Detecting the fluorescence emission wavelength using a spatially modulated color mask in a compact flow cytometer platform

Abstract

We will present an optical detection technique that delivers high signal-to-noise discrimination to enable a flow cytometer that can combine high performance, robustness, compactness, and low cost. The enabling technique is termed spatially modulated emission and generates a time-dependent signal as a continuously fluorescing (bio-) particle traverses an optical transmission pattern along the fluidic channel. Correlating the detected signal with the known transmission pattern achieves high discrimination of the particle signal from background noise. Additionally, the particle speed can be deduced from the correlation analysis. Our method uses a large excitation/emission volume along the fluidic channel in order to increase the total flux of fluorescence light that originates from a particle while requiring minimal optical alignment. Despite the large excitation/detection volume (~ 1mm), the mask pattern enables a high spatial resolution in the micron range. This allows for detection and characterization of particles with a separation (in flow direction) comparable to the dimension of individual particles. In addition, the concept is intrinsically tolerant of background fluorescence originating from fluorescent components in solution, fluorescing components of the chamber, and contaminants on the surface. We will present the characterization of different particles by fluorescence emission wavelength measurements. We encode a two color emission measurement into a time-dependent intensity signal on a single large-area detector. This is performed by filtering fluorescence emission of different fluorescence particles through a patterned color mask. Thereby the particles fluorescence emission spectrum is encoded in a time-dependent intensity signal when passing the emission mask. We will demonstrate our experimental results of micro-beads loaded with PE and Pe-Cy5 that is excited at 532 nm. Furthermore, we will show that the system is suitable for a commercial lymphocyte flow assay in whole blood, which utilizes PE and Pe-Cy5 to differentiate CD3-, CD4- and CD8-cells.