Below are some of the
common questions that we get asked regarding Optical Filters. If you
have a question that it not answered below, please e-mail us at .
Which way do I mount my filter?
Most of the
filters which you receive from us will have an arrow marked on the
ring. This indicates the direction that the light should pass through it.
If you have
a filter without such an arrow on it, the general rule of thumb is to
mount the reflective side facing the light source.
If I
purchase a filter
set and filter holder,
who will mount it and how much will it cost?
We will
mount your filter set into the filter holder for free. Please note
that if you have a required deadline to receive this, please allow at
least 24 hours for the mounting to be completed.
What if
I have my own filter holder and purchase a filter
set?
Who will mount it and how much will it cost?
Again, we
will mount the filter set into your own filter holder for free.
However, if it is determined that there is some difficulty in
removing old filters from a customer supplied holders, there will be
an additional mounting charge.
PLEASE
NOTE: Charges may be incurred for shipping your supplied holder.
Are
there any hidden costs in your quotes?
No. All
quotes take into consideration packing, carriage and insurance from
the US to Stanmore, all import duties, and exchange rate costs. The
only extra charge incurred is delivery to your premises, which ranges
from approximately £7 - £25 within the UK, depending on the
value of the goods and speed of delivery required.
Do you
take payment by Credit Card?
We will
take payment by Credit Card. However we do not accept American
Express or Diners cards.
Why do I
have poor signal or an
unexpected colour?
There are
many reasons why this may happen. Consider some of the following:
1) There
may be a problem associated with the light source.
i) The
light source may be inappropriate for the fluorophore. Ensure the
light source has spectral lines which will excite the fluorophore.
The line widths of the spectral prominences are typically 1-2nm
measured full width at half maximum.
ii) The
light source may be misaligned or no longer functioning properly due
to age. NOTE: Proper instrument set-up can be checked using
standards, such as fluorescent beads mounted on slides (commercially available).
iii) The
light source may be inappropriate of the wattage inadequate for
fluorescence microscopy. Tungsten (white light) bulbs are
inefficient; however, high power tungsten-halogen bulbs will work.
2) The
filters may be inappropriate for the fluorophore. Ensure the
specifications for the filters are compatible with the spectral
properties of the fluorophore. (When comparing the filter
specifications with the fluorophores specifications, ensure that you
have spectra appropriate for the application; for example, the
emission spectra of many fluorescent membrane probes in lipids are
different from the spectra of those same dyes in solvent)
3) The
filter block or filter slide may be inappropriately positioned, or
the filters within the filter set may have been mismatched. (mixing
and matching filters can be tricky; consult with before
trying to do so.) Try the following to determine if the filter
combination is incorrect:
i) With
just a glass slide placed on the sample stage, you should see almost
black through the microscope ocular. If the passband of the exciter
overlaps at all with the passband of the emitter, you will see lots
of background light - indicating an incorrect filter combination.
Change the filters and try again.
ii) The
excitation and emission filters may have been switched. For single
dye sets, the stage should be illuminated with light of a shorter
wavelength than the expected emission.
4) The
dichroic filter may be installed incorrectly, perhaps reversed
(flipped over). Be certain the light source is incident upon the
exposed, coated (unmarked) side of the filter.
5) The
filter may have a void in the coating:
i) Fringes
or rainbows may be an indication of a defective filter (also warped
cover glasses, immersion fluid, etc.) If you suspect the filter may
be defective, contact your vendor.
ii) Remove
the ocular and view a uniform sample. If you see unexpected bright
dots, there may be a void in the filter coating. To determine the
source of the void, rotate the sample, flip the dichroic over (this
may not be possible with all filter sets) or move the filters. The
filters can be shifted by moving the slider slightly or by rotating
the filters one at a time (for example, for a Nikon filter cube, turn
the filter 180 degrees; for a Zeiss filters, loosen the retaining
ring and holding it with a piece of lens paper, rotate the filter 180
degrees). If there is a void, the bright spot will shift with the
movement or the rotation of the filter.
Why do I
have low
signal or fluorescent artifacts?
There are
many possible reasons. Consider some of the following:
1) The
concentration of the fluorophore may be too low.
2) The
fluorophore may be photobleaching. The following instrument
adjustments may reduce photobleaching by reducing the excitation
light intensity:
i) Use
filters optimal for you fluorophore's spectral properties.
ii) You may
choose to use a neutral density filter to further reduce the
brightness of the excitation light, however this also reduces the
fluorescence signal.
iii) Use
low numerical aperture (NA) objectives or lower magnification. (Lower
NA reduces signal and may only be useful if you have adequate signal)
iv) Use
incandescent illumination, such as phase-contrast, to locate your
sample on the slide.
v) Minimise
the amount of time the sample is exposed to the excitation light.
vi) Try
using an efficient detection system - a low light level detection
systems, such as a CCD camera.
vii) For
long-term storage, minimise the exposure of the sample to ambient light.
Photobleaching
may also be reduced by adjusting the sample preparation:
i)
Experiment with altering the sample preparation. (ie the pH, solvent,
mounting media etc.)
ii) Try
using an antifading reagent.
iii) Some
recovery of the fluorescence after photobleaching may be achieved by
placing the sample in the dark at low temperature. It is also good to
store samples under these conditions to reduce fading during storage.
3)
Autofluorescence may be producing artifacts:
i) The
sample may be autofluorescent, swamping the fluorophore's signal.
Check for autofluorescence by viewing the sample in the absence of
the fluorophore. If the sample is autofluorescent, it may be
necessary to select another fluorophore. Choosing a fluorophore with
a longer excitation wavelength may solve the problem;
autofluorescence is normally excited most strongly by ultraviolet light.
ii) Fixing
samples with glutaraldehyde may cause problems with autofluorescence.
Agents such as sodium borohydride may block autofluorescence due to glutaraldehyde.
iii) The
materials used to prepare the sample may be autofluorescent, such as
the mounting media, the slide or the immersion fluid. View these
materials in the absence of the sample to ensure they do not fluoresce.
iv) Foreign
particles on the sample or in the optical system may be
autofluorescent, producing artifacts. Ensure the slides and
microscope lenses are kept scrupulously clean and free of all particulates.
The
microscope objectives or the mounting hardware in the light path may
be autofluorescent. Ask your microscope manufacturer.
4) The
fluorophore's spectral properties may not be compatible with the
specifications for the instrumentation. (See Light
Sources & Detectors)
|
