Cellular discrimination based on spectral analysis of instrinic fluorescence

Abstract

The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances, have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from higher density of spectral information and a more flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200 fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.