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What is FLIM - Fluorescence Lifetime Imaging Microscopy?

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FLIM exploits fluorescence lifetime which is a measure of how long a fluorescent molecule or fluorophore remains on average in its excited state before returning to the ground state by emitting a fluorescence photon. The emission of a fluorescence photon from a fluorophore does not always occur at an exact time after excitation. Instead, a distribution of times is observed which can be described by an exponential decay function. The characteristic time constant of this decay, the fluorescence lifetime, is in the range of a few picoseconds (1-10 ps) to several tens of nanoseconds (10-100 ns).

Fluorescence lifetime as a probe for the nano-environment

This lifetime is a characteristic parameter of each fluorescent dye or molecule that may change with its nanoscopic surroundings or its conformational state. Lifetime information probes the molecular environment for its composition, such as ion concentration, pH, lipophilicity, or the binding to other molecules.

Combination of lifetime measurement and imaging

FLIM combines lifetime measurements with imaging: lifetimes obtained at the pixel-level are color-coded to produce images. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its nano-environment. This way an additional dimension of information is obtained.

Principle of FLIM data acquisition and analysis

  1. Repeated measurement of the time between the laser excitation of the fluorophore and fluorescence photon arrival at each pixel of a detector (refer to figure 1A).
  2. Calculation of a histogram of photon counts versus arrival time after the laser pulse.
  3. Fit of the exponential decay curve, F(t) = A0 exp (-t/τ), to each histogram. The amplitude, F(t), reflects the total number of photons detected at a specific time and the time constant, τ, is called the fluorescence lifetime (refer to figure 1B).
  4. Lifetime images are displayed using an arbitrary color for each lifetime value (refer to figure 1C).
Fig.1: A) Diagram showing the repeated measurement of the fluorescence photon arrival time
Fig.1: A) Diagram showing the repeated measurement of the fluorescence photon arrival time, i.e. the time between the laser excitation and detection of photon emission. B) Calculation of a histogram of photon counts versus arrival time after the laser pulse. Fit of an exponential decay curve to the histogram where the amplitude reflects the total number of photons and the time constant, τ, is the fluorescence lifetime. C) Very simple example of a fluorescence lifetime image demonstrating how it is displayed using an arbitrary-color look-up table for the different lifetime values (τ1, τ2, etc.).

Solution for FLIM

Harness the power of FLIM with the STELLARIS 8 FALCON system. Efficiently investigate cellular physiology and explore dynamics in living cells.

Intensity and FLIM image of U2OS cells immunostained with α-tubulin (AF546) and vimentin (AF555) [1]. The scale bar represents 5 µm and the color bar 0–3 ns.
Intensity and FLIM image of U2OS cells immunostained with α-tubulin (AF546) and vimentin (AF555) [1]. The scale bar represents 5 µm and the color bar 0–3 ns.

References

L. Alvarez, B. Widzgowski, B. van den Broek, G. Ossato, K. Jalink, L. Kuschel, J. Roberti, F. Hecht: A novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM: With SP8 FALCON, Science Lab (2019) Leica Microsystems. 

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