S16_FCS

Abstract

We observe the diffusion of Rhodamine 6G (R6G) and fluorescent nanospheres in aqueous solution by focusing a 532nm laser through an objective lens to create a 1 μm3 observation volume. The laser excites the R6G molecules in this region, causing them to fluoresce. The photons released in this fluorescence pass through a confocal detection pinhole, which filters out light not originating from the specific observation volume, and are subsequently measured by a photomultiplier tube (PMT). The PMT is sensitive to single photons and outputs photon counts as TTL signal to the computer where the digital signal is processed. The data obtained is fluorescence intensity as a function of time. By auto-correlating this intensity data, we generate a correlation curve, from which the diffusion coefficient, concentration, and size of the molecules are determined.

Introduction

Fluctuation Correlation Spectroscopy (FCS) involves the observation of photons emitted by fluorescent molecules as they move in and out of a region of excitation (a focused laser beam). The movement of these molecules (or the particles they are attached to) is known as Brownian motion which describes the time-average rate of movement by the Diffusion coefficient (D). The larger the diffusion coefficient, the faster a particle moves, the smaller, the slower. When particles diffuse into the excitation region, they fluoresce and release a photon, and once they diffuse out of the volume they no longer fluoresce. This causes fluctuations in the fluorescence intensity over time as particles move in and out, and these fluctuations allow the diffusion coefficient of the particle (or particles in some cases) and the radius of the particle to be determined [1].

An important phenomenon that FCS relies on is the Stoke's shift. When the fluorescent molecule is excited (Figure 1), before the excited electron relaxes to the ground state, it goes through a series of small relaxations due to energy loss to the environment. Thus, when relaxation occurs, the emitted photon is of a lower energy (and longer wavelength) than the photon that excited it initially. In the case of Rhodamine 6G, when the molecule is excited by 532 nm light, it emits photons in the range of 560-650nm, allowing the separation of the fluorescent light from the excitation beam.