This proposal is for the purchase of a scanning laser image cytometer with multi color capabilities for evaluation of viable adherent or immobilized cells. The most appropriate commercially available instrument is the Meridian instruments ACAS 570 Interactive Laser Cytometer. With the newer options created for this instrument, it now incorporates both phase image and fluorescent image overlays. Thus, pseudo-enhanced fluorescence images can be superimposed against the morphological structure of the cell in real-time, providing a unique capability in kinetic analysis. Further, the sorting capabilities of this instrument can be applied within a culture plate allowing selective removal of particular cells via ablation, without disturbing the cellular matrix, something that the flow cytometer cannot do. In addition, using the confocal capabilities, 3D reconstructions can be made of fluorescent images, allowing identification of site-specific areas containing fluorescent metabolites resultant from cell activation receptor activation, or receptor binding. The projects listed come from diverse areas in biology, however, all can significantly benefit from the presence of such an instrument. No single investigator would be able to obtain the funding for such a costly instrument and it is not present at Purdue, or anywhere in this state. The instrument will become a part of the existing Purdue Cytometry Laboratories which is currently supported by over 30 faculty as well as the Purdue Cancer Center, an NCI funded research center. In this manner its availability to all interested faculty will be ensured, as is presently the case for the flow cytometry equipment currently within that facility. Users are presently fully accustomed to the capabilities of the flow cytometry facilities. Where appropriate, they already use these instruments. However, as explained in this proposal, the technologies are different and complimentary. Indeed, increased need for the ACAS type instrument has stemmed from the use and open availability of the flow cytometers.
