Time encoded multicolor fluorescence detection in a microfluidic flow cytometer.

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, 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 of multicolor 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 (Phycoerythrin) and PE-Cy5 that are excited at 532 nm.

Monitoring CD4 in whole blood with an opto-fluidic detector based on spatially modulated fluorescence emission.

We describe a new optical detection technique, termed "spatially modulated fluorescence emission," that delivers high signal-to-noise discrimination without precision optics to enable a flow cytometer that can combine high performance, robustness, compactness, low cost, and ease of use. The detection technique is demonstrated with measurements of absolute CD4+ and percentage CD4 counts in human blood. Benchmarking against a commonly used commercial instrument for this test yields excellent agreement for both absolute CD4 and percentage CD4.

Glucose concentration monitoring using a small Fabry-Perot etalon.

Accurate measurements of aqueous glucose concentrations have been made in a double-chamber Fabry-Perot etalon that can be miniaturized for subcutaneous implantation to determine the concentration of glucose in interstitial fluid. In general, optical approaches to glucose detection measure light intensity, which in tissue varies due to inherent scattering and absorption. In our measurements, we compare the spectral positions of transmission maximums in two adjunct sections of an etalon in order to determine the refractive index difference between these sections and therefore we can tolerate large changes in intensity. With this approach, we were able to determine aqueous glucose concentrations between 0 mg/dl and 700 mg/dl within the precision of our reference measurement (+/-2.5 mg/dl or 2% of the measurement value). The use of reference cavities eliminates interference due to temperature variations, and we show the temperature independence over a temperature range of 32 degrees C to 42 degrees C. Furthermore, external filters eliminate interference from large molecule contaminants.

Fluorescence spectrometer-on-a-fluidic-chip.

A chip-size spectrometer is realized by combining a linear variable band-pass filter with a CMOS camera. The filter converts the spectral information of the incident light into a spatially dependent signal that is analyzed by the camera. A fluidic platform is integrated onto the spectrometer for analyzing the fluorescence from moving objects. The target is continuously excited within an anti-resonant waveguide, and its fluorescence spectrum is recorded as the object traverses the detection area.

Performance of chip-size wavelength detectors.

Chip-size wavelength detectors are composed from a linear variable band-pass filter and a photodetector array. The filter converts the incident spectral distribution into a spatial distribution that is recorded by the detector array. This concept enables very compact and rugged spectrometers due to the monolithic integration of all functional components on a single chip. This type of spectrometer reveals its most convincing advantages through appropriate systems integration. We discuss the advantages of this concept for spectroscopy of light distributions that are hard to focus onto the entrance slit of a conventional spectrometer, namely large light emitting areas and moving point-like light sources. The excellent spectral performance of the system is demonstrated for both light input geometries.