Hi Everybody! We have been given a DAPI filter cube to trial prior to purchase. I have two questions regarding its use : 1. Different objective lenses give different images. Why? 2. General uses for a DAPI filter cube. Can we justify the cost? ------- 1. When we observe our specimen (macrophages within lung - cryostat sections) using a 40x Pl Fluotar objective 1.00-.50 oil (LEICA lens), our images are superb : clear, crisp, pretty. This is the case both with oil immersion and just dry viewing using this lens. The problem occurs when we view our sample with our 60x Pl Apo objective 1.4 oil (LEICA lens). The image becomes non-existent or ghost-like. A pale "shadow" only is seen, with no detail visible at all. However, viewing with green and blue excitation lines (fluorescence microscope) continues to give excellent (although non-specific fluorescent) images on both the 40x and 60x objectives. I discussed our problem with LEICA. At first thought, the explanation they proposed was that the 60x Pl Apo was giving autofluoresence from the glass components of the objective lens following excitation with ultraviolet light (selected by the DAPI filter). This then masked the sample fluorescence to the extent that no image was obtained. The longer wavelengths (green and blue lines) would not cause this autofluorescence. In comparison, the Pl Fluotar 40x objective could cope with a larger range of wavelengths, hence this problem was not observed with this lens. IS THIS A REASONABLE ASSUMPTION? HAVE OTHER PEOPLE EXPERIENCED SIMILAR DIFFICULTIES? ------- 2. Our application is visualisation of marcophage particles in lung tissue. Macrophages autofluoresce but we are trying to locate the presence of benzopyrene within the cells. Benzopyrene excites at 380nm and emits at 430nm. The DAPI filter seems a reasonable choice (and was recommended by this discussion list). WHAT ARE OTHER PEOPLE USING DAPI FILTERS/STAINS FOR? ARE THERE MANY OTHER GENERAL APPLICATIONS FOR DAPI THAT COULD HELP JUSTIFY THE PURCHASE PRICE OF ~$650 (AUSSIE DOLLARS)? Many thanks for your responses, Felicity Lawrence Analytical Electron Microscopy Facility, QUT Brisbane, Australia