Total Internal Reflection Fluorescence Microscopy

Axelrod, Daniel

doi:10.1007/978-1-4614-7513-2_21

Cited by 52 publications

(59 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Traditionally, background fluorescence reduction has been achieved by reducing the volume of sample that is illuminated, effectively lowering the number of fluorescing molecules present in the probed volume [41][42][43][44]. For example, TIRF microscopy confines laser excitation to a 100-nm thin layer above a glass surface, resulting in a drastic improvement of signalto-background ratios of single-molecule images [25].…”

Section: The Concentration Problemmentioning

confidence: 99%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

View full text Add to dashboard Cite

Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van Oijen, A. M. (2011). Single-molecule approaches to characterizing kinetics of biomolecular interactions. Current Opinion in Biotechnology, 22(1), 75-80. DOI: 10.101675-80. DOI: 10. /j.copbio.2010 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Available online at www.sciencedirect.com Single-molecule approaches to characterizing kinetics of biomolecular interactions Antoine M van OijenSingle-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.

show abstract

Section: The Concentration Problemmentioning

confidence: 99%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

View full text Add to dashboard Cite

show abstract

“…10,11 We have optimized oxygen-scavenging and emission stabilizer conditions that led to observation of single molecules for several minutes and collection of typically 1.4 million photons before photobleaching (Figure 1C). 12 To sensitively detect fluorescent photons, we have employed TIRF 13 that illuminates vicinity of the glass/ water interface, resulting in minimal fluorescent background. Moreover, TIRF is a wide field technique that allows simultaneous observation of many dyes on the surface.…”

Section: Fionamentioning

confidence: 99%

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

2005

View full text Add to dashboard Cite

We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within ∼1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

show abstract

“…The microscope was equipped with a modified total internal reflection condenser (TILL-Photonics) that allowed for switching between conventional Köhler and total internal reflection illumination. Assuming a refractive index for Jurkat cytosol of n ϳ1.38, a penetration depth of the evanescent field of 72 nm can be estimated (24).…”

Section: Total Internal Reflection Fluorescence (Tirf) Microscopy/frementioning

confidence: 99%

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

Paster

Paar

Eckerstorfer

et al. 2009

The Journal of Immunology

View full text Add to dashboard Cite

The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr 505 upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr 505 to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr 394 to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr 505 and Tyr 394 phosphorylation and conformational alterations.

show abstract

Total Internal Reflection Fluorescence Microscopy

Cited by 52 publications

References 24 publications

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

Contact Info

Product

Resources

About