Multicolor two-photon microscopy with a new red fluorescent protein

Two-photon microscopy can be used with multiple fluorescent markers to simultaneously observe multiple processes in vivo. Red fluorescent proteins (RFPs) are a popular choice because cellular autofluorescence is reduced at its emission region and it allows greater imaging depth thanks to less light scattering. In addition, longer wavelengths are less damaging to proteins and DNA.

However, using red fluorescent proteins with other colors such as GFP or cyan fluorescent protein (CFP) comes with some challenges. The Ti-Sapphire lasers used for two-photon microscopy are expensive, so most systems have only one laser. This makes it hard to efficiently excite RFPs at the same time as GFPs or CFPs. Also, Ti:Sapphire lasers have low power output in the excitation wavelengths for RFPs.

One way to overcome these problems is to shift the excitation spectrum of RFPs by making RFPs with a large Stokes shifts. One such RFP exists already. Called, mKeima, it can be used for dual-color two-photon microscopy, but its biochemical and photochemical properties leave room for improvement.

Researchers at the Albert Einstein College of Medicine in New York applied random and rational mutagenesis to the monomeric far-red mKate fluorescent protein and produced two RFPs with large Stokes shifts. The RFP variants have six amino acid mutations compared to mKate. Compare to mKeima, the new variants have higher pH stability, better photostability and faster chromophore maturation and monomeric behavior.

They used the new RFPs, named LSS-mKate1 and LSS-mKate2, for two-photon microscopy simultaneously with ECFP and EGFP. Their multicolor intravital imaging study of the motility and Golgi-nucleus alignment of tumor cells as a function of distance from blood vessels in a live mouse xenograft model of breast cancer revealed that breast cancer cells showed polarization towards vessels at a distance of 40 microns.

Research paper:
Monomeric red fluorescent proteins with a large Stokes shift
Kiryl D. Piatkevich, James Hulit, Oksana M. Subach, Bin Wu, Arian Abdulla, Jeffrey E. Segall, and Vladislav V. Verkhusha
PNAS doi:10.1073/pnas.0914365107

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1) two-photon microscopy 2) fluorescent proteins