Abstract
1779-Pos Board B671 FLIM Phasor Analysis for Time-Domain and Frequency-Domain Data Enrico Gratton, Michelle A. Digman, Chiara Stringari, Cosimo Arnesano. University of California, Irvine, Irvine, CA, USA. The phasor analysis of FLIM images provides a fit free global view of molec- ular species and their interaction in cells and tissues. Different techniques are used to collect the original data either in the time domain or in the frequency domain. The ‘‘phasor transformation’’ which is based on the calculation of Fourier components should in principle make the phasor plot independent of the domain of data collection. However, technical differences between the modalities of data acquisition in various instruments result in slightly different phasor calculations. In this poster we discuss the origin of the variations be- tween the different methods of data acquisition. In particular we compare data obtained with the classical analog frequency domain instrument, data obtained with the FLIMbox principle that is based on a digital equivalent of the frequency domain instrument and data obtained with the popular time- correlated single photon counting instrument. We discuss how to minimize these differences which could results in phasors plots that can be directly com- pared form data obtained with different instruments. We also discuss and com- pare methods of data filtering which can decrease the noise in the phasor plot without affecting the resolution of FLIM images. Finally we compare phasor plots obtained for different harmonics of the laser repetition frequency. We show that the phasor plot at high harmonics from autofluorescence tissue sam- ples can distinguish between various extracellular components such as the weak fluorescence from collagen and elastin. Work supported in part by NIH-P41 P41-RRO3155, 8P41GM103540 and P50-GM076516
Highlights
1778-Pos Board B670 Isotropic Resolution in Localization-Based Super-Resolution Microscopy by Single Objective Emission Interference Joerg Schnitzbauer
In order to test this hypothesis, we have developed both one-photon and two-photon fluorescence correlation spectroscopy (FCS) to measure the hydrodynamic radius of fluorescent particles in glucose solutions
We demonstrate that 4Pi detection for localization based super-resolution microscopy can be realized by using only one objective and a mirror, which eliminates the drawbacks of dual objective 4Pi detection, but still provides isotropic localization precision
Summary
1778-Pos Board B670 Isotropic Resolution in Localization-Based Super-Resolution Microscopy by Single Objective Emission Interference Joerg Schnitzbauer. 1779-Pos Board B671 FLIM Phasor Analysis for Time-Domain and Frequency-Domain Data Enrico Gratton, Michelle A. University of California, Irvine, Irvine, CA, USA.
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