCS especially in the case of zebrafish, where tissue thickness is an issue. Next, the applicability of FCS to study molecular processes is shown by the determination of blood flow velocities with high spatial resolution and the determination of diffusion coefficients of cytosolic and membrane-bound enhanced green fluorescent protein-labeled proteins in different cell types. This work provides an approach to study molecular processes in vivo and opens up the possibility to relate these molecular processes to developmental biology questions. Developmental Dynamics 238:3156-3167,

show abstract

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

Cited by 953 publications

References 11 publications

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

Rotational and Translational Diffusion of Peptide-Coated CdSe/CdS/ZnS Nanorods Studied by Fluorescence Correlation Spectroscopy

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy

Contact Info

Product

Resources

About