Functional super-resolution localisation microscopy by fluorescence lifetime imaging


Super-resolution and localization fluorescence microscopy techniques have attracted considerable attention in the past decade in particular (including the Nobel Prize in Chemistry, 2014), as they allow for localization of fluorophores on length scales below the optical diffraction limit, and elucidation of nanometer scale structural features in biological samples.

Academics at King’s College London have developed a novel method that will allow for functional superresolution imaging using differences in the fluorescence lifetimes of neighbouring molecules to distinguish them.

A major benefit of King’s technology is the possibility of determining whether there are highly localized variations in the local environment of a fluorophore using the fluorescence lifetime without the need to fit the time-resolved data to a model. The anticipated primary application of the method allows for functional superresolution by identification of protein-protein interactions between fluorescently labelled molecules by FRET. 



Technical Status


Proof of principle: The technology has been demonstrated computationally. 



IP Status


UK priority application



Commercial Status


Industry partners are being sought for licensing this technology. It can be combined into current microscope analysis software packages for enhanced performance and applications.



Key features

  • The proposed set-up will allow for dynamic super-resolution functional imaging of interactions, with identification of FRET without the necessity to fit the time-resolved data to a model, and using a single detection channel, with no chromatic aberrations.

  • Fluorescence lifetime measurements provide information on the local environment of the fluorophores on spatial scales below those of super-resolution imaging structure-function information rather than just structural information.

  • More than one molecule in a PSF can be detected and localized even if both are emitting simultaneously. This can potentially reduce the number of frames, and consequently the time necessary to acquire a superresolution image.

  • FRET can be identified by monitoring the trajectory of the PSF as a function of time.



Market Analysis


The techniques that are used currently can be broadly separated into three categories – (1) Photo-activation and photo-switching of molecules to image small subsets of individual emitters sequentially, frame-by frame, followed by reconstruction of the entire image from individual frames (STORM, PALM etc.), (2) spatio-temporal manipulation of interacting laser beams to modify the excited state emission of fluorophores whilst either the laser beams or the sample are scanned (STED) and (3) structured illumination (SIM) using patterned excitation light.

Whilst these methods allow for visualisation of biological structures with very high spatial resolution, close to or at the level of single molecules, uncovering the underlying biological function and dynamics of the system under study still represents a major challenge. Artefacts and uncertainties in localization microscopy also exist due to the stochastic nature of fluorophore blinking and switching.


In addition, multi-colour experiments which would be advantageous for monitoring colocalization of proteins labelled with different fluorophores, for example, suffer from complications due to chromatic aberrations in imaging systems, leading to compromised localization precision.  A problem in super-resolution microscopy is the persistence of fluorescence from molecules in successive frames during the acquisition which leads to an uncertainty in the number and position of molecules. Furthermore, measuring the number of molecules in a cluster of emitters mutually spaced at a distance much shorter than the optical resolution is also problematic. There are methods available, including single-molecule high-resolution imaging with photobleaching (SHRImP) and processing algorithms. However, the available algorithms are limited and generally require sparse distributions of emitters within an image to achieve high accuracy. Generally, if a number of identical emitters are present within an area defined by a point spread function (PSF), and are emitting simultaneously then asserting the number and position of those emitters is non-trivial.


The vast majority of super-resolution microscopy techniques rely only on spatio-temporal variations in fluorescence intensity as the contrast mechanism by which a complete image can be reconstructed. Individual molecules are activated or switched such that only a sparse subset of all the molecules in the sample emit in any one frame of acquisition. The fluctuations in intensity can be routinely measured using sensitive cameras.  However, fluorescence can be described by many parameters including the fluorescence lifetime, the average time spent in the excited state before emission. Indeed, of the fluorescence microscopy techniques capable of probing dynamic interactions, e.g. protein-protein interactions, fluorescence lifetime imaging (FLIM) is both well-established and very powerful. In the event that two neighbouring emitters are interacting via Förster resonance energy transfer (FRET), then the measured decrease in the fluorescence lifetime of the “donor” molecule can provide a measure of proximity to and degree of interaction with the “acceptor” molecule. 



Patent Information:
Physical Sciences
For Information, Contact:
Mugdha Joshi
IP & Licensing Manager
King's College London
Simon Ameer-Beg
Simon Poland