The differential polarization laser-scanning microscope (DP-LSM) combines the advantages of scanning optical microscope and spectropolarimeter. It can provide high precision pixel-by-pixel data on the anisotropic organization of biological samples in real time, during the recording of the intensity images. Differential polarization microscopy have been shown to supply unique information on the anisotropic organization of highly organized molecular macroassemblies (e.g. photosynthetic membranes, actin filaments, cell walls, membrane domains, protein aggregates), but its use is limited by the availability of DP-LSMs.
Earlier we designed and constructed differential polarization (DP) units for a Zeiss 410 laser scanning microscopes. Recently, we equipped an Olympus Fluowiev 500, with an easy to attach DP unit. This DP-LSM in its performance is similar to the one constructed earlier but its novel design allows us to readily transform a conventional LSM to DP-LSM, virtually without changing its optical and electronic units. For the modulation of the polarization state of the laser beam or of the fluorescence light, a high frequency photoelastic modulator (PEM) is used and the requested DP quantity can be obtained after demodulation of the signal of the photomultiplier, using a lock-in amplifier. The constructed DP-LSM allows fast and precise measurement of the following DP quantities: linear dichroism (LD), anisotropy of the emission dipoles upon non-polarized excitation (r, confocal), fluorescence detected linear dichroism (FDLD, confocal), the degree of polarization of the fluorescence emission (P, confocal), and birefringence (LB). By this work we demonstrate that a DP accessory unit can easily be attached to almost any LSM, making it capable of mapping anisotropy parameters in 2D or 3D.