This application is for a dual beam multiphoton microscope to allow non-invasive simultaneous imaging of multiple fluorophores deep in tissues of live specimens. Such real-time observations will enable the applicants to gain new insights into the regulation of cell behaviour through the interplay of genetic programs, cell-cell interactions and environmental cues. The applicants will use the microscope to study embryonic and adult zebrafish and mice, developing chick and Xenopus embryos and human cells cultured in bioengineered microenvironments. They are investigating diverse biological problems: how the spinal cord, brain, ear, skull and skin form during embryogenesis, how tumour growth is influenced by macrophages, how adult epidermal and muscle stem cell renewal is regulated and how bone and cartilage differentiation can be optimized in vitro. The requested equipment will enable the applicants to achieve the following objectives: - detect fluorescence in live tissue to a depth of 1 mm - continuously record cell behaviour for up to 2 days with miminal phototoxicity - make repeated recordings from individual anaesthetised mice and zebrafish - simultaneously image up to three different fluorophores and non-fluorescent tissues (4-colour imaging) in the same specimen - use post-processing software to generate and interpret 3D and 4D data.