Molecular Imaging by FLIM

Measurement of Molecular-Environment Parameters

The fluorescence lifetime of most fluorophores more or less depends on the molecular environment. FLIM can be therefore used to measure local environment parameters, such as pH, oxygen concentration, concentration of physiologically important ions, binding to RNA, DNA, proteins or lipids, local viscosity, and local refractive index. Compared with intensity-based methods fluorescence-lifetime detection has the advantage that the result gets de-coupled from the concentration of the sensor, possible absorption in the sample, intensity of the laser, focusing, detector gain and other instrumental effects. A few examples are described below.

pH Imaging

Lifetime image of skin tissue stained with BCECF. The lifetime is an indicator of the pH. Right: Fluorescence decay curves in an area of low pH (top) and high pH (bottom)

Microscopic pH imaging by FLIM can be achieved by staining the sample with a pH-sensitive fluorescent probe. These probes usually have a protonated and a deprotonated form. There is an equilibrium between both forms that depends on the pH of the local environment. If both forms have different fluorescence lifetimes the average lifetime is a direct indicator of the pH. A typical representative of the pH-sensitive dyes is 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). An example of pH imaging of skin tissue is shown in the figure below. A single-exponential lifetime image is shown left, decay curves from different areas of the image are shown right.

Calcium Imaging

Ca2+ ions are involved in a large number of cell functions, such as intracellular transport, membrane potential, muscle contraction, gene expression, and cell differentiation. The mechanism of the Ca2+-dependent lifetime of Ca2+ sensors is that the fluorophore has a Ca-bound and an Ca-unbound form of different fluorescence quantum efficiency and thus different fluorescence lifetimes. The fluorescence lifetime of the bound form is higher than that of the unbound form. Consequently, the net fluorescence lifetime depends on the Ca2+ concentration. The traditional Ca2+ dyes, such as Calcium Green and Oregon Green Bapta, display large lifetime changes and work beautifully for lifetime-based Ca2+ measurement. Examples are shown in the figures below

The Ca2+ concentration in cells can change within remarkably short periods of time. The Ca2+ response of live neurons to stimulation occurs within milliseconds. Nevertheless, the effects can be measured by special TCSPC FLIM techniques, such as FLITS and Temporal Mosaic FLIM. An example is shown in the figure below.

Ca2+ transient in cultured neurons. Oregon Green Bapta, data recorded by temporal mosaic FLIM, 38 ms per mosaic element.

Chloride Imaging

The chloride concentration is important to the function of the neuronal system. MQAE (a quinin derivate) is used as a fluorescent probe. MQAE is quenched by Cl, and the concentration can be calculated from the lifetime change via the Stern-Volmer relation. The figure below shows a spinal ganglion of a mouse stained with MQAE. Short lifetimes indicate high Cl concentration and vice versa.

Spinal ganglion of a mouse stained with MQAE. Short lifetime indicates high Cl- concentration.

Local Viscosity
Local viscosity has a large influence on the rates of biochemical reactions in a cell. Changes in viscosity on the sub-cellular level have been shown to accompany a wide range of cell mal-functions. Changes in viscosity have been found for hematoligic disorders, diabetes, Alz-heimer disease, liver malfunction and cancer. Viscosity on the molecular scale can be determined by ‘molecular rotors’. These are fluoro-phores which have two more or less rigid parts connected by a single bond. The two parts can rotate one against the other. Rotation is an additional relaxation pathway which competes with radiative relaxation. Ability of internal rotation therefore shortens the fluorescence lifetime. When the rotation is hindered by high viscosity of the molecular environment this relaxation pathway becomes less efficient, and the fluorescence lifetime increases. An example of a vis-cosity measurement by a Bodipy-based sensor is shown in the figure below.

Viscosity measurement by a Bodipy-based viscosity sensor. Long lifetime means high viscosity, and vice versa.

Protein Interaction

Protein interaction is investigated by using Foerster-Resonance Energy Transfer (FRET) between a donor molecule and an acceptor molecule. The donor molelcule is attached to one protein, the acceptor molecule to another. If the proteins are connected (interacting) energy is transferred from the donor to the acceptor. The causes a decrease in the fluorescence lifetime of the donor. FRET experiments by FLIM are more quantitative than by staedy-state imaging because FLIM is independent of the concentration. Moreover, FLIM delivers not only the classic FRET efficiency but also the fraction of interacting proteins and the FRET efficiency of the interacting donor alone. FRET distances from this FRET efficiency are not offset by variable fraction of interacting proteins. Please see Protein-Interaction Experiments by Förster Resonance Energy Transfer (FRET).


W. Becker, The bh TCSPC Handbook

W. Becker (ed.), Advanced Time-Correlated Single-Photon Counting Applications

Selected References Related to Molecular Imaging by FLIM

For more references please see W. Becker, The bh TCSPC Handbook, 9ed. (2021).

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