The value of qualitative research in randomized controlled trials involving drugs and medical devices: a systematic mapping review.

Genetically encoded calcium indicators (GECIs) have revolutionized our ability to visualize neural activity by translating intracellular calcium transients into dynamic fluorescent signals. Two-photon (2P) microscopy has become the gold standard for in vivo calcium imaging, due to its ability to achieve optical sectioning and deep tissue penetration in brain tissue with minimal photodamage. When paired with GECIs, 2P imaging enables high-speed recording of neural activity in intact brain tissue at the single-cell level. Traditionally, two-photon excitation has relied on tunable Ti:Sapphire lasers. However, these systems are expensive, power-limited, and require routine maintenance that constrain adoption in many laboratories. In contrast, fixed-wavelength lasers, such as ytterbium-doped fiber lasers (YbFLs), offer a promising alternative that operate at wavelengths in the range of 1000–1080 nm, are cost-effective, high-powered, and increasingly integrated into modern imaging platforms. However, GECIs that perform optimally under these excitation wavelengths remain limited. The excitation wavelength of GECIs is closely tied to the fluorescent protein scaffold from which they are derived. GFP-based sensors, such as GCaMP6 and jGCaMP8 provide the highest levels of sensitivity, kinetics, and in vivo applicability, but exhibit poor two-photon excitation above 1000 nm due to their declining cross-section beyond ~950 nm. Red-shifted indicators, typically engineered using mApple-, mRuby-, or mKate- scaffolds, including R-GECO1, jRCaMP1a, and K-GECO extend excitation beyond 1000 nm but exhibit trade-offs such as poor photostability, slower kinetics, photoswitching, and lysosomal accumulation. Newer variants like mApple-based XCaMP-R and RCaMP3 have improved response kinetics and dynamic range, but remain sensitive to pH (apparent Ca-bound p of 5.3 and 6.1, respectively), and exhibit poor photostability and photoswitching.