Unveiling the Dynamics of Protein-Protein Interactions: Fluorescence Lifetime Imaging Microscopy (FLIM)

Table of content:

1. What is FLIM, and what does it stand for?
2. What are FLIM's possible Applications?
3. Insights Unveiled by FLIM
4. How the typical FLIM Setup can be made?
5. Understanding FLIM Measurement
6. Variations of FLIM
7. Revolutionizing Biological Research with FLIM

What is FLIM, and what does it stand for?

Fluorescence Lifetime Imaging Microscopy (FLIM) is a cutting-edge imaging technique that offers unique insights into the dynamics of protein-protein interactions at the molecular level. By measuring the fluorescence lifetime of fluorophores, FLIM provides a powerful approach to studying these interactions and investigating the microenvironment of biological samples.

At least Becker & Hickl had protein-protein interaction in mind when they invented FLIM back in 1993. Today, there are two intentions for the use of FLIM. The first is the originally conceived molecular FLIM imaging, where the deep e.g. biological function of a system is to be investigated. The second emerging goal is video rate FLIM to follow processes in real time. For video rate FLIM, a very photostable sample is needed that can withstand the required laser power. Normally this is not about cells or processes within cells, because cells die at so much laser light. Often it is about systems that have been stained, in which case FLIM can increase the contrast. 

What are FLIM's possible Applications?

FLIM finds widespread applications in various scientific disciplines, with a particular focus on understanding protein-protein interactions. These interactions play a vital role in biological processes, and FLIM allows researchers to explore their dynamics, spatial distribution, and binding kinetics within living cells and tissues. FLIM can also be used to measure pH or temperature even within a cellular system. On the other hand, FLIM is also used for contrast enhancement. Also video rate FLIM employing a laser scanner is now well established. 

Insights Unveiled by FLIM

FLIM offers invaluable insights into protein-protein interactions. By measuring the fluorescence lifetime of labeled proteins, researchers can extract critical information such as association and dissociation rates, shedding light on the strength and specificity of these interactions. Additionally, FLIM can identify different interaction states and conformational changes, providing a deeper understanding of the functional states and regulatory mechanisms of proteins.

How the typical FLIM Setup can be made?

A standard FLIM setup involves a fluorescence microscope equipped with a pulsed laser for excitation and a single photon counting detector capable of capturing fluorescence decay kinetics. The sample, containing fluorescently labeled proteins, is illuminated by the laser, and the emitted fluorescence is collected and directed to the detector.

The detector records the fluorescence decay kinetics, which are then analyzed to extract the fluorescence lifetime information. This analysis often involves fitting the decay curve to a mathematical model, such as a single or multi-exponential function. The resulting fluorescence lifetime values are used to generate lifetime maps, visualizing regions of distinct protein-protein interactions or conformational states.

Understanding FLIM Measurement:

FLIM measurements rely on the principle that the fluorescence decay of a fluorophore is influenced by its microenvironment, including interactions with neighboring molecules. The fluorescence lifetime represents the average time taken for a fluorophore to return to its ground state after excitation.

To measure fluorescence lifetime, FLIM analyzes the fluorescence decay kinetics. This process typically involves fitting the decay curve to an appropriate mathematical model. By doing so, the fluorescence lifetime information is extracted, facilitating the creation of lifetime maps that highlight different protein-protein interactions or conformational states within the sample.

Variations of FLIM:

FLIM has undergone advancements, resulting in specialized techniques that expand its capabilities and application range. Becker & Hickl invented the Time-Correlated Single-Photon Counting FLIM (TCSPC-FLIM), which leverages single-photon detection to measure the arrival time of individual photons. TCSPC-FLIM boasts excellent temporal resolution and works well in low-light conditions as well as with bright samples. 

FLIM can also be combined with other imaging modalities, such as Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). These combinations enable simultaneous investigation of molecular interactions, spatial distributions, and dynamics, providing comprehensive information about protein-protein interactions.

A comparably low performance method is Frequency-Domain FLIM (FD-FLIM). FD-FLIM utilizes modulated excitation light and analyzes the phase and modulation of the emitted fluorescence to determine fluorescence lifetime. Due to the many advantages of TCSPC FLIM, we recommend the users to apply the power of TCSPC FLIM.