Upright two-photon microscope for intravital fluorescence imaging and photostimulation

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Two-photon microscopy has become an established method for the investigation of a rich variety of processes in the life sciences. Due to the high penetration depth in biological tissue and the relatively simple mode of operation, this method is particularly suited for live imaging in the central nervous system. However, in recent years, two-photon microscopy has also been increasingly used in other fields of biological research. Applications for two-photon microscopy include fluorescence imaging of fast, subcellular processes, such as calcium dynamics in neuronal processes or synaptic transmission, calcium imaging of large cellular populations, or chronic imaging of cell morphology and motility. With the continuous improvement and development of genetically encoded fluorescent indicators for a large number of signaling molecules and metabolites, two-photon microscopy continues to gain significance for the investigation of biological processes. In addition to imaging, two-photon excitation can also be used to stimulate light-sensitive proteins. Also, in this case, the technology has reached a state of maturity that it can be used to reliably control the function of cellular processes at various spatiotemporal scales. Given the importance of this technology in biomedical research, we aim to establish an upright two-photon microscope in the Life Imaging Facility Mannheim and make it available to the research community at the Medical Faculty Mannheim. Applications will include long-term monitoring of immune responses to brain tumors, interaction of brain cancer cells with neurons, brain cell dynamics during pathological states, such as neuroinflammation, optogenetic investigation of nociceptive and somatosensory periphery neurons, cardiovascular (patho)physiology and all-optical investigations of cellular networks using optogenetic manipulation and functional read-out of cellular and subcellular function in the brain. The system will allow two-photon imaging of neuronal dynamics in vivo and in slices in conjunction with holographic two-photon stimulation. The two-photon system we apply for has the following properties and capabilities: 1) Upright microscope compatible with intravital imaging in small animals (mostly rodents), 2) a removable stage for imaging of tissue samples such as organoids or brain slices, 3) a versatile scanning system with a large field-of-view enabling fast scanning of large areas as well as precise configuration of the scan path, 4) a fast, 3D focusing system for fast volumetric imaging in thick tissue, 5) two femtosecond-pulsed infrared laser lines for simultaneous dual-color imaging, 6) capability of holographic, two-photon stimulation in 3D with an appropriate high-energy pulsed infrared laser.