A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

Abstract

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a novel sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. A new engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids.

A general method for the development of multicolor biosensors with large dynamic ranges

“…In the future, it will be therefore interesting to explore whether ChemoD-based biosensors can be improved to reach comparable dynamic ranges. On the other hand, ChemoD-NAD showed fluorescence lifetime changes of at least similar magnitude than current state of the art fluorescence lifetime biosensors [53][54][55] . Based on synthetic far-red fluorophores (maximum emission wavelengths ≥ 628 nm), the intensiometric and FLIM-based modalities of ChemoD biosensors bring multiple advantages in term of brightness, photostability, phototoxicity and auto-fluorescence compared to FP-based biosensors.…”

Cell-based optimisation and characterisation of genetically encoded, location-based biosensors for Cdc42 or Rac activity

“…We noticed that spinning disk microscopy and TRIF microscopy were beneficial for the imaging of relocation sensor, as did others (Graessl et al, 2017). Additionally, it is essential to be aware of the fact that morphology changes such as local protrusions, especially in ventral orientation, can cause intensity increases that are not related to relocation (Dewitt et al, 2009; van der Linden et al, 2021). That issue could be addressed with a reference signal for the plasma membrane intensity (Mahlandt et al, 2021).…”

Smart genetically-encoded biosensors for the chemical monitoring of living systems

“…Because this ratio is independent of the amount of biosensor present, this so-called 'ratiometric' imaging enables conceptually straightforward quantification of the stimulus activity. As we will discuss further in this work, recent developments have also seen other approaches to deliver quantitative readout for such sensors, based on fluorescence lifetime imaging (FLIM) 30 and on photochromism, 44 the lightinducible on-off switching of the fluorescence emission.…”

“…49 Even though the instrumentation for fluorescence lifetime microscopy (FLIM) is generally more complex and expensive, reports of lifetime sensing with single reporter biosensors are also appearing. 30,50 Another approach is to use identical reporters for both donor and acceptor, thus relying on energy transfer based on homo-FRET. Such a strategy can be used to generate sensors that are read out by detecting the fluorescence anisotropy, 51,52 which is likewise quantitative and does not require the use of two different spectral bands (Fig.…”