We do a LOT of fluorescent microscopy in our immunology lab, so I have a bit of experience with fluorescence.
The answer to your question depends on what type of filters you have in your microscope, and what type of light source is on the microscope. Older fluorescent systems used absorption filters, which were simply discs of coloured glass. These filters had a fairly wide band-pass, so the fluorescence tended to be less than we see with interference filters. The good news with these filters is that they are nearly indestructible (unless you drop and break them.) Interference filters are produced by vacuum deposition of a thin film of metal vapour on high-quality glass. These filters usually have much sharper band pass characteristics than absorption filters. They are also considerably more expensive. If handled properly, these filters will last for decades, but improper cleaning and handling of the filters can shorten their lifespan. I have also heard that prolonged exposure to solvent vapours (such as we find in a histology lab), can damage the filters, although I have not seen any filters that suffered this type of damage. I have seen interference filters that show "delamination" of the metal film over time. In my experience these are older filters that were not produced with the current technologies, and filters that have been mishandled. Interference filters made in the past 20 years should last as long as the microscope, if they are properly handled. If you are seeing reduced fluorescence, I would suspect the light source as the most likely problem. Halogen lamps have less intensity than mercury vapour lamps, which are less intense than metal halide lamps, which are less intense than LED sources. We have converted all of our microscopes to LED sources. If you are using an older HBO or halogen lamp, the age of the lamp, the initial wattage of the lamp, and the alignment can all affect the fluorescent output. As you identified, perhaps the most important aspect of immunofluorescence is the skill and experience of the person who reads the slides. Let me know if you have further questions. Eric Hoy =============================================== Eric S. Hoy, Ph.D., SI(ASCP) Clinical Associate Professor Department of Medical Laboratory Sciences The University of Texas Southwestern Medical Center Dallas, Texas Email: eric....@utsouthwestern.edu =============================================== On 6/5/12 9:37 AM, "Gudrun Lang" <gu.l...@gmx.at> wrote: > Hi! > > Filters for fluorescencemicroscopy tend to "burn out" after a certain > duration of usage. What duration? > > We have filters for FITC, TRITC, Dapi and a triplefilter. The working-hours > are about 150 per year. > > > > What do you think? Is it time to change them. > > I have often bad feedback about weak signals, and I would not be surprised > if the microscope is the culprit and not our protocol. > > > > Weak signals refer last times to ALK-FISH on lung biopsies. Well fixed but > tumourcells mixed within collagenfibers. > > - and unfortunately unexperienced doctors on reading of this special probe. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet