IN SITU TWO PHOTON IMAGE CORRELATION STUDIES OF ADHESION RECEPTORS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Continued efforts are being made the study the use of image correlation spectroscopy(ICS) and image cross-correlation spectroscopy (ICCS). The regulation of the aggregation state and transport properties of adhesion receptors is believed to play a significant role in the underlying mechanisms that control cellular adhesion in migratory cells. We continue to study the dynamics of cellular adhesion, structure formation, and disassembly by combing two-photon microscopy and ICS methods for live cell measurements. The use of ICS offers a way to simultaneously monitor the aggregation state of fluorescently labeled bio-molecules within the plasma membrane as well as their transport properties. Additionally, two-color image cross correlation spectroscopy (ICCS) permits the direct measurement of dynamic co-localization phenomena in live cells by imaging non-identical species in two wavelength channels. These are then analyzed by spatial and temporal cross-correlation. The combination of ICS/ICCS and two-photon microscopy allows these measurements to be performed in a minimally invasive way on living cell specimens using less damaging infrared wavelength laser excitation. To study the dynamics and aggregation of focal adhesion components, we have prepared GFP fusions of the a5 integrin subunit, paxillin and a-actinin. When expressed ectopically in CHO or WI-38 cells, they localize to focal adhesions and do not perturb migration, spreading or formation of focal adhesions. We have studied the dynamic regulation of adhesion structures in living fibroblasts cultured on various activating and non-activating extra cellular matrix (ECM) coated substrates by the unique approaches offered by two-photon ICS and ICCS. Furthermore, we have applied ICS techniques to light and electron microscopy images to determine spine densities in brain tissue of laboratory animals. Initial progress using image correlation spectroscopy at NCMIR has produced the following publications: Wiseman PW, Squier JA, Ellisman MH, Wilson KR. (2000) Two-photon image correlation spectroscopy and image cross-correlation spectroscopy. J Microsc. 200 ( Pt 1):14-25. Wiseman PW, Capani F, Squier JA, Martone ME. (2002) Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis. J Microsc. 205(Pt 2):177-86.