Description
FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique inconfocal microscopy, two-photon excitation microscopy, and multiphoton tomography. The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.
Since the fluorescence lifetime of a fluorophore depends on both radiative (i.e. fluorescence) and non-radiative (i.e. quenching, FRET) processes, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor. Thus, FRET measurements using FLIM can provide a method to discriminate between the states/environments of the fluorophore. In contrast to intensity-based FRET measurements, the FLIM-based FRET measurements are also insensitive to the concentration of fluorophores and can thus filter out artifacts introduced by variations in the concentration and emission intensity across the sample.
How this lab uses this technology
Fluorescence Lifetime Imaging Microscopy (FLIM) is an advanced imaging technique that measures the average time a fluorophore remains in its excited state before emitting a photon. Unlike conventional fluorescence microscopy, which is based mainly on fluorescence intensity, FLIM generates spatial maps of fluorescence lifetime values within a specimen.
Fluorescence lifetime is largely independent of fluorophore concentration and excitation intensity, but it is highly sensitive to the local molecular environment. Changes in pH, ion concentration, oxygen levels, viscosity, molecular binding, protein interactions and metabolic state can therefore be detected with high spatial resolution.
Principle
After excitation with a short or modulated light source, the fluorescence decay of the sample is recorded at each image pixel. These decay profiles are analysed to calculate fluorescence lifetime values, producing a quantitative lifetime map of the specimen.
FLIM can be performed using:
Time-domain approaches, such as Time-Correlated Single Photon Counting (TCSPC).
Frequency-domain approaches based on modulation of the excitation light.
Single- or multi-exponential decay analysis.
Phasor plot analysis for rapid and model-free interpretation of fluorescence lifetime distributions.
Main Applications
FLIM is particularly useful (among others) for:
Förster Resonance Energy Transfer (FRET-FLIM) studies.
Analysis of protein–protein interactions.
Monitoring molecular binding and conformational changes.
Measurement of intracellular pH, calcium, oxygen and other environmental parameters using lifetime-sensitive probes.
Metabolic imaging based on endogenous fluorophores such as NADH and FAD.
Differentiation between free and protein-bound NADH.
Evaluation of cellular metabolism, stress and viability.
Analysis of tumour cells and tissue metabolism.
Monitoring drug response and treatment-induced metabolic changes.
Characterisation of fluorescent biosensors.
Discrimination between fluorophores with overlapping emission spectra but different lifetimes.
Reduction of interference caused by fluorescence intensity variations and photobleaching.
FRET-FLIM
FLIM is widely used in combination with Förster Resonance Energy Transfer. In FRET-FLIM experiments, energy transfer from a donor fluorophore to an acceptor produces a reduction in the donor fluorescence lifetime.
This approach enables the detection of molecular interactions at nanometre-scale distances and provides a robust alternative to intensity-based FRET measurements.
Metabolic FLIM
Autofluorescence lifetime imaging of endogenous molecules such as NADH and FAD provides label-free information about cellular metabolism.
FLIM can distinguish between:
Free NADH, mainly associated with glycolytic metabolism.
Protein-bound NADH, mainly associated with mitochondrial oxidative metabolism.
This makes FLIM particularly valuable for investigating metabolic changes associated with cellular differentiation, oxidative stress, hypoxia, cancer progression and therapeutic response.
Advantages
Quantitative imaging independent of fluorophore concentration.
Reduced dependence on excitation intensity and detector sensitivity.
High sensitivity to the local molecular environment.
Suitable for live-cell and fixed-sample imaging.
Compatible with fluorescent proteins, organic dyes and biosensors.
Enables label-free metabolic imaging.
Provides reliable analysis of molecular interactions through FRET-FLIM.
Can be combined with confocal and multiphoton microscopy.
Allows three-dimensional and time-resolved imaging.
Sample Types
FLIM can be applied to:
Living and fixed cells.
Cell cultures and organoids.
Tissue sections.
Plant tissues.
Microorganisms.
Animal models.
Fluorescently labelled biological samples.
Samples containing endogenous fluorescent molecules.
Sample Preparation Considerations
Sample preparation depends on the fluorophore, probe and experimental objective. For reliable FLIM measurements, it is important to:
Use fluorophores with suitable brightness and photostability.
Minimise background fluorescence and spectral overlap.
Include appropriate positive and negative controls.
Avoid mounting media or fixation procedures that alter fluorescence lifetime.
Maintain consistent acquisition conditions between samples.
Select appropriate reference samples for lifetime calibration.
Reduce photobleaching and phototoxicity during live-cell imaging.
FLIM provides quantitative information that cannot be obtained from fluorescence intensity alone, making it a powerful technique for studying molecular interactions, cellular metabolism and changes in the physicochemical environment of biological samples.