In vivo cell tracking with video rate multimodality laser scanning microscopy

Studies of biological processes, such as disease progression and response to therapy, call for live imaging methods that allow continuous observation without terminating the study subject for histological tissue processing. Among all current imaging modalities, optical microscopy is the only method capable of probing live tissue with cellular and subcellular resolution. We present a video-rate (30 frames/s), multimodality imaging system that is designed specifically for live animal imaging and cell tracking. In vivo depth-sectioned, high-resolution images are obtained using confocal and nonlinear optical techniques that extract structural, functional, and molecular information by combining multiple contrast mechanisms, including back scattering, fluorescence (from single- and two-photon excitation), second harmonic generation, and coherent anti-Stokes Raman scattering. Simultaneous use of up to three modalities is possible and eliminates the need for coregistration, especially on large-scale images. A real-time movement correction algorithm was developed to extend integration times in cases where the image needs to be stabilized against subject movement. Finally, imaging of fast moving leukocytes in blood vessels is made possible with a modification that permits operation at 120 frames/s over a smaller area. Sample imagery obtained in vivo with the microscope is presented to illustrate the capabilities.