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)

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)