Fluorescência por absorção de dois fótons em corantes com aplicações biológicas

Abstract

We use the technique of fluorescence by two photon absorption to study some dyes that are important for applications in biological imaging. Some biological samples are autofluorescent, i.e. they have a high absorption cross section and absorb and emit radiation. However mostsamples are not autofluorescent and need to be marked by a fluorescent dye for visualization. The aim of our study was to investigate some of these dyes for biological labeling. We studied the analog of the Lambert Beers law for a process of two-photon absorption and present the techniques of fluorescence microscopy and spectroscopy that we used to obtain the results. We implemented an experimental setup for the two-photon absorption fluorescence spectroscopy using a pulsed Ti-sapphire laser. The laser is tunable in the wavelength range of 680 to 1080 nm and allows ultra short pulses, on the order of 140 fs, high repetition rates, high peak power and large spectral bandwidth. The fluorescence is collected by a lens and through an optical fiber is directed to a spectrometer coupled to a CCD camera for spectral analysis. We used a programmable acousto-optic modulator control the temporal dispersion of the pulse. The dispersion is responsible for the time delay of the spectral components on the pulse bandwidth and consequently introduces a pulse broadening as the beam passes through a dispersive medium. We measured the fluorescence spectra for various dyes and obtained their two-photon excitation photoluminescence spectra (TPE). For rhodamine 6G we compared the measured TPE spectrum with data on the literature. We also obtained the one photon absorption spectra(1PA) for rhodamine 6G dye, DAPI and ER-Tracker Red, and we show a comparison between the 1PA and TPE spectra for rhodamine 6G. We show also results for the dependence of the fluorescence with the excitation power, for the dyes rhodamine 6G and ER-tracker red. Using multi-photon microscopy, I collaborated in the imaging of biological tissue sections, as part of the Masters research work of Paulo Campos-Junior, on studies of how the Leydig cells are distributed in the testis of a wild pig, Caititu, Tayassu tajacu.