New Tools for Biophotonics

Microfluidics and Time-Resolved Fluorescence for the Investigation of Out-of-Equilibrium biomolecular interactions / S. Haacke, J. Léonard.

Biomolecular interactions are characterized by structural heterogeneity and dynamics, the resolution of which would allow a mechanistic understanding of biomolecular interactions at the molecular scale. This in turn would enable the rational design of innovative therapeutic strategies targeting specifically these interactions. For instance, targeting specifically protein-DNA interactions would be a valuable clue to fight against a variety of viruses and cancer diseases.

We developed a new experimental approach combining Time-Resolved fluorescence (TRF) spectroscopy and droplet microfluidics to investigate the structural dynamics of biomolecular systems in so-called double-kinetics experiments. Droplet microfluidics is used to produce with minimal material consumption an out-of-equilibrium biomolecular complex by rapid mixing of two reagents within water-in-oil droplets. TRF detection (ps to ns time scale fluorescence kinetics) is used as a reporter of the conformational heterogeneity along the relaxation of the system (ms to seconds reaction kinetics) during its propagation inside the microfluidic channel. In contrast to conventional stopped-flow experiments, which monitor the time-averaged fluorescence intensity, that is a signal averaged over all conformations, this approach does reveal conformational heterogeneity along the relaxation process through the distribution of fluorescence lifetimes. The set-up uses a streak camera to measure fluorescence decay kinetics with a 10-ps time resolution, along the microfluidic channel in which droplets propagate (Figure). Beyond the proof-of-principle experiment [1], the approach is being applied to the investigation of biomolecular interactions in structurally heterogeneous and dynamic biomolecular systems.

Figure caption. Left: using a wide-field fluorescence microscope, the fluorescence of the droplets circulating in the main channel is imaged along the photocathode (PC) wire of a streak camera. 2D datasets are acquired in which the dimension along the PC wire is the chemical relaxation time axis (propagation in the microfluidic chip over seconds) and the second dimension is the fluorescence decay kinetics, which encodes the structural information (= distribution of conformations). Right: set of fluorescence decay kinetics revealing structural relaxation over a few hundred ms in a test bimolecular complex of bovine serum albumin (BSA) with Patent Blue V: a very good signal-to-noise ratio is achieved, which allows one to identify a distribution of fluorescence lifetimes interpreted as a distribution of conformations. The experiment thus reveals the time evolution of the population in the different conformations over ms to seconds [1]. 

Funding: ANR FEMTOSTACK (2011-14); Région Alsace; Institut Carnot MICA.

Collaborations: Yves Mély (LBP, Uni. Strasbourg); Wilfried Uhring, (Icube, Strasbourg France);

1. Maillot, S., A. Carvalho, J.-P. Vola, C. Boudier, Y. Mely, S. Haacke, and J. Leonard, Out-of-equilibrium biomolecular interactions monitored by picosecond fluorescence in microfluidic droplets.Lab on a Chip. 14(10): p. 1767-1774 (2014).

STED Microscopy

Traditional optical microscopy is suffering the diffraction limit. Recent innovations in the field of microscopy have helped to overcome this limitation.

Among them, STED (Stimulated Emission Depletion) is a technique based on fluorescence laser scanning and where the resolution increase is based on a purely physical principle. STED is based on a stimulated emission on the periphery of the excitation spot. This stimulated emission is conducted by superimposing to the excitation beam a second laser beam slightly red shifted and previously shaped to obtain a Laguerre Gauss type profile. Consequently, the fluorescence emission is maintained at the center of the excitation spot whereas the stimulated emission process is favored at the periphery of its spot.

The resolution of this type of assembly is dependent on the depletion beam (i.e. its profile but also its intensity). Stefan Hell, a pioneer of this kind of microscopy, showed that in theory there exists no limitation in spatial resolution as far as the depletion beams intensity is not limited and the studied material does not suffer degradation.

Nevertheless, it is common to obtain resolutions of about tens of nanometers, around 10 times better than in traditional confocal microscopy.

Figure : Intensity profiles of the excitation beam in blue, the depleting beam in red and the resulting fluorescent spot (green). Scheme of the setup.