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The imaging technique known as FLIM, or fluorescence-lifetime imaging microscopy, is based on variations in the exponential decay rate of a fluorophore’s photon emission from a material. It can be applied as an imaging method in multiphoton tomography, two-photon excitation microscopy, and confocal microscopy.
The image in FLIM is produced using the fluorophore’s fluorescence lifetime (FLT), not its intensity. In order to prevent inaccurate measurements of fluorescence intensity caused by changes in the brightness of the light source, background light intensity, or restricted photo-bleaching, fluorescence lifetime depends on the local microenvironment of the fluorophore.
The advantage of this method is that it lessens the impact of photon scattering in dense layers of sample. Lifetime measures have been employed as a gauge for pH, viscosity, and chemical species concentration since they are reliant on the microenvironment.
Based on the rates of decay by a variety of various (radiative and/or nonradiative) decay routes, a fluorophore excited by a photon will drop to the ground state with a specific probability. One of these methods must involve the spontaneous emission of a photon in order to observe fluorescence.
Even if two materials fluoresce at the same wavelength, fluorescence-lifetime imaging produces images where the intensity of each pixel is determined by, allowing one to view contrast between materials with different fluorescence decay rates. Fluorescence-lifetime imaging also generates images that reveal changes in other decay pathways, such as in FRET imaging.
Nowadays, protein-protein interactions and intracellular ion changes are two examples of the dynamic assessments of signalling events inside single living cells that are frequently carried out using fluorescence lifetime imaging microscopy (FLIM).
The Global Fluorescence Lifetime Imaging Microscopy Market accounted for $XX Billion in 2023 and is anticipated to reach $XX Billion by 2030, registering a CAGR of XX% from 2024 to 2030.
For quantitative live-cell imaging, fluorescence lifetime imaging microscopy (FLIM) techniques are emerging as a well-established technology. When combined with optical microscopy’s great temporal and spatial resolution, lifetime imaging can deliver fresh data from unaltered cellular settings.
When the fluorescent probes being scanned are spectrally similar, this becomes especially useful. The FLIM approaches can offer different lifetimes characteristic of the individual fluorophores linked to each protein and are relatively insensitive to artefacts that can impact the spectroscopic features of fluorophores (e.g., donor and acceptor).
Over the past ten years, numerous FLIM techniques have been created to measure various intracellular parameters by tracking changes in fluorescence excited-state lifetime.
The detectors utilised in these systems are mostly to blame for the typical temporal resolution of a few hundred picoseconds (ps) of conventional time-domain FLIM approaches like multigate detection and single-photon counting.
Recently, a new multiphoton FLIM system with improved photon detection efficiency and good spatiotemporal resolution was created. This system can be used to quickly determine lifetimes in live-cell applications.
In optically transparent tissue, fluorescence lifetime imaging microscopy (FLIM) allows for the subcellular-resolution identification of complex molecular assemblies inside a single voxel for studies of cell function and communication.
