Overview

Successful fluorescence microscopy depends on matching filters with the source of illumination and the fluorophore to be excited. It is important to ensure that optimal filters have been selected for your experimental set up, and here we have provided recommendations.

Drag the bar to see how optimised filters enhance image contrast

CoolLED specialises in LED illumination systems for microscopy. We know that to get the best results from your microscope you need to match the right filters to your illumination system and samples.

Although LEDs produce a narrow spectral peak of light, there are often regions of shorter and longer wavelength illumination which may lead to increased background in the image or interfere with the excitation of another fluorophore if no excitation filter is used. We therefore always recommend that excitation filters are used with pE-Series LED illumination systems for fluorescence microscopy.

When selecting an LED illumination system which is based on a single, or few wavelength peaks, it is essential that care is taken to match these with the excitation filter in the microscope. Alternatively, a new filter can be fitted which is matched to the illumination system and the fluorophore.

Broad spectrum illumination systems are less sensitive to this optimisation requirement but it is still beneficial to check the filters. For example, with systems such as the pE-4000, the range of available wavelengths means that LED wavelengths can be selected to match the existing filters in the microscope.

It must also be taken into account that many standard filter sets have been optimised for mercury and metal halide peaks rather than for LEDs, which can lead to sub-optimal experimental conditions and images.

To overcome this, we have designed a set of filters perfectly matched to the spectra of CoolLED LEDs and common fluorophores. For example, new excitation bandwidths now harvest maximum power from LED peaks, and emission filters reduce bleed-through during multi-colour imaging. Single band filters for DAPI, GFP, mCherry and Cy5 can be found as part of our recommendations in the tool below, and are now available through the supplier or local microscope distributors.

Our products will work with most filter sets out there, but for optimum results we have made some recommendations below. There is never a definite right answer as depending on the demands of your setup you may want more brightness, more contrast or to satisfy some other requirement.

Find the Optical Filter sets by using the menus above.

Above are recommended filter sets from Chroma and Semrock for popular fluorophores. In most cases performance is similar, but those looking for particular brightness or contrast are urged to examine the manufacturer’s specifications further or ask us for help.