Fluorescence Imaging with Laser Illumination

A typical fluorescence imaging or microscopy system is composed of three major components: an illumination source, a photo-activated fluorophore sample, and a detector. Any light incident on the sample within the sample’s excitation band will cause the fluorophore to emit light; typically the sample absorbs photons within its excitation band and emits photons at lower energy and longer wavelengths in its emission band. Two-photon fluorescence is another common fluorescence mechanism and may occur when a fluorophore is excited by a photon with a wavelength that is two times longer than a wavelength from its excitation band. Fluorescence that occurs this way requires two photons to reach the minimum energy needed to fluoresce because photons from this doubled-wavelength region have half of the energy.

System performance can be improved by using two filters: one between the illumination source and the sample and a second between the sample and the detector. The purpose of filtering is to limit all light to within the excitation or emission bands. System performance can be further improved by using a narrow band illumination source that is fully contained within an excitation band, like a laser, eliminating the need for an excitation filter.

The figures below give examples of popular fluorophores and match an appropriate illumination laser and emission filter that could be used within a high performance fluorescence imaging or microscopy system. The two blue curves represent the fluorophore’s excitation and emission bands, while the green curve shows the transmission profile of a filter that would adequately isolate the peak transmission from the emission band.