Here are three papers that are worth reading over. No time for full reviews.
New Single-FP GECIs
The Russian fluorescent protein team has come out with some new single fluorescent protein G-CaMP/pericam-like sensors. They fiddled with the linker sites at the 145 and 148AA insertion points and found a great deal of fluorescence sensitivity to the amino acid composition at those sites. They note two new sensor constructs Case12 and Case16 that have 12-16.5x maximal changes in fluorescence upon calcium binding, a significant improvement over G-CaMP2. The tradeoff appears to be that they are dimmer. They show calcium responses in HeLa, PC-12 and cortical neuron cells, but no direct head-to-head with other sensors in cells.
Multipoint multiphoton microscopy
In this technical paper, an MIT group led by Peter So examines some issues surrounding multipoint excitation multiphoton microscopy. In theory, multipoint excitation will dramatically increase image acquisition rate through parallelization. However, this comes at the expense of large increases in background scattered light, which reduces optical resolution and penetration depth. They present an imaging system using multianode photomultiplier tubes that lets them acquire an 8×8 grid of multiphoton excitiation points. This technique, plus post-hoc deconvolution allows them to approach the resolution and depth of single point multiphoton systems, with a parallel array.
Effects of linker length and stiffness on FRET
This paper from Harold Erickson’s group carefully examines the role of linker length and stiffness in determining the amount of FRET transfer between CFP and YFP derivatives. They show that intrinsically unstructured domains of identical amino acid length produce significant differences in FRET between tethered FPs. They propose a formula for estimating the stiffness of linkers from the degree of FRET, which corroborates a 2006 study by Evers et al. They also demonstrate that the reported enhancement of FRET between the CyPet and YPet pair is due to an enhanced tendency for the proteins to dimerize. This reinforces my thinking that the most needed and generalizable improvement to two-FP FRET systems is an enhanced photostability of the FRET acceptor. Somebody please screen for a bleach-resistant YFP! Props to Michael Lin for pointing out the paper.