Application of fluorescence correlation spectroscopy for drug delivery to tumor tissue

Quantitative fluorescence microscopy methods can provide valuable insight into drug delivery and pharmacokinetics. We are investigating the use of single photon fluorescence correlation spectroscopy (FCS) to measure particle concentration and mobility in living tissue. In this study we examined whether a relatively large illumination volume (laser spot radius of approximately 20 μm) could be used to probe file state of macromolecules in free solution and in tissue. The FCS set-up is based upon an upright research microscope, diode laser with 635 nm wavelength, an avalanche photodiode/single photon counting module, and PC based correlation electronics. Diffusion coefficients were extracted from measured autocorrelation functions. We used fluorescent monodisperse beads with diameter 20 and 200 mn to calibrate the excitation volume. Particle diffusion coefficients measured by FCS were compared with conventional light-scattering measurements. We then applied the technique to measure fluorescently labelled liposome distribution in tissue and tissue models. We found that the difference in quantum brightness and diffusion times of liposomes and free dye may be used to detect changes due to liposome interaction with living cancer cells.