| Method |
Atomic Force Microscopy-AFM provides
increased power of magnification/resolution coupled
to real-time imaging of living samples—capabilities
not shared with any other single imaging modality.
AFM also provides the investigator with an unequaled
measurement capability for minute samples (Snyder &
White, 1992), permitting precise computation of surface
areas, volumes, and linear distance. Researchers punctured cell and measured volume changes: Immediately after membrane puncture, the cell began
to spill its cytoplasm onto the substrate, forming
an enlarging pool. Thus, the liquid pool that formed
around the punctured neuron was of a higher specific
gravity than the extracellular medium, and unlikely
to be simply cytoplasm as we discuss below. In every
case, this extracellular pool enlarged coincident with
the collapse of the punctured cell body. This is shown
in Figure 4, the initial height of the soma was determined
to be 1.5 µm and plummeted to 0.3 µm in just
over 15 minutes. The volume of the cell body was reduced
from 127.6 µm3 to 28.2 µm3 as the volume of
the pool of extruded material increased from 45.8 µm3
to 103.8 µm3 over this same period of time. The calculation
of the volume of extruded cytoplasm are actually
underestimated since the enlarging liquid pool
extended past the field of view in some images. With
time after injury, compromise of the membrane spread
progressively across the soma from the minute and local
puncture, eventually degrading even the proximal
neurites (Fig. 4c). |