Flow Optical Tomography (FOT) is a novel technology, currently in early stage development by Vision Gate Inc., for rapidly imaging individual stained cells in a high-throughput flow stream. Multiple point projection sources and detectors generate 2D and 3D densitometric images and chromagen quantitation at subcellular resolution. In this pilot study, we build on our past experience and collaborate with Vision Gate to evaluate the promising ability of FOT through three Aims:- First, we will select from commercially available nuclear stains those most compatible with FOT technology.- Second, we will use our existing imaging technology to create and validate a digital library of pre-malignancy associated features from nuclear stained and immuno-cytochemical markers.- Finally, we will determine whether FOT can capture these 2D morphologic and densitometric features of lung cancer and pre-cancerous cells from a high throughput flow of archived sputum specimens produced by individuals with known clinical outcomes from the ongoing Moffitt lung cancer screening study.In the proposed investigation, we will evaluate the ability of FOT to capture, in a high-throughput manner, the relevant diagnostic information for lung cancer early detection using both morphology and densitometry to enable the development of a platform for mass screening. Vision Gate technology also supports this project by developing, in a separate study, a high throughput specimen-processing device. This provides specimens for the FOT that will be enriched by removal of confounding debris and uninformative cell types, former limitations to flow analysis of sputum specimens. The system, comprised of the specimen processor and the FOT analysis instrument, is expected to have applicability in high throughput testing environments.