TCSPC upgrade of a confocal FCS microscope

Abstract

We extended the measurement capabilities of the Carl Zeiss ConfoCor 1 FCS microscope by (a) using pulsed picosecond diode lasers instead of a continuous wave (CW) laser excitation, (b) introducing a fast single photon avalanche diode detector, and (c) exploiting the capabilities of the PicoQuant TimeHarp 200 board. When the time-tagged time-resolved (TTTR) mode of the TimeHarp is utilized, the complete fluorescence dynamics are recorded. That is, the time-evolution of the fluctuations and the fluorescence decay kinetics are captured simultaneously. Recording individual photon events (without on-the-fly data reduction like in hardware correlators) preserves the full information content of the measurement for virtually unlimited data analysis tasks and provides a much more detailed view of processes happening in the detection volume. For example, autocorrelation functions of dyes in a mixture can be separated and/or their cross-correlation can be investigated. These virtual two-channel measurements are performed utilizing a single detection channel setup. The time-resolved FCS is a powerful tool in biological studies and is demonstrated here on unilamellar vesicles giving clear evidence for Bodipy dye exchange between them. The described upgrade scenario is applicable to other confocal microscopes as well. In principle, any FCS system so far utilizing conventional CW lasers can benefit from pulsed excitation and the original functionality of the setup is fully preserved.