Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) using a single-photon avalanche diode (SPAD) array

Datum

04.06.2012

Zeit

14:00 - 15:00

Sprecher

Jan Krieger

Zugehörigkeit

German Cancer Research Center (DKFZ), Heidelberg, Germany

Sprache

en

Hauptthema

Biologie

Andere Themen

Biologie

Host

Peter Pitrone

Beschreibung

Measuring the time- and position-dependent intensity distribution of fluorescent probes is a promising approach to characterize the mobility of molecules insideliving cells and the accessibility of cellular compartments. Typically Fluorescence Correlation Spectroscopy (FCS) based on confocal microscopy is used for such measurements, which implies high time-resolution but only for a single-position measurement. In order to achieve a high time-resolution and parallel multi-position measurements, we built a Single Plane Illumination Microscope (SPIM) equipped with a high-speed image acquisition device and high-NA detection optics. The image sensor is either a fast EMCCD camera, or a 32×32 pixel array of single-photon avalanche photodiodes (SPAD). The read-out time of the SPAD array is 10µs for a complete 32×32 pixel frame. We also developed readout electronics that allows to calculate up to 1024 autocorrelation functions for data from this array in real time. It is based on reconfigurable logic chips (FPGAs - Field Programmable Gate Arrays). The SPAD array is two orders of magnitude faster than current EMCCD cameras and will thus allow to resolve the typical motion time-scale of small molecules (like eGFP) moving inside living cells. A comparison of the performance of these two image sensors for application in SPIM-FCS will be given.

Letztmalig verändert: 05.06.2012, 09:35:02