Since GFP (green fluorescent protein) and other green emitters (e.g. FITC) are the most widely used fluorochromes, our Royal Blue excitation/emission sets are the most popular option for the Stereo Microscope Fluorescence Adapter. We are often asked for advice in selecting between the yellow longpass and green bandpass barrier filter options. This article will address the pros and cons of each.
In practical terms, that means that the bandpass transmits only green wavelengths, while the longpass transmits greens, yellows, oranges and reds.
What the barrier filters are for
The purpose of any barrier filter in a fluorescence application is to increase the viewing contrast of what you want to see (the ‘signal’). The main job is to block reflected light from the excitation source and transmit the fluorescence emission. The next source of potential interference (‘noise’) is fluorescence from other substances in the viewing area that can mask the signal. This might be background fluorescence from things like growth medium (common when working with C. elegans) or parts of the subject itself (e.g., chlorophyll in plants or the yolk of a developing zebrafish).
Both the longpass and bandpass filters do a fine job of blocking the excitation light.
While you might think that it would be better to choose a filter that is closely matched to the expected emission, this is not always the case.
If the noise that you want to eliminate does not overlap (spectrally) with the signal then the bandpass filter is a good choice. This is the case, for example, with the red fluorescence from chlorophyll in Arabidopsis or other plants, as in the images below*. Chlorophyll emits in the far red, with its peak at about 685 nm. In the image on the left, made with the longpass filter in place, the red fluorescence makes it hard to distinguish the green GFP fluorescence. The green bandpass filter eliminates this, making it easy to see the GFP fluorescence in the leaves.
(Click image for larger view)
The bandpass filter can potentially cause confusion if the noise has spectral overlap with the signal. With the longpass filter you have two potential ways to distinguish the signal from the noise – intensity and color. With the bandpass filter you remove the color (spectral) dimension and leave only the intensity dimension. The images below of a fluorescent transgenic zebrafish illustrate this. This line of fish** expresses GFP in the heart and mCherry in the blood cells. The image below is a composite of two images – one taken using the longpass filter and the other with the green bandpass filter in place.
In the top image you can see the green-fluorescent heart, the red-fluorescent blood cells, and the natural yellow fluorescence of the yolk. These are easy to distinguish. In the lower image everything appears in varying intensities of green. The heart is bright but the yolk is not that much dimmer. It would be incorrect to say that all green is associated with GFP expression. In this case you would be better off viewing the subject with the longpass filter.
There are cases where the choice is less obvious. The growth medium for C. elegans has some background green fluorescence. The green bandpass filter does seem to add some enhanced clarity and contrast compared to the yellow longpass. In a number of tests we have had several observers look at the same specimens. Some have preferred the longpass because they feel it actually helps to see some of the background for context, while others preferred the bandpass for the same sample. In this case, and there may well be others, the choice is not completely obvious and may come down to a matter of personal preference.
And if you are exploring fluorescence in nature? Longpass is definitely the way to go. There is no way you could capture this image of an Ageratum flower with a bandpass filter.
For forensic science and most industry applications we recommend the longpass filter.
* Arabidopsis courtesy of Dr. John Celenza, Boston University.
** Zebrafish courtesy of Dr. Martha Marvin (Williams College), transgenic line bred by Dr. Lara Hutson (University of Buffalo).