Advances in microscopy techniques applied to biology have led to the conviction that the cell membrane is not merely a sea of lipids and proteins. Indeed, the membrane is a more complex system consisting out of a vast collection of lipids and proteins pre-organized into spatially distinct compartments at the nanoscale. This nanoscale membrane organization is not fully understood, but focuses around three main hypotheses of domain organization: lipid rafts, protein islands and actin corrals. Lipid rafts as one of the mechanisms leading to compartmentalization have been conceptualized as organizing principle of membrane heterogeneity. These lipid rafts are enriched in cholesterol, sphingolipid, such as the ganglioside GM1 and glycosylphosphatidylinositol anchored proteins (GPI-AP). Although lipid rafts have been found important for cellular function they have been challenging to investigate due to their small nanometric sizes of 20-200 nm. To address these length scales by optical means we applied superresolution near-field scanning optical microscopy (NSOM). Unlike standard lens-based fluorescence techniques that are diffraction limited to about 300 nm, the resolution for NSOM is dependent on the aperture diameter of the subwavelength probe (typically 50-100 nm). The research performed during this thesis can be split up in two parts. The first part concerns the investigation of cell membrane compartmentalization using state-of-the-art NSOM. The second part of this research was focused on technical advances in optical probe microscopy for their application in biology. Regarding progress in the field of membrane biology we were able to directly map nanoscale connectivity mediated by cholesterol on the cell membrane. Dual color superresolution images revealed that GM1 nanodomains and GPI-APs organized as separate nanoassemblies on the cell membrane but remained proximal to each other. This non-random raft-based connectivity was re-organized upon receptor activation. The observed nanoscale hierarchical organization and it subsequent evolution likely corresponds to a generic mechanism to provide cells with privileged areas that can act to generate rapid cellular function and responses to the outside world.

In the second part of the thesis we have applied concepts from the field of optical nanoantennas that confine and enhance the optical field in ultra small regions to increase the optical resolution up to 20-30 nm on intact biological membranes. In addition, we combined the confined illumination area provided by NSOM with fast dynamic measurements offered by measuring the fluorescence fluctuations by means of fluorescence correlation spectroscopy (FCS). As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale while the use of optical antennas will open the way for true optical nanometric microscopy.


Monday November 21, 17:00. ICFO Auditorium

Thesis Advisor: Prof. María García-Parajo