FLIM & PLIM Module

Many biological processes — including synaptic plasticity — involve dynamic interactions between proteins. Unfortunately, neither classical biochemical approaches nor normal diffraction-limited fluorescence microscopy provides the required sensitivity or resolution to study these intimate and sometimes transient interactions (1).

Contrasting these methods,Fluorescence Lifetime Imaging Microscopy (FLIM)delivers information about changes in the binding, conformation, and composition of biological samples that are not accessible using other techniques. It stands out as the most rigorous method for measuring:

• Molecular environment parameters;
• Protein-interactions by Fӧrster resonance energy transfer (FRET) (2); and
• The metabolic state of cells and tissue via their autofluorescence (3).

To do this, FLIM-based FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. Similarly, Autofluorescence FLIM differentiates changes in the biology of objective cells and tissues based on the altered decay parameters of relevant endogenous fluorophores.

Likewise,Phosphorescence Lifetime Imaging Microscopy (PLIM)has become increasingly popular due to the recent development of two-photon-excitable oxygen-sensing probes (4). PLIM allows for quantitative in vivo oxygen measurements at high spatial and temporal resolution with high specificity, which makes it uniquely useful for live-cell and metabolic imaging.