[Histonet] Fluorescence-filters

Eric Hoy Eric.Hoy <@t> UTSouthwestern.edu
Tue Jun 5 15:30:19 CDT 2012

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.Hoy <@t> UTSouthwestern.edu

On 6/5/12 9:37 AM, "Gudrun Lang" <gu.lang <@t> 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.

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