Giulia Ossato, product manager, Confocal, Leica Microsystems discusses how advances in imaging techniques could improve drug discovery research.
Drug Discovery
Acquired immunodeficiency syndrome (AIDS) is the most advanced stage of infection by human immunodeficiency virus (HIV). While AIDS is a secondary immunodeficiency syndrome that develops because of a persistent viral infection, there are many other immunodeficiency syndromes that are primary diseases because they are caused by inherited genetic defects. One such primary immunodeficiency syndrome was discovered last year, which has shed more light on how human immune systems work. To better understand the genetic cause of this new disease and connect it to cellular functions in the human immune system, researchers utilised advanced tools such as fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET).
FLIM produces images based on lifetime, the time it takes a molecule to return to the ground (lower energy) state by emitting a photon. Fluorescence lifetime depends on the fluorophore and its micro-environment and it is independent of concentration. The lifetimes obtained for each image pixel are colour-coded to produce the contrast and the corresponding FLIM image (even if the fluorophores emit in the same spectral range). The advantages of FLIM make it ideal for functional imaging and biochemical sensing, enabling measurements of parameters such as the pH and temperature of the local molecular microenvironment.
FRET is a well-established technique used to study molecular interactions. Photon energy emitted from a one fluorophore can be absorbed by an adjacent fluorophore, so long as the two fluorophores are spatially sufficiently close together, the emission spectrum of the donor and the absorption spectrum of the acceptor molecule overlap, and donor and acceptor dipoles are almost parallel. This direct energy transfer from the donor to the acceptor molecule occurs without the emission of light and is referred to as FRET. As energy is transferred, FRET alters the wavelength of the photons being emitted– thus, changing the colour of the fluorescence in the resulting image. As FRET requires a very short distance between fluorochromes, it is a useful tool for visualising and verifying interactions between molecules. FRET imaging is typically used to detect the co-localisation of molecules with an accuracy within a few nanometres, thus informing on potential molecule-molecule interactions within the same scale.
FLIM can measure FRET (FLIM-FRET) and therefore can visualise molecular interactions in live biological samples. It can track dynamic changes in structural and functional states, providing researchers with a versatile tool to observe living processes at a microscopic scale. However, despite these powerful capabilities, FLIM and thereby, FLIM-FRET, have not been widely applied. Several factors account for this, such as the intrinsically slow hardware and complex implementation of the traditional FLIM or time-correlated single photon counting (TCSPC) solutions, particularly for complex imaging workflows. As a result, FLIM imaging was used mostly in specialised laboratories and, even with expert knowledge, traditional TCSPC has been unable to deliver the speed needed to observe biological processes occurring at time scales below tens of seconds. To address these challenges, companies such as Leica Microsystems are continuing to develop microscopes, such as the SP8 FALCON (FAst Lifetime CONtrast), which is able to perform FLIM and FLIM-FRET ten times faster than with traditional TCSPC. SP8 FALCON is a fully integrated FLIM solution. The speed and full integration mean that complex FLIM experiments can now be accessible for life scientists on a daily basis – for many, adding a new dimension of contrast to their imaging and enabling them to perform new kinds of research.
Such advances in fluorescent microscopy for live imaging open up new possibilities for application in basic biomedical research and bio/pharmaceutical drug discovery and development. Not only will broader application of FLIM-FRET facilitate the development and use of biosensors for life sciences research, such as that conducted regarding the recently discovered immunodeficiency syndrome, but it also holds great potential for accelerating drug discovery and development in the bio/pharmaceutical industry. With FLIM-FRET biosensors, scientists will be able to accelerate research to understand the causes and mechanisms of disease, and so reveal new potential treatment targets. FLIM-FRET biosensors can also be used to help screen compound libraries to discover which ones are able to hit these treatment targets to trigger a potentially therapeutic change. These biosensors can continue to be used, to test lead compounds as they progress through to development and manufacture. As such, it is anticipated that imaging and techniques such as FLIM-FRET will play a much greater role in the elucidation of human health and disease, as well as in the acceleration of drug discovery and development to produce new medicines to ameliorate these disorders.