Our workhorse instrument is a single-capillary laser-induced fluorescence detector based on a sheath-flow cuvette. These cuvettes are most commonly employed by the flow-cytometry community to study single mammalian cells by fluorescence and light-scatter. We have been using the cuvettes as electrophoretic and chromatographic detectors for many years.
       The cuvettes are designed to generate the best possible detection limits in fluorescence analysis. In any analytical method, the detection limit is related to the signal-to-noise ratio. The signal in fluorescence is the detect photons emitted from an illuminated sample. We employ a high numerical aperture microscope objective, which provides for the detection of a significant fraction of photons.

       The noise associated with the detection limit is the noise in the background or blank signal. In fluorescence, there are five background signals: Raman scatter from solvent, Rayleigh scatter from solvent, Rayleigh scatter from the cuvette windows, Raman scatter from the cuvette windows, and detector dark count. The sheath flow cuvette eliminates all scatter from the cuvette windows, which eliminates a major source of background signal and which improved detection limits.
       The cuvette consists of a square flow chamber, usually made from quartz but sometimes from other transparent material. In our design, the electrophoresis capillary is inserted into the flow chamber, where the inner dimension of the flow chamber matches the outer diameter of the capillary.

       Buffer is pumped in the top of the cuvette, which draws analyte from the capillary as a thin stream. Fluorescence is excited by a focused laser beam, collected with a microscope objective, imaged onto a pinhole to block scattered laser light, filtered to block Raman scatter, and detected with a photomultiplier tube or avalanche photodiode. Because the flow chamber is much larger than the sample stream, it is trivial to block light scattered from the cuvette walls and prevent it reaching the photodetector.
       Care is required in the design of the spectral filter. The filter must block as much of the Raman scatter as possible while passing as much of the fluorescence as possible. For most dyes in common use, there is a relatively small Stokes-shift between the excitation and emission wavelengths. In this case, it is vital to design the filter with care to optimize the detection limit. In the example below, the red curve is the Raman scatter generated when water is illuminated with light at 488 nm. The scatter maximizes in the yellow portion of the spectrum, and is visible as the horizontal streak in the photograph of the 16-capillary cuvette. The blue curve is fluorescence generated by the dye fluorescein, which is the same dye used in yellow highlighter pens. A spectral filter must be used that only passes light in the 475-525 nm range, to block as much Raman as possible while passing much of the fluorescence.