Time encoded multicolor fluorescence detection in a microfluidic flow cytometer

Abstract

We describe an optical detection technique that delivers high signal-to-noise discrimination to enable a multi-parameter flow cytometer that combines high performance, robustness, compactness and low cost. The enabling technique is termed “spatially modulated detection” 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 expected transmission pattern achieves high discrimination of the particle signal from background noise. Additionally, the particle speed and its fluorescence emission characteristics are 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 non-encoded background fluorescence originating from fluorescent components in solution, fluorescing components of the chamber and contaminants on its surface. The optical detection technique is illustrated with experimental results on multi-color detection with a single large area detector by filtering fluorescence emission of different particles through a patterned color mask. Thereby the particles’ fluorescence emission spectrum is encoded in a time dependent intensity signal and color information can be extracted from the correlation analysis. The multicolor detection technique is demonstrated by differentiation of micro-beads loaded with PE and PE-Cy5 that are excited at 532 nm.

Optofluidic wavelength division multiplexing for single-virus detection

“…Emitted photons are captured and routed to a detector if the channel also acts as a liquidcore optical waveguide (19)(20)(21)(22). This type of multispot excitation can be used to significantly enhance the signal-to-noise ratio of a fluorescence assay (13,23). The SEM image in Fig.…”

“…Optical detection, by fluorescence or "label-free," is extremely sensitive, allowing for single-molecule and even single-dye detection under appropriate conditions (5)(6)(7)(8)(9)(10)(11). The availability of over a dozen dyes across the visible spectrum opens the door to implementing the desired multiplexing capability with multiple dyes, i.e., spectral channels (12)(13)(14). Secondly, diagnostic tests are rapidly transitioning toward integrated laboratory-on-chip platforms on which small volumes of biological or chemical samples can be rapidly analyzed.…”

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

“…Because we address common technical concerns with microfluidic detection systems, previous investigators have developed methods to address similar concerns. For instance, Martini et al frequency-encode multicolor fluorescence recorded by a single detector by projecting an excitation source through uniquely patterned "bar code" filters 12 .…”

The intersection of flow cytometry with microfluidics and microfabrication

“…For example, in flow cytometers designed for routine use in the field where service is difficult and usage may be rough, it may be desirable to include a robustly aligned optical pathway or alignment free detection at the expense of sensitivity and resolution. This has been done both with patterned optical masks and fixed fiber optics 145, 148, 149 . Another common approach is to simplify an instrument through the use of field based or inertial particle focusing.…”