Characterization and Alignment of the STED Doughnut Using Fluorescence Correlation Spectroscopy

Abstract

<p>This report primarily focuses on effectively obtaining a Stimulated Emission Depletion fluorescence (STED) microscope, while using Fluorescence Correlation Spectroscopy (FCS) as a guide for the alignment of the system. STED is a super-resolution microscopy technique that has gained favour in the biological sciences due to its ability to successfully resolve sub-diffraction structures within live cells. Moreover the ease with which it can be combined with FCS has extended the applications of this technique to the study of the dynamics within a system as well. The central premise of this work focuses around building a STED-FCS system and developing an alignment tool for obtaining a symmetric STED doughnut. Since the point spread functions (PSF) seen in confocal microscopy can be generally approximated by a Gaussian function, we approximate the doughnut PSF with a difference of Gaussian functions. We calculated an autocorrelation function (ACF) corresponding to the simplified Gaussian form of the doughnut PSF and we found that this ACF contained three very similar diffusion times, all inversely proportional to the dye diffusion coefficient. In agreement with the fact that the doughnut PSF is spread out compared to the purely Gaussian PSF, the doughnut ACF amplitude is lower and its average diffusion time large. Lastly we calculated the quality factor, which is the product of the amplitude of the correlation function with the average intensity, Q=G(0)*I, for the purposes of alignment of the system. When translating the confocal pinhole along an axis of the doughnut we were able to identify the centre of the doughnut due to the presence of a minimum in Q which can be very handy for alignment of the doughnut with respect to the pinhole. This operation is essential when aligning the excitation and STED beam. For future work, a road map for alignment of the two beams in the focal plane is also presented utilizing the cross correlation function between the two beams.</p>