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Biophys J, July 2000, p. 536-549, Vol. 79, No. 1

Time-Resolved Polarization Imaging By Pump-Probe (Stimulated Emission) Fluorescence Microscopy

Ch. Buehler,* C. Y. Dong,dagger P. T. C. So,dagger T. French,Dagger and E. Gratton§

 *Novartis Pharma AG, Pharma Research, CTA, LFU/NAT S-360.4.16, CH-4002 Basel, Switzerland;  dagger Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA;  Dagger LJL Biosystems, Sunnyvale, California 94089 USA; and  §Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 USA

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-µm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from ~30 to ~150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.

Biophys J, July 2000, p. 536-549, Vol. 79, No. 1
© 2000 by the Biophysical Society   0006-3495/00/07/536/14  $2.00


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