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IOCB Prague

Josef Lazar Group

Advanced Optical Microscopy
Research Group
Junior
PHYS cluster

About our group

The Laboratory of Advanced Optical Microscopy develops advanced techniques of optical microscopy and uses them to gain information about mechanisms of molecular processes taking place in living cells and organisms. The Laboratory has developed the technique of two-photon polarization microscopy, which allows sensitive observations of changes in conformation of membrane proteins. Such conformational changes can occur, for example, in response to a therapeutic drug or to changes in cell membrane voltage. The laboratory is equipped by a state-of-the-art laser scanning confocal/two-photon microscope (Olympus FluoView 1200MPE-IX83) adapted for single- and two-photon polarization microscopy. The microscope's versatile design enables accommodating a wide range of microscopy techniques and custom-made solutions. The Laboratory's multidisciplinary expertise in biochemistry, molecular and cell biology, biophysics, optics, electronic engineering, computer programming and mathematical modeling allows tackling of a wide range of difficult scientific questions.
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Publications

All publications
Nonlinear Optical Properties of Fluorescent Dyes Allow for Accurate Determination of Their Molecular Orientations in Phospholipid Membranes
Nonlinear Optical Properties of Fluorescent Dyes Allow for Accurate Determination of Their Molecular Orientations in Phospholipid Membranes
Štěpán Timr
J. Brabec
Alexey Bondar
T. Ryba
M. Železný
Josef Lazar
Pavel Jungwirth
Journal of Physical Chemistry B 119 (30): 9706-9716 (2015).
Several methods based on single- and two-photon fluorescence detected linear dichroism have recently been used to determine the orientational distributions of fluorescent dyes in lipid membranes. However, these determinations relied on simplified descriptions of nonlinear anisotropic properties of the dye molecules, using a transition dipole-moment-like vector instead of an absorptivity tensor. To investigate the validity of the vector approximation, we have now carried out a combination of computer simulations and polarization microscopy experiments on two representative fluorescent dyes (DiI and F2N12S) embedded in aqueous phosphatidylcholine bilayers. Our results indicate that a simplified vector-like treatment of the two-photon transition tensor is applicable for molecular geometries sampled in the membrane at ambient conditions. Furthermore, our results allow evaluation of several distinct polarization microscopy techniques. In combination, our results point to a robust and accurate experimental and computational treatment of orientational distributions of DiI, F2N12S, and related dyes (including Cy3, Cy5, and others), with implications to monitoring physiologically relevant processes in cellular membranes in a novel way.