Equipment — Leica TCS SP2 Confocal Microscope
This instrument facilitates confocal microscopy of slide-mounted cells and tissue sections stained with fluorescence dyes. Since this microscope is upright, it is excellent for fixed cells and tissues, but less well-suited for live cell microscopy.
The microscope has a motorized stage (Z) as well as a galvanometer-driven, high precision Z-stage, which facilitates rapid imaging in the focal (Z) plane. In addition to epifluorescence, the microscope supports transmitted light contrast methods, including brightfield and differential interference contrast (DIC), and is equipped with 10x, 20x, 40x (oil), and 63x (oil) Leica objective lenses.
The system has six laser lines: 458, 476, 488, 514, 543 and 633. Instead of Hoechst/DAPI as nuclear stain, which require excitation below 458, use Draq5 (a far red dye). The CTIF Hillman Satellite has free samples of this dye, of which only few microliters are needed (works well at 1:2,000).
The system can handle excitation and detection of three fluorochromes simultaneously. A transmission light detector makes it possible to obtain a transmitted light image, which can be overlaid with the fluorescence images.
The system is controlled by the Leica Confocal Software, which is relatively easy to use. In addition to controlling image acquisition, the program also allows for 3D reconstruction, time-lapse, FRAP (fluorescence recovery after photo-bleaching) and FRET (fluorescence energy transfer), as well as spectral unmixing.
A ‘Lite’ version of the Leica Confocal Software can be downloaded for free and used on any computer, but this software version only allows viewing of image files, some volume projections, and simple image analysis. Many of these tasks may be better handled by MetaMorph (available on our workstation in Hillman G.25), Photoshop, or the freeware ImageJ.