Fluorescence photobleaching recovery spectroscopy in a dye doped nematic liquid crystal

Abstract

The in situ study of the diffusion dynamics of fluorescently tagged proteins or dextrants within cells or biologically relevant fluids is a customary practice in the field of biophotonics @1,2#. Normally, dyes which bind to specific macromolecules or proteins are employed as tracers to follow the particular dynamics of the latter. The dyes can be resonantly pumped by an external laser which is usually focused by a microscope objective. The fluorescence emitted by the tagged molecules can be collected by the same objective, to be subsequently filtered and detected. If photobleaching of the dyes takes place within the focal region of the objective, a decay of the fluorescence signal is observed in the early stages of laser illumination. The decay time produced by photobleaching depends on the input power, specific laser wavelength, and peculiarities of the absorption spectrum of the dyes. On the other hand, if the laser is turned off for a given period of time, a recovery of the fluorescence signal can be observed when the laser illumination is reinstated, for diffusion of new molecules takes place from the neighboring regions into the focal volume of the laser spot. In this manner, the diffusion process of the tagged molecules themselves can be studied. Diffusion of biomolecules is, in fact, one of the most basic an important mechanisms in cell biology @3#, and can be microscopically studied in this manner. The term fluorescence photobleaching recovery spectroscopy ~FPRS! has been coined as a general description of this method. The technique has different variations according to the details of the specific experiment. In particular, the geometrical aspects of the illuminating optics and the imaging method to monitor the fluorescence emission ~normal, confocal, etc.! determine the type of experiment under consideration. Very recently, the usage of two-photon scanning microscopy in addition to FPRS has been proposed @4# as a method for creating a flat, almost two-dimensional, distribution of photobleached molecules, allowing a better control of the initial conditions before diffusion takes place. Liquid crystals ~LC’s ! are complex fluids with intrinsic long range orientational order and intricate hydrodynamic properties @5#. Dye molecules can be diluted within a LC to take advantage, in this manner, of the intrinsic molecular order of the liquid crystalline phases, and become oriented along particular directions. The induced macroscopic order of the dyes was called guest-host interaction by Heilmeier, Castellano, and Zanoni, who observed it for the first time@6#. Since the absorption of the dyes is normally very anisotropic, dye doped LC’s are ideal systems where dichroism can be controlled by external parameters like temperature and magnetic or electric fields. Furthermore, the addition of dyes to nematic liquid crystals has been shown to be the source of several very interesting and complex nonlinear optical properties. In particular, the observation of enhanced optical torques @7,8#, unusual reorientation dynamics upon ultrashort laser illumination @9#, and permanent holographic recording of patterns @10# are a few incomplete examples of the rich optical properties of these systems. From the viewpoint of applications, dye doped LC’s seem to be promising substances for electro-optic devices with enhanced contrast ratios in the visible @11#. The diffusion process of dyes in LC’s has heretofore been poorly studied. In this paper, we shall concentrate on the study of dye diffusion through both oriented and unoriented nematic liquid crystal hosts. In particular, the application of FPRS, the effect of temperature, and a direct view into the diffusion process by means of digital imaging of the fluorescence emission will be demonstrated. Dye diffusion within an ordered nematic LC cell takes place in an anisotropic manner, which ensures the long term preservation of the dichroism. A direct view into the diffusion process of photobleached dye molecules will be shown by virtue of a twobeam FPRS technique. The interplay between photobleaching and diffusion will be explicitly shown, as well as the effect of temperature on a planar oriented LC cell where dye molecules are locally photobleached. Moreover, we show that Raman rather than fluorescence signals could be used to study the interdiffusion of transparent substances like binary mixtures of nematogens, where intrinsic fluorescence cannot be used. The paper is organized as follows: Section II presents the experimental setup for the forthcoming experiments, the sample preparation, a description of the interpretation of the data, and the experimental findings. The necessary theoretical background is also supplied in Sec. II, whenever it is relevant for the interpretation of the data. Finally, in Sec. III, a brief discussion is presented and a few conclusions are