Keith Frost

Extended depth of field imaging for high speed object analysis

Fluoresence microscopy is an extremely useful tool to analyze the intensity, location and movement of fluorescently tagged molecules on, within or between cells. However, the technique suffers from slow image acquisition rates and limited depth of field. Confocal microscopy addresses the depth of field issue via “optical sectioning and reconstruction”, but only by further reducing the image acquisition rate to repeatedly scan the cell at multiple focal planes. In this paper we describe a technique to perform high speed, extended depth of field (EDF) imaging using a modified ImageStream® system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 μm focal range.

Sensitivity measurement and compensation in spectral imaging

The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented.