Two-Photon Imaging of the Immune System: A Custom Technology Platform for High-Speed, Multicolor Tissue Imaging of Immune Responses

Modern imaging approaches are proving important for addressing contemporary issues in the immune system. These approaches are particularly useful for characterizing the complex orchestration of immune responses in vivo. Multicolor, two-photon imaging has been proven to be especially enabling for such studies because of its superior tissue penetration, reduced image degradation by light scattering leading to better resolution and its high image quality deep inside tissues. Here, we examine the functional requirements of two-photon imaging instruments necessary for such immune studies. These requirements include frame rate, spatial resolution and the number of emission channels. We use this discussion as a starting point to compare commercial systems and to introduce a custom technology platform that meets these requirements. This platform is noteworthy because it is very cost-effective, flexible and experimentally useful. Representative data collected with this instrument is used to demonstrate the utility of this platform. Finally, as the field is rapidly evolving, consideration is given to some of the cutting-edge developments in multiphoton microscopy that will likely improve signal strength, C, depth penetration and/or the experimental usefulness of this approach.