Oral cancer incidences have been increasing in recent years and late detection often leads to poor prognosis. Raman spectroscopy has been identified has a valuable diagnostic tool for cancer but its time consuming nature has prevented its clinical use. For Raman to become a realistic aid to histopathology, a rapid pre-screening technique is required to find small regions of interest on tissue sections <sup></sup>. The aim of this work is to investigate the feasibility of hyperspectral imaging in the visible spectral range as a fast imaging technique before Raman is performed. We have built a hyperspectral microscope which captures 300 focused and intensity corrected images with wavelength ranging from 450- 750 nm in around 30 minutes with sub-micron spatial resolution and around 10 nm spectral resolution. Hyperstacks of known absorbing samples, including fluorescent dyes and dried blood droplets, show excellent results with spectrally accurate transmission spectra and concentration-dependent intensity variations. We successfully showed the presence of different components from a non-absorbent saliva droplet sample. Data analysis is the greatest hurdle to the interpretation of more complex data such as unstained tissue sections.
Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)3]2+ characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.
Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging
Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing
methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised
approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution
and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up.
Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride
hexahydrate [Ru(bipy)<sub>3</sub>]<sup>2+</sup>, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card
with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated
chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this
technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.