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Label-free flow-cytometry on a microfluidic chip based on native fluorescence

 

The detection and characterization of single cells without the need for sample preparation is highly desirable. Flow cytometry based on native fluorescence spectroscopy is a promising approach that does not require specific binding or tagging of the analyte. However, the variety of cells is large compared to the number of basic molecular building blocks. Therefore, the fluorescence spectra of different species are often very similar, and sophisticated detection methods are required to reveal differences. The specificity of this approach can be improved by implementing high spectral resolution and using multiple excitation wavelengths. We have developed a compact platform that combines a microfluidic quartz channel with chip-size wavelength-selective detection which records the fluorescence of particles as they traverse the channel. The interaction between the excitation light and the analyte is enhanced by anti-resonantly guiding the excitation light within the analyte-containing fluid. We have recorded the intrinsic fluorescence of single cells (e.g., yeast, e-coli and BT) as they transit the detection area even at high speed. Simultaneously monitoring total intensity and spectrally-resolved emission yields accurate spectra for particle discrimination. Knowing the particle speed and the physical dimensions of the observation window, we are able to determine particle positions with microscopic (<10 microns) resolution. A novel modulation technique allows us to achieve a high signal-to-noise ratio even at high particle speeds. Our platform can readily incorporate both the detection of scatter signals as well as cell sorting techniques to enable on-the-chip characterization and sorting of untagged cells. This work is partially funded under ONR contract N00014-05-C-0430 and by the Alexander-von-Humboldt-Foundation.

 
citation

Beck, M.; Kiesel, P.; Bassler, M.; Johnson, N. M.; Schmidt, O. Label-free flow-cytometry on a microfluidic chip based on native fluorescence. Joint Meeting of the European Society for Clinical Analysis (ESCCA) and the German Society of Cytometry (DGfZ); 2008 September 16-20; Bremen, Germany.