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.
We describe a novel method by which it is possible to apply and measure torque directly on particles grabbed in optical tweezers. It can be used to orient particles of micron size or even on single molecules, biopolymers by the use of test particles.
The procedure is based on the observation that flat objects are oriented in an optical trap formed by linearly polarized light. The orienting torque originates from the anisotropic scattering of polarized light by the trapped particle. The phenomenon is characterized in detail, the physics is analysed.
A tool is developed that exploits this effect to manuipulate biological macromolecules. Microscopic particles are produced by photopolymerisation that exploit this orientation effect and by which the torque is applied upon the biological object. In our system the applied torque can be turned on and off, it is controlled independently of the grabbing force of the tweezers during the manipulation process.
The capabilities of the method are demonstrated. The method has great promise for application on DNA, DNA-protein complexes, actin filaments and other biopolymers.