March 2020: Publication from the Xu lab
'Multicolor three-photon fluorescence imaging with single-wavelength excitation deep in mouse brain' has been published in Science Advances.
Hontani et al. (2020) demonstrate that many red proteins have a larger, often >10-fold larger 3-photon cross-section at ~1300 than at ~1700 nm. Fluorophores with large 1300 nm cross-sections include small dye molecules (Texas Red, SR 101, Alexa 546) and genetically-encoded indicators (DsRed). Interestingly, not all red indicators have large cross-sections at ~1300 nm. mCherry, for example, does not. The underlying mechanism appears remains incompletely characterized, but resonance-enhanced excitation is a likely candidate.
The significance of this discovery is unclear. Certainly it opens the door to simultaneous imaging of red and green indicators with a single ~1300 nm excitation wavelength, but what implications does the result have for the 3-photon depth limit? Have red indicators, with their long emission wavelengths and lesser emission attenuation length than green indicators suddenly become the best indicators for imaging near the 3-photon depth limit?