Increasing Viewing Contrast in Fluorescence Microscopy: Longpass vs Bandpass Barrier Filters

GFP (green fluorescent protein) and other green emitters, such as Fluorescein isothiocyanate (FITC), are the most widely used fluorochromes in fluorescence microscopy. This means that the associated Royal Blue excitation/emission sets are the most popular option for microscope fluorescence adapters, such as the SFA Stereo Microscope Fluorescence Adapter from NIGHTSEA. Barrier filters are used to increase the viewing contrast of what you want to see in the fluorescence microscopy application (the ‘signal’). This article will address the differences and the pros and cons of selecting between the yellow longpass barrier filter and the more recently introduced green bandpass barrier filter options, from these sets.

Barrier filter applications
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 filter’s main job is to block reflected light from the excitation source, or specimen, and transmit the fluorescence emission. The next source of potential interference (‘noise’) is fluorescence from other substances in the viewing area, which can mask the signal, for example, background fluorescence from growth medium or from parts of the subject itself (e.g. chlorophyll in plants or the yolk of a developing zebrafish).

Both the yellow longpass and green bandpass filters work well to block the excitation light.

What are the spectral differences?
Longpass – transmits all wavelengths longer than ~500nm

Bandpass – centered at ~530nm, bandwidth ~60nm at FWHM (full width at half maximum)

In simpler practical terms, the bandpass transmits only green wavelengths, while the longpass transmits greens, yellows, oranges and reds.

Which filter, which application?
If the noise that you want to eliminate does not overlap with the signal then the bandpass filter is a good choice. For example, chlorophyll in Arabidopsis or other plants emits in the far red, with its peak at about 685 nm; the green bandpass filter eliminates this, making it easy to see the GFP fluorescence in the leaves that is otherwise hard to distinguish.

However, 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; by intensity and color. With the bandpass filter you remove the color (spectral) dimension and leave only the intensity dimension.

In some instances, the choice of barrier filter 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. NIGHTSEA has performed a number of tests on this, with several
observers looking at the same specimens. Some have preferred the longpass because they feel it 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.

(Based on the NIGHTSEA blog: ‘Green Fluorescence – Longpass vs Bandpass Barrier Filter’)