Fluorescence Imaging Cryomicrotome (Barlow Scientific):
An imaging cryomicrotome is available for acquiring high resolution (10-30 mm) fluorescence images of frozen tissue sections up to rodent-sized specimens (maximum specimen size: 11 x 24 cm). This system consists of a light-tight cryomicrotome, a high intensity broad-band excitation source filtered by one of five computer-selectable filters on one wheel, a second wheel that filters the image with one of five computer-selectable filters, and CCD camera to record images. The excitation source emits over a broad spectrum up into the near infra-red and is thus suitable for in vivo studies with a range of fluorophores, quantum dots, and fluorescent proteins. The excitation light passes through an excitation filter wheel that illuminates the exposed face of a frozen block of tissue. The returning fluorescence from the specimen passes through the emission filter that selects the fluorescence wavelength of interest and a Nikon F-mount lens zoom lens. An Apogee U32 CCD camera with2184×1472 pixels pixels (3 Mpixels) detector records each image. The system can image blocks as large as 5 x 5” (blockface surface) by 10” long. Image resolution is 18 microns/pixel at a 4cm field of view. The microtome part of the system removes successive slices from the frozen specimen but instead of saving them, as in a conventional microtome, the slices are discarded. The system images the exposed face of the frozen specimen and proprietary software takes the images from successive slices to deduce the actual location of the fluorescence; the software takes into account that the fluorescence imaged originates from a range of depths into the specimen that depend on the tissue optical properties at the excitation and fluorescence wavelengths. Multiple images can be taken between slices. This procedure enables imaging of multiple fluorophores each on a separate image but all automatically registered.