Camera-based single-molecule FRET detection with improved time resolution

Farooq, Shazia; Hohlbein, Johannes

doi:10.1039/c5cp04137f

Cited by 50 publications

(56 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Live root imaging was performed on a home-build microscopy setup described in [57]. The setup is equipped with a 100x/1.49NA TIRF objective (Nikon) and an Ixon Ultra 897 emCCD camera with 512 x 512 pixel (Andor) for imaging.…”

Section: Methodsmentioning

confidence: 99%

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

et al. 2017

Self Cite

View full text Add to dashboard Cite

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation—fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.

show abstract

Section: Methodsmentioning

confidence: 99%

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…34 Briefly, our microscope is equipped with a laser engine (Omicron, Germany), a 100× oil immersion SR/HP objective with NA = 1.49 (Nikon, Japan), and a Zyla 4.2 plus sCMOS camera for image acquisition (Andor, UK). 2 × 2 binning was used during acquisition, which resulted in a pixel size of 128 × 128 nm.…”

Section: Single-molecule Microscopymentioning

confidence: 99%

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Martens

Bader

Baas

et al. 2017

The Journal of Chemical Physics

Self Cite

View full text Add to dashboard Cite

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 10 6 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x-and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

show abstract

“…This limits the time scale of the dynamics that can be captured by lattice occupancy analysis. A detector with a faster frame rate31, stroboscopic laser excitation32 and 3D single-molecule tracking techniques33 may further expand the applicability of the analysis to wider time scales.…”

Section: Discussionmentioning

confidence: 99%

Conserved linear dynamics of single-molecule Brownian motion

Serag

Habuchi

2017

Nat Commun

View full text Add to dashboard Cite

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

show abstract

Camera-based single-molecule FRET detection with improved time resolution

Cited by 50 publications

References 54 publications

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

Conserved linear dynamics of single-molecule Brownian motion

Contact Info

Product

Resources

About