Figure 4. Custom two-photon microscope provides advanced reflectance capture capabilities.

(A) The beam from a titanium-sapphire laser (ts) passes through a polarizing beamsplitter (pb) and on to a rotating polygonal mirror (pm) driving the fast scanning axis. The beam is relayed to a galvanometer mirror (gm) providing the slow scanning axis. Together, the scanners generate a frame rate of 22 frames per second (fps) at 512×512 pixels. The fluorescence signal generated in the tissue is reflected towards four photomultiplier tubes (pmt) separated by dichroic filters (df) to emitter filters for CFP, GFP, YFP, and a red marker (propidium iodide). Backscattered 910 nm light is also collected by the objective lens, retracing the optical path and collected through the confocal detector aperture (ca) onto the avalanche photodiode (rld). Depth scanning is provided by a piezoelectric translator (pz) that positions the objective lens (ol). A programmable XY motorized stage (xt, yt) is used to form tiled composite images. A full detailed description is available in Sup Fig. S3. (B) (LEFT) White arrows in left image demonstrate the location of blood vessels detected in the reflectance; insert yellow box (Right) is 3D composite image. (RIGHT) Reflectance also can detect unlabeled cells, such as red blood cells shown by white arrows. (C) B&W panels show: Collagen fibers detected through second harmonic generation, reflectance imaging and GFP-Pmel-1 T cells. Pseudo-color overlay (RIGHT) shows co-localization of collagen (blue), with reflectance (red), Pmel-1 T cells are shown in green.