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Proceedings Paper

Differential polarization laser scanning microscopy: biological applications
Author(s): G. Steinbach; F. Besson; I. Pomozi; G. Garab
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Paper Abstract

With the aid of a differential polarization (DP) apparatus, developed in our laboratory and attached to our laser scanning confocal microscope, we can measure the magnitude and spatial distribution of 8 different DP quantities: linear and circular dichroism (LD&CD), linear and circular anisotropy of the emission (R and CPL, confocal), fluorescence detected dichroisms (FDLD&FDCD, confocal), linear birefringence (LB), and the degree of polarization of fluorescence emission (P, confocal). The attachment uses high frequency modulation and subsequent demodulation, via lock-in amplifier, of the detected intensity values, and records and displays pixel-by-pixel the measured DP quantity. These microscopic DP data carry important physical information on the molecular architecture of anisotropically organized samples. Microscopic DP measurements are thought to be of particular importance in biology. In most biological samples anisotropy is difficult to determine with conventional, macroscopic DP measurements and microscopic variations are of special significance. In this paper, we describe the method of LB imaging. Using magnetically oriented isolated chloroplasts trapped in polyacrylamide gel, we demonstrate that LB can be determined with high sensitivity and good spatial resolution. Granal thylakoid membranes in edge-aligned orientation exhibited strong LB, with large variations in its sign and magnitude. In face-aligned position LB was considerably weaker, and tended to vanish when averaged for the whole image. The strong local variations are attributed to the inherent heterogeneity of the membranes, i.e. to their internal differentiation into multilamellar, stacked membranes (grana), and single thylakoids (stroma membranes). Further details and applications of our DP-LSM will be published elsewhere.

Paper Details

Date Published: 13 October 2005
PDF: 10 pages
Proc. SPIE 5969, Photonic Applications in Biosensing and Imaging, 59692C (13 October 2005); doi: 10.1117/12.639362
Show Author Affiliations
G. Steinbach, Hungarian Academy of Sciences (Hungary)
F. Besson, Hungarian Academy of Sciences (Hungary)
I. Pomozi, PI Vision Bt. (Hungary)
G. Garab, Hungarian Academy of Sciences (Hungary)

Published in SPIE Proceedings Vol. 5969:
Photonic Applications in Biosensing and Imaging
Brian C. Wilson; Richard I. Hornsey; Warren C. W. Chan; Ulrich J. Krull; Robert A. Weersink; Kui Yu, Editor(s)

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