Long Working Distance Time Resolved Pump-Probe (Stimulated Emission) Fluorescence Anisotropy Imaging
Ankur Gogoi1*, Subir Das1, Yi-Chih Liang1, Che-Lun Hsu1, Fu-Jen Kao1
1Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan
* Presenter:Ankur Gogoi
Time resolved fluorescence anisotropy imaging (TRFAI) is a powerful tool to investigate the molecular rotational dynamics, which depends on the size and shape of the rotating unit (molecule) as well as the temperature and viscosity of the biophysical micro-environment. Notably, TRFAI measures the decay of fluorescence depolarization caused by the rotational Brownian diffusion of the fluorophores in the excited state upon excitation with linearly polarized light. It can be used to extract sensitive information about cell biology, such as, local micro-viscosity, nano-scale clustering of proteins and lipids, molecular interactions (binding events), etc. Form the instrumentation point of view, there are several fluorescence lifetime imaging microscopy (FLIM) modalities, e.g., time gated wide-field time domain, wide-field frequency domain, streak camera FLIM, etc., which can be used to measure fluorescence anisotropy measurements.

In this contribution, we report the successful implementation of the intensity modulated ultrafast lasers with integrated electronic delay control and lock-in detection for long working distance fluorescence lifetime anisotropy measurements. The method employed a pair of synchronized lasers at wavelengths 635 nm and 780 nm as the pump and probe beams, respectively, for the ATTO647N labelled samples. The use of electronic time delay trigger provided an advantageous way in controlling the relative pulse separation to probe fluorescence lifetime of ATTO647N dye in nanosecond scale, whereas low NA optics, used in the experiment, allowed long working distance measurements. The pump-probe (stimulated emission) signal was acquired by using a lock-in amplifier, the output of which is connected to the A/D converter of the galvano-mirror based laser scanning system (Olympus FV300) to reconstruct the image. Polarization resolved fluorescence decay curves, and subsequently the fluorescence lifetime anisotropy, were obtained by imaging the parallel and perpendicular polarization components of the fluorescence emission. The scheme presented here proved to be a highly efficient alternative to measure relatively long fluorescence lifetime anisotropy of the order of a few nanoseconds.

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Keywords: Time resolved fluorescence anisotropy, Fluorescence lifetime imaging, Pump-probe microscopy, Stimulated emission microscopy, Lock-in detection