Tips:
Fluorescent secondaries
In general, most systems can image your fluorochromes, however we recommend preselecting based on the system that you plan to use (eg. ensure the imaging system can efficiently excite and detect the reporter). Keep in mind that many of the newer generation fluorochromes have a high output and show minimal bleaching. Mounting media with anti-fade reagent can help enhance and preserve your fluorochrome signals. You can start with Cy2, Cy3 and Cy5 (Jackson Immunochemicals) or Alexa-488 -568 and -633 secondaries from Molecular Probes.
Imaging multiple dyes
Most of the imaging systems can handle multiple fluorochromes, however a common problem is that cross-talk between channels can occur and is often ignored; if the bleed-through is not addressed, you will have a false-positive (and incorrectly conclude co-localization of probes). Generally, for multi-labelled imaging, choose fluorochromes with minimal emission wavelength overlap, and set up the system to reduce false positives. A few of our systems can build an emission curve from your sample, which can help you understand how your fluorochrome behaves in your sample environment and also can be used for spectral separation purposes in multi-labelled applications. Finally, another consideration is axial chromatic aberration in which dyes with great wavelength differences (eg. 450nm-uv versus 650nm-infrared) are focused at different distances from a (non-corrected) lens, and thus the signals will not appear in the same imaged plane.
Preparation and imaging
The optimal method requires a method that preserves sample morphology while maximizing excitation and detection of your probes. Once you determine how to prepare your sample (including fixation, labelling, mounting), you must choose an appropriate microscope system by considering whether your signal is weak/intense, the need for low/high magnification (including type of lens: dry, water, oil) and finally the xy as well as z-resolution required. Generally you should avoid imaging through plastic, and include anti-fade reagent if necessary.
Optimal cover glass
Virtually all oil high-NA lenses for high-resolution microscopy are manufactured for use with a #1.5 (0.16-0.19 mm) glass coverslip; note that #1 (0.13-0.16 mm) coverglass is found commonly in the marketplace, and while it can give you more working distance the resulting images reveal a spherical aberration and reduced resolution/intensity.
