High resolution optics combined with high spatial reproducibility in flow.

Abstract

Accurate sizing in flow using optical methods generally requires high resolution optics and specially designed flow systems. Flow systems developed by this group have following features: (a) double sheath configuration for optical index match, (b) no curved optical surface in the sensing area, (c) gradual hydrodynamic focusing over a long distance to minimize mechanical shearing, (d) precision spatial positioning of cells by reducing suspension fluid diameter to a cell diameter or less, (e) total thickness between outer surfaces of the flow chamber at the viewing area of 1.5 mm or less. Cells intersect a laser light beam focussed go circular as well as elliptical cross-sections or 1 micron or less in diameter. Cellular extinction is monitored during transit through the beam. Cell length is derived from the time for flight measurement and corrected for absolute values by continuous velocity reference using a second laser beam intersecting the cell stream at a predetermined distance. This second spot may be circular or elliptical, of a different polarization and/or frequency. Simultaneous fluorescence intensity and diameter measurements were performed on test particles using different optical geometries. The influence of the particle structure on fluorescence measurements is demonstrated where high resolution sizing is required at the same time.