Far-red fluorescent genetically encoded calcium ion indicators

Abstract

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ (ΔF/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.

Fluorescent and Bioluminescent Calcium Indicators with Tuneable Colors and Affinities

“…Nevertheless, its brightness of ∼15 mM –1 cm –1 is in the same order of magnitude as genetically encoded red-shifted indicators (brightness FR-GECO1c: 9.3 mM –1 cm –1 ). 29 As HaloTag binding reduced the calcium turn-on observed in the free dye ( F max / F 0 = 8–24×; Table S1 ), we tested if it also affected the calcium-binding kinetics and the selectivity of the indicators against other cations. Stopped-flow measurements of HaloTag-bound MaPCa revealed high k off values of above 248 s –1 , which are significantly higher than those observed for GCaMP6f with 4 s –1 .…”

A Genetically Encoded Far-Red Fluorescent Indicator for Imaging Synaptically-Released Zn2+

“…FRISZ exhibited peak excitation and emission above 600 nm and is among the most red-shifted FP-based indicators. A recent preprint described far-red fluorescent Ca 2+ indicators based on circularly permuted FP, mKelly2 ( 51 ). Still, the excitation and emission of FRISZ are more red-shifted than these Ca 2+ indicators by ∼ 10 nm, allowing the effective excitation of FRISZ with common ∼ 630 nm lasers or LEDs.…”

“…The cross-section σ 2 ,A was measured at two close wavelengths, 1060 and 1064 nm. The measurement was performed using Rhodamine 6G (Rh6G) in methanol at λ = 1060 nm (for which σ 2 , A = 10 ± 1 GM) ( 66 ) as a reference standard, and, independently, using Rhodamine B (RhB) in alkaline ethanol at λ = 1064 nm (for which σ 2 , A = 13.3 ± 2.8 GM) ( 51 ) as an additional reference standard. The two-photon fluorescence signals of the sample and reference solutions in the same excitation and collection conditions were measured.…”

Circularly Permuted Far-Red Fluorescent Proteins

“…It is highly possible that circular permutation at this site may perturb the environment required for far-red chromophore formation and stabilization, causing the loss of fluorescence. However, recently, a preprint appeared reporting the development of several far-red fluorescent calcium indicators with excitation and emission maxima at ~596 nm and ~644 nm, respectively, based on circular permutation of another FrFP, mKelly2, within β-strand 7 [ 43 ]. Thus, it may still be possible to rescue the fluorescence of cpmMaroon145-148 and cpmCarmine145-148 through further protein engineering efforts.…”