Fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy

Measuring a fluorescence lifetime image (flim) with a lsm

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“Fluorescence lifetime imaging microscopy: fundamentals and developments in instrumentation, research, and applications,” by Rupsa Datta, Tiffany M. Heaster, Joe T. Sharick, Amani A. Gillette, and Melissa C. Skala, J. Biomed. Opt. 25(7) 071203 (13 May 2020) 10.1117/1.JBO.25.7.071203 (https://doi.org/10.1117/1.JBO.25.7.071203)

Interview: development of fluorescence lifetime imaging

The fluorescence lifetime of molecules is measured using the FLIM technique. The average time a molecule spends in an excited state before returning to the ground state is described as this. It’s often used with fluorescent molecules in cells, tissues, and whole animal models to determine molecular dynamics with nanoscale resolution.
Changes in the local environment and conformation state, such as ionic concentration, pH, and lipophilicity, affect the lifetime of fluorescence. There are two main FLIM implementations:
Fluorophore lifetimes used to be measured in tens of nanoseconds or longer. As a result, commercially available amplified CCD (ICCD) cameras with slow-gate and fast-gate image intensifiers capable of gate widths on the order of nanoseconds were adequate for measuring and observing such phenomena. Using tens-of-nanosecond gating, scientists were able to obtain many… Read the Complete Article
FLIM stands for fluorescence lifetime imaging microscopy, and it refers to a group of techniques for mapping the spatial distribution of excited-state lifetimes (fluorescence decay times) of emitting molecular species with nanosecond and microsecond temporal resolution. As a result, images created by fluorescence microscopy via FLIM are focused on the acquisition of decay-time data.

Fluorescence lifetime imaging (flim) with a focus on

The image generated by Fluorescence Lifetime Imaging (FLIM) is based on variations in the excited state decay rate of a fluorescent sample. FLIM is a fluorescence imaging technique that contrasts individual fluorophores based on their lifetime rather than their emission spectra. The fluorescence lifetime is the total amount of time a molecule spends in an excited state before releasing a photon to return to its ground state.
Fluorescence lifetime is more stable than intensity-based approaches since it is independent of concentration, sample absorption, sample thickness, photo-bleaching, and/or excitation intensity. At the same time, the fluorescence lifetime is influenced by a wide range of environmental factors such as pH, ion or oxygen concentration, molecular binding, and the proximity of energy acceptors, making it a common technique for functional imaging.
The fluorescence lifetime is calculated using Time-Correlated Single Photon Counting (TCSPC). The time between sample excitation by a pulsed laser and the arrival of the emitted photon at the detector is measured in TCSPC. TCSPC needs a given “start” signal, which is generated by electronics directing the laser pulse or a photodiode, as well as a defined “stop” signal, which is realized by single-photon sensitive detectors (e.g. Single Photon Avalanche Diodes, SPADs). To account for the statistical nature of fluorophores emission, the calculation of this time delay is repeated several times. After the excitation pulse, the delay times are sorted into a histogram that charts the occurrence of emission over time.

Introduction to flim-fret techniques

The FLIM method is a technique for calculating the distance between two

Frequency domain fluorescence lifetime imaging microscopy

The fluorescence lifetime is a measurement of how long a fluorophore stays excited on average before releasing a fluorescence photon and returning to the ground state. A fluorescence photon is not emitted at a constant rate from a fluorophore. Instead, an exponential decay function can be used to define a distribution of periods. The fluorescence lifetime, which is the characteristic time constant of this decay, ranges from a few picoseconds (10-12 s) to several tens of nanoseconds (10-9 s).
Fluorescence lifespan as a nanoenvironmental probe
This lifetime is a property of each fluorescent dye that varies depending on its microenvironment and conformational state. The molecular environment is probed for its composition, such as ion concentration, pH, lipophilicity, and binding to other molecules, using lifetime information.
Imaging and lifespan estimation combined FLIM blends lifetime measurements with photography, producing images by color-coding lifetimes obtained at the pixel level. As a result, FLIM provides knowledge about a fluorescent molecule’s spatial distribution as well as its nano-environment. In this way, a new level of knowledge is obtained.