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.

A prototype EDF ImageStream system incorporating a Wavefront Coded™ element was used to capture imagery from fluorescently labeled beads. Bead imagery was quantitatively analyzed using photometric and morphological features to assess consistency of feature values with respect to focus position. Jurkat cells probed for chromosome Y using a fluorescence in situ hybridization in suspension protocol (FISHIS™) were used to compare standard and Wavefront Coded‐based EDF imaging approaches for automated chromosome enumeration.

Qualitative visual inspection of bead imagery reveals that the prototype ImageStream system with EDF maintains focus quality over a 16 μm focus range. Quantitative analysis shows the extended depth field collection mode has approximately ten‐fold less variation in focus‐sensitive feature values when compared with standard imaging. Automated chromosome enumeration from imagery of Jurkat cells probed using the FISHIS protocol is significantly more accurate using EDF imaging.

The use of EDF techniques may significantly enhance the quantitation of cell imagery, particularly in applications such as FISH, where small discrete signals must be detected over a wide focal range within the cell. © 2007 International Society for Analytical Cytology