Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles

Abstract

Background: Light scattering is an essential parameter in flow cytometry, facilitating functions such as size measurement, discrimination of cell types on the basis of shape and morphology, detection of fluorescence-negative cells, and gating of fluorescence measurements. Light scattering measurement of viruses is generally not feasible with current flow cytometers due to their small size. The problem is aggravated by the fact that the light scattering of particles in this size range (< 200 nm) falls off with roughly the sixth power of their linear dimensions.

Methods: A new optical layout using darkfield illumination and detection has been developed. A 532-nm laser was used for excitation, and scattered light was collected with large aperture optics.

Results: Light scattering histograms of polymer particles with diameters of 70-300 nm were recorded without gating by other parameters. By extrapolation, a detection limit of about 50 nm was obtained. Different species of virus with sizes of approximately 100 nm also were recorded.

Conclusions: Flow cytometric light scattering measurement of submicroscopic particles, in a size range that includes many viral species, is now feasible. The results indicate that it may be practically impossible to measure by flow cytometry the light scattering of particles smaller than 40 nm.