Fluorescence and fluorescence optical microscopy are widely used to evaluate organic molecules, including extensive use in the imaging of photoluminescent dyes in labeled biological cells and tissues. However, the spatial resolution is restricted to ~500 nm because of the optical diffraction limit when using visible light. In principle, the electron beam can overcome this limitation exciting cathodoluminescence (CL) with nano-sized probes.
Luminescence is generated based on the relaxation of electrons from the lowest unoccupied molecular orbital (LUMO) to the highest occupied molecular orbital (HOMO), analogous to the bandgap of inorganic crystals. The transition of electrons across the HOMO-LUMO energy gap can result in the emission of characteristic photons in the visible wavelength range that enable wavelength spectroscopy to differentiate organic molecules and map their distribution with nanoscale resolutions. Unlike fluorescence microscopy, the incident electron beam can be considered as a broadband excitation source without the limitation of needing to match the incident wavelength with the absorption spectra of the studied material.
CL imaging of biological samples is extremely challenging due to technological limitations in the efficient collection of usually very low-intensity optical signals and quenching of signal as many organic molecules disintegrate under the electron beam. Nevertheless, CL is an attractive tool for several applications: