Despite very high resolution, single molecule localisation microscopy techniques, which typically deliver resolutions down to 10’s nanometres, lack the ability to report the nanometre scaled protein-protein interactions inside living cells. This is a crucial challenge vital to understanding cellular function. The ability to report protein complex formation, with high spatial resolution in cells is extremely powerful, especially combined with all the other molecular imaging approaches. Förster Resonance Energy Transfer (FRET) has evolved as a powerful tool to access protein-protein interactions and molecular configurations on the 3-10 nm scale. When combined with Time-Correlated Single Photon Counting - Fluorescence Lifetime Imaging Microscopy (TCSPC-FLIM) FRET is a robust technique that can be applied to accurately quantify interaction between two molecules within a live or fixed sample by measuring a change in the fluorescence lifetime. To explain the theory behind this technique it has been broken down into three sections; FLIM Time-correlated single photon counting (the importance of fluorescence lifetime), TCSPC (how we measure the fluorescence lifetime) and FRET (how we measure a change in interaction). Using TCSPC-FLIM to measure FRET provides a quantifiable and highly informative approach to study interactions within live cells. This technique can be performed within ESRIC on the Leica SP5 SMD gSTED microscope equiped with a TCSPC Module and picosecond event timer and single photon avalanche detectors (SPADs). This article will stufy each component behind this powerful technique and outline the theory that makes it possible.

Time-correlated single photon counting (TCSPC)

If we ignore the rapid absorption and relaxation processes the fluorescence lifetime is simply the time between a fluorophore absorbing and emitting a photon. However, this is a statistical process at the level of single photons and to record an accurate representation of the molecules behaviour the time at which a photon is emitted needs to be recorded at the single photon limit and over many multiples of emission events. This is possible with time correlated single photon counting (TCSPC). TCSPC is essentially a single photon stopwatch, recording the time difference between the excitation and emission event on the nanosecond timescale. TCSPC uses a high repetition rate pulsed laser, a single photon detector and sophisticated counting electronics. The time between exciting the sample with the pulsed laser, and collecting the emitted photon with a single photon detector, is recorded as a single event on a histogram, see below. As the time between these two events is both statistical (as described by an exponential decay) and stochastic many thousands of measurements are repeated to construct the typical exponential decay used to quantify the lifetime (see graph below). The histogram can be fitted to well understood exponential decay functions and the representative fluorescence lifetime extracted with high reliability and accuracy.

FLIM Histogram Ali

TCSPC-FLIM is performed on a confocal laser scanning microscope (CLSM) and so the data is acquired pixel-by-pixel and the histograms of emission photon arrival times, correlated to the laser pulse responsible for each event, are built up for each pixel. This is relatively easy with a laser-scanning microscope and suitable fast electronics and single photon detectors. The disadvantage is that it is slow - it takes at least 1 minute to acquire sufficient data to make accurate fits estimating the decay times.

Fluorescence Lifetime Imaging Microscopy (FLIM)

Fluorescent lifetime is a robust, fundamental parameter that describes a key mechanism of the fluorescence cycle, essentially the time it takes a single molecule to emit a photon after excitation. As described in Principles of Fluorescence a fluorescent molecule will absorb and then subsequently emit light. Absorption, and relaxation into the lowest energy excited state, is a extremely quick process occurring on the picosecond timescales. Once in the lowest excited state however, the molecule will reside for several to tens of nanoseconds. This physical time final state absorption and emission is the fluorescence lifetime and is specific to each fluorophore. This is shown in the diagram below:

Jablonski FLIM3

As the fluorescent lifetime of a fluorophore is often unique to that specific fluorophore lifetime imaging can be used to produce a mutli-fluorophore image similar to traditional intensity based images from normal microscopy, this is FLIM. When combined with molecular interactions of closely adjacent molecules a change in interaction between two molecules can be measured by a change in fluorescent lifetime as described in the FRET section. These techniques have several advantages over intensity-based measurements, for example increased sensitivity or ability to separately probe spectrally identical fluorophores, but required advanced acquisition methods such as Time Correlated Single Photon Counting (TCSPC). TCSPC-FLIM has been applied across the disciplines; the biological and physical sciences as well as biomedicine. Below is an example of how the fluorescence lifetime (upper right panel) of a fluorophore can be more informative than simply measuring the intensity (upper left panel) and its application in studying proteins involved in cell communication in neuroendocrine cells (upper panels) as well as primary neurones (lower panel).

FLIM Image1

Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a remarkable energy transfer phenomenon that occurs between pairs of specific fluorescence molecules in close proximity. Specifically FRET is a resonant dipole-dipole energy transfer between one excited molecular state and an equivalent state in an adjacent molecule. In practice this means that for two FRET pair molecules in close proximity, optically exciting one molecule (the donor) transfers that energy to the second molecule (the acceptor), which subsequently emits fluorescence. FRET efficiency is hugely sensitive to donor-acceptor separation (on 3-10 nm length scale) such that measuring this emitted fluorescence allows for a quantification of the donor-acceptor spacing’s. If donor and acceptor are attached to interacting proteins, this allows for the quantification of protein-protein interactions on the nanometre scale. Donor-acceptor choice is crucial for FRET; the FRET pair must have overlapping excitation and emission spectra, such as GFP and mCherry or Cerulean and YFP.

FRET Ali10

Intensity based FRET measurements are simplest to perform, however FRET measured through this method is dependent on the protein concentration and proves highly problematic when trying to quantify the degree of interaction. Fluorescence lifetime is a significantly more robust measurement of FRET, increased FRET transfer reduced donor lifetime, which can simply be recorded with TCSPC-FLIM. This type of measurement is advantageous as it requires low excitation light levels, it is oblivious to donor concentration, and in addition it provides information about the ratio of interacting versus non-interacting proteins in a system.