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UID:69f244b284178
DTSTART:20201127T100000Z
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TRANSP:OPAQUE
LOCATION:ICFO Auditorium and Online (Teams)
SUMMARY:ICFO | PAMINA WINKLER
CLASS:PUBLIC
DESCRIPTION:The cell membrane is the encompassing protective shield of ever
 y cell and it is composed of a multitude of proteins\, lipids and other mo
 lecules. The organization of the cell membrane is inextricably intertwined
  with its function\, and sensitive to perturbations from the underlying ac
 tin cytoskeleton and the extracellular environment at the nano- and the me
 soscale. Elucidating the dynamic interplay between lipids and proteins dif
 fusing on the cell membrane\, forming transient domains and (re)organizing
  them according to signals from the juxtaposed inner and outer meshwork\, 
 is of paramount interest in fundamental cell biology. The overarching goal
  of this thesis is to gain deeper insight into how lipids and proteins dyn
 amically organize in biological membranes at the nanoscale.\nPhotonic nano
 -antennas are metallic nanostructures that localize and enhance the incide
 nt optical radiation into highly confined nanometric regions (&lt\; 20 nm)
 \, leading to greatly enhanced light-matter interactions. In this thesis\,
  we exploit an innovative design of planar gold nano-antenna arrays of dif
 ferent gap sizes (10-45 nm) and embedded in nanometric-size boxes. To eluc
 idate nanoscale diffusion dynamics in biological membranes with high spati
 otemporal resolution and single-molecule detection sensitivity\, we furthe
 r combine our nanogap antenna arrays with fluorescence correlation spectro
 scopy (FCS) in a serial and multiplexed manner.\nIn this dissertation\, we
  first describe the fabrication process of these planar gold nanogap anten
 nas and characterize their performance by means of electron microscopy and
  FCS of individual molecules in solution. We demonstrate giant fluorescenc
 e enhancement factors of up to 104-105 times provided by our planar nanoga
 p antennas in ultra-confined detection volumes and with single molecule de
 tection sensitivity in the micromolar range.\nSecond\, we apply these plan
 ar plasmonic nano-antennas in combination with FCS for assessing the dynam
 ic organization of mimetic lipid membranes at the nanoscale. For a ternary
  composition of the model membranes that include unsaturated and saturated
  lipids together with cholesterol\, we resolve transient nanoscopic hetero
 geneities as small as 10 nm in size\, coexisting in both macroscopically p
 hase-separated lipid phases.\nThird\, we add a Hyaluronic Acid (HA) layer 
 on top of the model lipid membranes to emulate the effect of the extracell
 ular environment surrounding native biological membranes. We extend our na
 no-antenna-FCS approach with atomic force microscopy and spectroscopy. We 
 reveal a distinct influence of HA on the nanoscale lipid organization of m
 imetic membranes composed of lipids constituting the more ordered lipid ph
 ase. Our results indicate a synergistic effect of cholesterol and HA re-or
 ganizing biological membranes at the nanoscale.\nFourth\, we apply our pla
 nar nano-antenna platform combined with FCS to elucidate the nanoscale dyn
 amics of different lipids in living cells. With our nanogap antennas we we
 re able to breach into the sub-30 nm spatial scale on living cell membrane
 s for the first time. We provide compelling evidence of short-lived choles
 terol-induced ~10 nm nanodomain partitioning in living plasma membranes.\n
 Fifth\, we demonstrate the multiplexing capabilities of our planar gold na
 nogap antenna platform combined with FCS in a widefield illumination schem
 e combined with sCMOS camera detection. Our approach allows recording of f
 luorescence signal from more than 200 antennas simultaneously. Moreover\, 
 we demonstrate multiplexed FCS recording on 50 nano-antennas simultaneousl
 y\, both in solution as well as in living cells\, with a temporal resoluti
 on in the millisecond range. The dissertation finishes with a brief discus
 sion of the main results achieved in this research and proposes new avenue
 s for future research in the field.
DTSTAMP:20260429T174938Z
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