Since green fluorescent protein (GFP) and other green emitters (for example, FITC) are the most extensively used fluorochromes, NIGHTSEA’s Royal Blue excitation/emission sets are the most popular option for the Stereo Microscope Fluorescence Adapter. NIGHTSEA is frequently asked for advice in choosing between the green bandpass and yellow longpass barrier filter options. This article will look at the advantages and disadvantages of each.
- Longpass – Transmits all wavelengths longer than ~500 nm
- Bandpass – Transmits only in the wavelength range from approximately 500 – 560nm
Longpass and bandpass filters for use with Royal Blue excitation
Practically, that means that the bandpass transmits only green wavelengths, while the longpass transmits greens, oranges, yellows and reds.
What the Barrier Filters are for
The purpose of any barrier filter in a fluorescence application is to boost the viewing contrast of what you want to see (the ‘signal’). The key 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 things in the viewing area that can mask the fluorescence you want to see.
This could be background fluorescence from things like growth medium (common when working with C. elegans) or parts of the subject itself (for example, chlorophyll in plants or the yolk of a developing zebrafish).
Both the bandpass and longpass filters are good at blocking the excitation light. While one might think that it would be better to select a filter that is closely matched to the estimated emission, this is not the case every time. If the noise that one wants to remove does not overlap (spectrally) with the signal, then the bandpass filter is a good selection.
This is the case, for instance, with the red fluorescence from chlorophyll in Arabidopsis or other plants, as illustrated in the two images below*. Chlorophyll fluoresces in the far red, with emission peak at about 685 nm.
In the first image below, made with the longpass filter, the red fluorescence makes it difficult to differentiate the green GFP fluorescence in the leaf vasculature. The green bandpass filter removes this, making it easy to view the GFP fluorescence.
Arabidopsis fluorescence imaged with longpass filter (c) NIGHTSEA.
Arabidopsis fluorescence imaged with bandpass filter (c) NIGHTSEA
The bandpass filter can potentially cause confusion if the ‘noise’ has spectral overlap with the signal. With the longpass filter, there are two potential ways to differentiate the signal from the noise – intensity and color. With the bandpass filter, the color (spectral) dimension is removed and only the intensity dimension remains.
The composite image below of a fluorescent transgenic zebrafish** exemplifies this. The specimen expresses GFP in the heart and mCherry in the blood cells. The image shows two views of the same fish – one captured using the longpass filter (top) and the other with the green bandpass filter in place.
Transgenic fluorescent zebrafish photographed through longpass (top) and bandpass filters
The red-fluorescent blood cells, the green-fluorescent heart, and the natural yellow fluorescence of the yolk can be seen easily in the top image. In the lower image, everything appears in different intensities of green. The heart is bright, but the yolk is not that much dimmer. The bandpass filter removes the longer wavelength portion of the yolk emission that gives it the yellow color. It would be inaccurate to say that all of the green fluorescence in this image is indicative of GFP expression. In this case, it would be better to view the subject with the longpass filter.
There are cases where the choice is less apparent. The growth medium for C. elegans has some amount of background green fluorescence. The green bandpass filter does seem to improve the clarity and contrast compared to the yellow longpass. In several tests, NIGHTSEA asked observers to look at the same specimens through both filters. Some favored the longpass, while others favored the bandpass. In this case, and there may well be others, the choice is not totally obvious and may amount to a matter of personal liking.
And if one is exploring fluorescence in nature, Longpass is unquestionably the way to go. There is no way one can capture this colorful image of an Ageratum flower with a bandpass filter.
Ageratum under the microscope, fluorescence (c) Charles Mazel
* 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).
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