Biological Applications
Fluorescence intensity informs us about the relative abundance of the light emitting objects. Fluorescence lifetime reveals information about the immediate biochemical environment (such as the redox state and local pH), the molecular interactions (the binding or release of small molecules, ligand-receptor interactions), and molecular conformational changes.
Using quantitative analysis for complex fluorescence lifetime data, we can characterize fluorescence dynamics of fluorescent protein-based biosensors and derive information related to tissue homeostasis, disease mechanisms, and drug effects in the tissue.
Fluorescence Lifetime as Target Indicator
Fluorescence lifetime can serve as an indicator to find a fluorescent target of interest.
For example, when a rodent is placed under an immobile condition, an optical fiber probe can be placed in a precise x- and y-location. As the fiber tip is moved in the z-axis from the surface of the brain down toward the target where the fluorescent protein sensor is expressed, the fluorescence decay reflects the tissue environment where the fiber tip is.
A complex exponential decay is indicative of a composite of many types of biomolecules that have native fluorescence in the brain tissue (blue trace). As the fiber tip moves to the target site, the fluorescence decay kinetics (green trace) closely resembles that of a mono-exponential decay (Akerboom, 2012) . This feature has proven to make experiments highly efficient because it helps to quickly identify those rodents that have properly expressed the fluorescent protein sensor through a viral infection.
Fluorescence Resonance Energy Transfer (FRET)
Many fluorescent sensors have been designed to have the property to allow the transfer of energy from a fluorescent donor to an accepter within a macro molecule such as a fluorescent protein. Examples that are relevant to neuroscience are many and include FLIM-AKAR (PKA), Tq-Ca-FLITS (intracellular calcium), iGlucoSnFR-TS (glucose), and sDarken (serotonin).
A ChiSquare system has been deployed in a study of in vivo PKA activity in BNST Guansembles (Brown, JA, 2023). The lifetime changes of the donor fluorophore were measured showing decreased donor lifetime and were indicative of increased PKA activity.
Fig. 3J from Brown, JA et al. Neuropsychopharmacology, 2023 Jul;48(8):1133-1143.
Fluorescence Intensity at Two Locations in the Brain
A ChiSquare instrument can be built to have two or more optical fiber probes, each connected to a photon detector. For example, by implanting two optical fiber probes at different locations in the brain of a rodent, one can monitor the dynamic changes in the fluorescence intensity of the fluorescent calcium sensor population as the mouse moves under a particular behavioral paradigm.
