8.2 2P fluorescence microscopy
Another approach to reducing sample autofluorescence and excitation
light scatter is 2P fluorescence microscopy. 2P microscopy relies on the
excitation of fluorophores with two photons of infrared light instead of
a single visible light photon (Figure 2n) (Denk, 1994). The main
advantages of 2P microscopy include its penetration depth allowing
imaging a few millimetres deep inside living tissue, inherent sectioning
ability due to fluorescence excitation only in a small focal volume (2
nanolitres), and a considerable reduction of autofluorescence. The
limitations of 2P microscopy include the requirement for an expensive
pulsed infrared laser light source and a low efficiency of excitation
and hence a low intensity of emitted fluorescence. In GPCR-related
research, 2P microscopy was used, for example, for the imaging of
Gi-dependent neutrophil accumulation in response to S. aureusinfection in live mice (Liese et al., 2013). 2P microscopy has been
combined with other imaging modalities, such as FRET-FLIM for the
imaging of protein kinase A activation by GPCRs in brain slices (Chen et
al., 2014) or STED for the intravital imaging of glucagon-like peptide-1
receptor signalling (Ast et al., 2020). Several existing photoswitchable
ligands for 2P microscopy can be used for the regulation of dopamine
(Araya et al., 2013) and metabotropic glutamine receptor (Carroll et
al., 2015) activity in the brain.