Chemistry Reference
In-Depth Information
imaging is restricted to the area of substrate contact. Reducing the incident angle
(measured relative to the optical axis) to slightly below the critical angle for total
internal re
ection produces oblique illumination propagating into the cell
(Figure 3.2A, blue light ray near the critical angle). For favorable cases with low
auto
uorescence, this arrangement has been used to image individual signaling
molecules [167].
Fast diffusion or active translocation of a labeled macromolecule, can spread its
fluorescence emission over many pixels, causing reduced intensity above the cellular
auto
uorescence. This problem has been tackled for markers of gene expression by
immobilization in the cell membrane [168], but that route would not be appropriate
for functioning molecular motors. Techniques that have been used with other
systems to capture images of moving targets might be promising for molecular
motor studies. A very brief pulse of exciting light acts like a stroboscope and localizes
the
fluorescence to the spatial region occupied by the
fluorophore during the pulse,
thereby improving the signal-to-noise ratio [169]. Lastly,
fluorescence lifetime
imaging microscopy (FLIM) is a method using repeated sub-nanosecond excitation
pulses and time-gated photon counting to resolve and form an image from the
nanosecond decay rates of the excited
fluorescent species. The emission wavelength
and lifetime of the
fluorophore of interest may differ from those of the non-speci
c
background emission, enabling separation of the probe photons from the back-
ground luminescence [170].
The sub-diffraction localization of individual
fluorophores, developed for single-
molecule studies can also be used to obtain images with dramatically improved
spatial resolution of multiple cellular structures. A promising route to achieve this
goal is to use
fluorophores that can be repeatedly switched on and off [171, 172]. Both
organic
fluorophores and modi
ed
fluorescent protein analogs have been developed
that can be repeatedly transferred by pulses of light from
uorescent and non-
fluorescent states, or between two different-color states [173
-
177]. These light-
controlled
fluorophores are useful in studying the dynamics of cell processes,
transfer between compartments, and mobility [178].
Examples of using photoactivatable
fluorescent proteins in sub-diffraction
microscopy are shown in Figure 3.15 [179]. The authors used blue (405 nm) laser
pulses to switch on sparse populations of a photoactivatable GFP and then imaged
and photobleached them under green (561 nm) light. The individual
uorescent
GFP molecules were located within
20 nm by
fitting two-dimensional Gaussian
functions, as in FIONA. After photobleaching, a new set of
uorophores were
activated by the 405-nm laser pulse and imaged at 561 nm. After several thousand
photo-activation, imaging, and photobleaching cycles, the summed information
gave an image representing the likelihood of a GFP molecule being located at each
position, but with spatial resolution given by the nanometer-scale uncertainty of
individual molecule localization (Figure 3.15 lower panels).
Several other approaches using switchable
fluorophores [171, 172] and excitation
elds structured by optical interference [180] or stimulated emission [181] have
been described. This area of hyper-resolution microscopy is rapidly evolving for
imaging molecular structures in cells.
