Spatially-offset fluorescence spectroscopy (SOFS) using ring illumination and point collection for sub-surface measurements in layered tissue

Abstract

PurposeTo report development of a depth-sensitive fluorescence spectroscopy system based on the configuration of point collection fixed at the centre of an illumination ring on the surface of a target sample. Methods The system makes use of an axicon for converting the collimated laser beam into ring shaped illumination and achieves the ability of subsurface interrogation by varying the radii of the illumination rings thereby introducing spatial offsets between collection and illumination. The system was validated on a non-biological phantom comprising a thin tissue paper overlaying a thick dye card and also on a biological tissue sample that was a chicken leg tissue consisting of a thin epithelial membrane on the top of a much thicker muscle tissue. ResultsIt was found that while the fluorescence spectra corresponding to zero spatial offset are dominated by the fluorescence emission bands associated with the top layer of the layered samples, the fluorescence spectra measured with spatial offsets contain higher contribution of fluorescence signatures characteristics of the bottom layer as compared to the spectrum measured with the zero offset. Conclusions The SOFS based system is able to interrogate subsurface depths beyond the reach of the conventional confocal fluorescence by simply varying the position of the axicon in the excitation arm. Due to the simplicity in its instrumentation, the approach has the potential to be developed into a portable system for in situ measurement in a tissue.