Abstract Total
internal reflection fluorescence (TIRF) microscopy is used to
measure the dissociation kinetic rate of fluorescein-labeled epidermal
growth factor from its specific receptors on the surface of intact
but mildly fixed A431 human epidermoid cells in culture. Prior
applications of TIRF microscopy have been limited to nonreceptor
binding or to model membrane systems. The evanescent field excites
fluorescence selectively at the surface of the cell proximal to
the coverslip. "Prismless" epiillumination TIR is employed
to avoid space limitations and is achieved by passing the excitation
laser beam through a high (1.4)-aperture objective so that the
light is incident at the glass/water interface beyond the critical
angle. Long-term focus is maintained by a special feedback system.
Of the possible effects that can influence the time course of
the postbleach fluorescence recoveries—the EGF/receptor
dissociation rate
k2, the bulk solution diffusion
rate of EGF, and the cell surface motion of the receptors—we
infer that the dissociation rate
k2 dominates.
Several fitting schemes are compared and indicate the presence
of a multiplicity of values for
k2, ranging
from about 0.05 to 0.004 s
–1, with an average
value of about 0.012 s
–1. These results compare
well with values previously obtained by radiolabel/washing techniques.
The significance of the results in terms of kinetic models and
the advantages of the TIRF technique for these sorts of measurements
are discussed.
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