Sensitive detection via the time-resolved fluorescence of circularly permuted yellow fluorescent protein biosensors

Genetically encoded fluorescence lifetime biosensors: overview, advances, and opportunities

“…However, the fractional amplitudes (α 1 , α 2 and α 3 ) corresponding to each lifetime component changed significantly, and the fractional intensities α 2 τ 2 and α 3 τ 3 showed opposite changing trends with increasing histidine concentrations. 33 The ratio of fractional intensities R (=α 3 τ 3 /α 2 τ 2 ) remarkably decreased about 8-fold, which was larger than the results obtained with the average fluorescence lifetime as well as the excitation ratio (Fig. 5g).…”

“…FHisJ also appeared to be the case, where its lifetime changed from 2.8 ns to 1.6 ns but the excitation ratio changed 5.2-fold upon histidine saturation. 33 To improve the sensitivity and expand the dynamic range, we proposed a ratiometric analysis method by using the fractional intensities of the time-resolved fluorescence of single FP-based biosensors. 60 After incubation with histidine, three lifetime components (τ 1 , τ 2 and τ 3 ) in FHisJ remained almost constant.…”

“…3c). 33 FHisJ was developed by inserting cpYFP into the protein HisJ and capable of binding histidine in vivo. 34 FHisJ existed in three forms (A, I and B), and the fluorescence mainly comes from the excited states I* and B*.…”

“…The dynamic range of ∼0.38 ns was much smaller than that of fluorescence lifetime biosensors Tq-Ca-FLITS, LiLac and FHisJ (∼1.3 ns, 1.2 ns and 1.2 ns, respectively). 29,33,88 Although iGlucoSnFR-TS is capable of the quantitative imaging of glucose concentration in cells and brain tissue with FLIM (Fig. 6k and l), it requires further optimization by strategies such as substitution of cpT-Sapphire with circularly permuted mTurquoise2 (cpmTurquoise2) or cpYFP.…”