Glare Points in Laser Flow Cytometry

Abstract

When images are recorded using the elastically side-scattered light of spherical micro-particles (“beads”) in an imaging flow cytometer one can often see two or more bright spots rather than an image resembling the outline of the particle, like this would be seen in a bright-field or dark-field microscope. The cause of this effect is not clear - at least in the flow cytometry community. Some common explanations include them being the entry and exit points of the laser beam, possibly in connection with either fluorescence or parasitic scattering at a rough particle surface. Here we show that these bright spots are in fact so called glare points (or glare spots) that are fully explained by the elastically scattered light from a smooth, homogeneous particle and have been know in other fields of optics for many years. However, the common theoretical framework (particles with large size parameter $x > 1000$, small optical apertures) needs to be extended in order to quantitatively describe the glare points seen with high numerical aperture objectives and particle size parameters $x\leq 100$ that are common in flow cytometry. The characteristics of the glare-point image (number, shape, position and brightness of points) depend on particle diameter and refractive index, as well as the optics used to form the image. Particularly for small particles $(x\leq 50)$, the glare points seem to lie outside of the particle, as we discuss. We show measurements and simulations for polystyrene particles, where this effect is clearly visible and - if not properly taken into account - would significantly affect the results of glare-point based particle sizing.