Observation of single‐cell fluorescence spectra in laser flow cytometry

Abstract

We report the first demonstration of directly recording fluorescence spectra of single cells in flow cytometry. An intensified, 512-element photodiode array was used in conjunction with a dispersing prism to capture the fluorescence emission spectra of Coulter ImmunoCheck calibration beads and Dictyostelium discoideum spores stained with the indocarbocyanine derivative CY3, fluorescein isothiocyanate, or R-phycoerythrin. The demonstration was made feasible by enhancing the signal-to-noise ratio of the detection process by using a fast gating technique applied to the detector. Results show that the complete fluorescence spectra of individual stained cells contain information that is normally not captured by conventional flow cytometers. By using the spectrographic flow cytometer, all this information is recorded, allowing small features and shifts in the fluorescence spectra of labelled particles to be studied.

Go with the flow - use of flow cytometry in environmental microbiology

“…Modifications to lasers and detectors can also be used to enhance the discriminatory power of flow cytometry. Gauci et al [50] demonstrated the use of spectral fingerprinting, a technique to determine spectral properties of particles at tens to hundreds of wavelengths simultaneously. Particle types are recognised by reference to previously assigned optical fingerprints.…”

Capillary electrophoresis with wavelength-resolved laser-induced fluorescence detection

“…The IPDA is a solid-state diode array coupled to an image intensifier via a fiber-optic bundle. With the image intensifier as an amplifier for photons, a gain of 3000 to 9000 is typically obtained, thus the signal-tonoise ratio of the IPDA detector is significantly higher than that of the PDA and the IPDA is thus highly sensitive under low-light conditions [58,59]. In comparison, the CCD has extremely low noise, generally under 5 e-and has a dark current less than 1e-per hour if properly cooled, and so is commonly used without intensification in this application [51,57].…”

Current and future applications of flow cytometry in aquatic microbiology

“…Neural networks can be trained to recognise sub‐populations of microorganisms from previous data to examine and sort aquatic microbes [171]. Spectral fingerprinting can be used to determine the spectral properties of particles at hundreds of wavelengths simultaneously [172] and lectins can help to improve discrimination of cells from abiotic particles [173]. The exposure of front‐line soldiers or civilian populations to biological warfare agents needs extremely rapid detection.…”