HIT

MOLECULAR BIOPHYSICS

Protein-protein and other solute-solute interactions (binding)

Goal: Analysis of molecular binding in solution.

Methods:
– Absorbance and fluorescence spectroscopy;
– Time-correlated single photon counting;
– Fluorescence anisotropy (steady-state and time-resolved).
– Surface plasmon resonance;

Deliverables: Binding site and stoichiometry. Affinity constants. Association/dissociation rates. Rotational correlation times.

Solute-lipid interactions (lipophiliticy) / binding to lipid membranes

Goal: Analysis of molecular interactions with lipids using lipid membrane model systems (liposomes, deposited lipid surfaces).

Methods:
      – Absorbance and fluorescence spectroscopy;
      – Time-correlated single photon counting;
      – Fluorescence anisotropy (steady-state and time-resolved).
      – Surface plasmon resonance;

Deliverables: Partition constants. Dissociation constants. Retained solute fractions. Saturating lipid to solute ratio. Lipid membrane mixing, fusion and permeabilization. Intra-membrane depth localization and rotational correlation times. Membrane dipole potential variations.

Molecular structure

Goal: Characterization of protein and peptide secondary structure and structure stability.

Methods:
      – Absorbance and fluorescence spectroscopy;
– Circular dichroism spectroscopy;
– Fourier-transform infrared spectroscopy.

Deliverables: Secondary structure motif identification and relative content. Denaturation midpoints (temperature, chemicals).

Nanoparticles and aggregation dynamics

Goal: Characterization of nano-sized particles and aggregates in solution.

Methods:
      – Dynamic lights scattering;
      – Absorbance and fluorescence spectroscopy;
      – Transmission electron microscopy (facility service).

Deliverables: Critical micellar concentrations. Aggregation kinetics. Aggregate core accessibility. Morphology. Multimodal particle size distribution. Hydrodynamic radius. Polydispersity index. Zeta-potential.

CELLULAR AND TISSUE BIOPHYSICS

Cellular morphology and biomechanics

Goal: Characterization of single cell physical and mechanical properties.

Methods:
      – Atomic force microscopy;
      – Transmission electron microscopy (facility service).

Deliverables: Live and fixed cell topographs. Cell height and roughness. Indentation force parameters. Cell-surface and cell-cell adhesion force parameters.

Cellular membrane penetration

Goal: Analysis of molecular internalization into cells and characterization of the associated mechanism.

Methods:
      – Fluorescence spectroscopy;
      – Flow cytometry (facility service);
      – Confocal microscopy (facility service).

Deliverables: Internalization kinetics and mechanism (endocytosis, direct translocation). Intracellular localization.

Blood-barrier model translocation

Goal: Evaluation of molecular translocation efficiency across a blood-brain, blood-placental and/or blood-ocular barrier models.

Methods:
      – Transwell cell culture;
      – Fluorescence spectroscopy;
      – ELISA.

Deliverables: Translocation kinetics and mechanism (transcytosis, direct translocation). Molecule fractions in external aqueous phases and in the plasma membrane.

Intra-vital vessel imaging (rodent models)

Goal: Evaluation of circulating leucocyte dynamics in response to a stimulus.

Methods:
      – Bi-photon confocal fluorescence microscopy.

Deliverables: Intra-vital microscopy time-lapse recordings. Rolling vs adherent leucocyte proportions.