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Abstract:

Förster resonant energy transfer measured by fluorescence lifetime imaging microscopy (FRET-FLIM) is the method of choice for monitoring the spatio-temporal dynamics of protein interactions in living cells. To obtain an accurate estimate of the molecular fraction of interacting proteins requires a large number of photons, which usually precludes the observation of a fast process, particularly with time correlated single photon counting (TCSPC) based FLIM. In this work, we propose a novel method named pawFLIM (phasor analysis via wavelets) that allows the denoising of FLIM datasets by adaptively and selectively adjusting the desired compromise between spatial and molecular resolution. The method operates by applying a weighted translational-invariant Haar-wavelet transform denoising algorithm to phasor images. This results in significantly less bias and mean square error than other existing methods. We also present a new lifetime estimator (named normal lifetime) with a smaller mean squared error and overall bias as compared to frequency domain phase and modulation lifetimes. Overall, we present an approach that will enable the observation of the dynamics of biological processes at the molecular level with better temporal and spatial resolution.

Registro:

Documento: Artículo
Título:pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments
Autor:Silberberg, M.; Grecco, H.E.
Filiación:Departamento de Física, FCEyN, UBA and IFIBA, CONICET, Pabellón 1, Ciudad Universitaria, 1428 Buenos Aires, Argentina
Año:2017
Volumen:5
Número:2
Página de inicio:024016
DOI: http://dx.doi.org/10.1088/2050-6120/aa72ab
Título revista:Methods and applications in fluorescence
Título revista abreviado:Methods Appl Fluoresc
ISSN:20506120
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_20506120_v5_n2_p024016_Silberberg

Citas:

---------- APA ----------
Silberberg, M. & Grecco, H.E. (2017) . pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments. Methods and applications in fluorescence, 5(2), 024016.
http://dx.doi.org/10.1088/2050-6120/aa72ab
---------- CHICAGO ----------
Silberberg, M., Grecco, H.E. "pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments" . Methods and applications in fluorescence 5, no. 2 (2017) : 024016.
http://dx.doi.org/10.1088/2050-6120/aa72ab
---------- MLA ----------
Silberberg, M., Grecco, H.E. "pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments" . Methods and applications in fluorescence, vol. 5, no. 2, 2017, pp. 024016.
http://dx.doi.org/10.1088/2050-6120/aa72ab
---------- VANCOUVER ----------
Silberberg, M., Grecco, H.E. pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments. Methods Appl Fluoresc. 2017;5(2):024016.
http://dx.doi.org/10.1088/2050-6120/aa72ab