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.