[Histonet] Fluorescence-filters

Mark Tarango marktarango <@t> gmail.com
Tue Jun 5 16:47:58 CDT 2012

Hi Gudrun,

i read about 15 ALK cases a week.  If you are seeing a lot of collagen
fibers around the tumor cells, I'd try increasing the digestion time of
your pepsin (especially if they were fixed for longer than usual).  Before
altering the pretreatment though, you would want to make sure that these
slides were not exposed to light for any period of time.  The discrete
signals of the probes can quickly fade (much faster than with IF stained
slides).  I'd also make sure the door is closed in your dark room.  If
there is light in the corner of your eye the signals can be hard to see.
 ALK FISH should be scored at high power under oil immersion (60-100x
objective).  If they are scoring at 40x, it could be a problem.

good luck!

Mark Tarango

On Tuesday, June 5, 2012, Eric Hoy wrote:

> 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 <javascript:;>> 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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