Viral entry into a host cell requires coordination of both viral and cellular proteins in a precise order to trigger membrane fusion. Here we present two examples from the field of HIV research to demonstrate how advanced confocal microscopy techniques enable time-resolved stoichiometric analysis of HIV fusion events in live cells  and how fluorescence lifetime imaging (FLIM) facilitates the understanding of the infection changes to the host. This result is achieved with molecular reporters that give a fluorescent readout of physiological changes within the cell. Today’s new generation of confocal microscopes—with the ability to perform lifetime-based imaging—has facilitated use of multiple fluorescent biosensors in the same experiment. Application of FLIM to Förster Resonance Energy Transfer (FRET) determination also allowed the collection of more quantitative data. This approach has been highly informative in elucidating the role of cellular metabolism in viral susceptibility, as well as the subsequent post-infection effects on cellular metabolism .
Getting a handle on viral entry mechanisms
HIV entry into immune cells, such as macrophages and T cells, requires a complex and coordinated cellular response that is initiated by interaction of the viral particle with cell surface receptors. Immature HIV particles are not able to enter a host cell because of the rigidity of their glycoprotein spikes . Before becoming infective, viral particles require Gag proteins to be cleaved. This step enables a structural reorganization that results in fluidity of the viral receptor binding protein Env at the viral membrane. Once the Env proteins can move freely, they are able to form Env trimers in the membrane, thus providing the functional membrane cluster needed for host membrane fusion (for details see the review Jakobsdottir et al. 2017) . This interplay between the viral glycoprotein and the host receptors is the key initiation step that determines an effective infection event. A more complete understanding of the molecular mechanics of this infection process is thus highly desirable, as it could be exploited for the development of novel intervention strategies to prevent viral infections.
Gaining insight with quantitative photon counting and fluorescence lifetime information
Fluorescence Lifetime Imaging Microscopy (FLIM/FALCON ) and photon counting-based advance analysis are all powerful techniques for measuring molecular dynamics and mechanisms. Recent advances in photon counting introduced with the STELLARIS platform increase the ability for further discrimination of photon events and improve the fidelity of the measured fluorescence . Taking advantage of fluorescence-lifetime based information to distinguish multiple biosensors in the same experiment is an effective means of elucidating the complex, multi-stage, mechanisms involved in viral entry. Recent advances in confocal microscope technology present in the STELLARIS platform allow fluorescence lifetime information to be captured on a confocal microscope ,. FLIM can also be used to measure FRET which is an important technique for understanding dynamic spatial relationships between molecules. FLIM-FRET is a gold standard technique, because it provides more robust and quantitative data than other FRET methods, such as sensitized emission or photobleaching.
Tracking single particles and molecules in real time
When studying viral entry, a critical goal is to understand how the virus particle physically interacts with the host cell. Microscopy techniques have now advanced to the point where it is possible to analyze these interactions at the molecular level. Within any given illumination volume in a fluorescence microscopy experiment, the maximum fluorescence intensity of a given molecule is directly proportional to the total number of that molecule . In addition, the corresponding amplitude of the intensity fluctuations will vary depending on whether the molecules are in a monomeric or a dimeric state . These fluctuations require time-resolved microscopy techniques in order to accurately quantify them. Using this information, it is possible to track single viral particles as well as single molecules on the cell surface to get an accurate picture of the stoichiometry of virus receptor and co-receptor binding.
There are three microscopy methods in particular that have been key to the study of viruses in real time:
- Single viral particle tracking (SVPT) – individual viral particles are tracked and their spatiotemporal interactions with host cells receptors are monitored. Viral particle tracking is used as the anchoring point for the cellular analysis. It also allows for the cellular signal, which does not correlate with particle tracking, to be used as an internal control measure.
- Number & Brightness (N&B) - – time-dependent fluctuation of fluorescent signals can be monitored to determine the oligomeric state of a given protein in real time using fluorescence correlation spectroscopy (