NEAR-FIELD SCANNING OPTICAL MICROSCOPY OF BIOLOGICAL MEMBRANES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 190

with the near-ield approach. Light that is emitted by the aperture locally
excites luorescent markers attached to the biological molecules under
investigation (proteins and/or lipids). The emitted luorescence emerging
from the imaging zone must be collected with the highest possible eficiency.
For this purpose, high numerical aperture (oil immersion) microscope
objectives are usually employed. The collected light is directed to sensitive
detectors, such as avalanche photodiodes or photo-multiplier tubes, via
suitable dichroic mirrors for spectral splitting or through a polarizing
beam splitter cube for polarization detection. Filters are also commonly
used to select the spectral regions of interest removing unwanted spectral
components. In this sense, inverted optical microscopes are an advantageous
solution for light collection, redistribution and iltering.

9.3 APPLICATION OF NSOM TO MODEL AND CELL
MEMBRANES

9.3.1 Model Membranes Inspected by NSOM

Model membranes have been used for a long time to investigate the
segregation behaviour of lipids and different proteins in predetermined lipid
mixtures, while reducing the complexity of the cell membrane. The typical
binary or ternary lipid mixtures used to mimic the lipid composition of cell
membranes indeed phase-segregate into liquid-condensed (LC) and liquid-
expanded (LE) phases. By transferring monolayers of a lipid mixture on a
substrate using standard Langmuir-Blodgett techniques, Hwang used

NSOM in dry and buffer conditions to reveal previously unresolved features
of around 50 nm. 45,46 When a higher pressure was used to form the monolayer,
the domains of the LC phase appeared to decrease in size, and an increasingly
complex ine web structure of the LE phase emerged. 45,46 Cholesterol addition,
typically enriching the LC phase, resulted in the formation of elongated
thin LC domains. From these morphology changes, it was concluded that
cholesterol reduced the line tension between the domains in regions of
LC/LE coexistence. Likewise, the addition of the ganglioside GM1, again a
LC constituent, affected the monolayer morphology signiicantly. Moreover,
GM1 induced a more pronounced segregation between the LC and LE
phases. These results suggested the formation of genuine distinct domains,
thus favouring the occurrence of a lipid raft type of phenomenon on model
membranes. The lipids typically enriching the LC phase are signiicantly more
saturated than lipids constituting the LE phase. Thus, when all lipids pack

et al.