Biology Reference
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(a)
(b)
Figure 9.2.
(a) Schematic layout of a combined confocal/near-ield optical set-up.
Laser light is focused onto the sample using a high NA objective (confocal excitation)
or alternatively by the use of a subwavelength aperture probe. Fluorescence is
collected by a conventional inverted microscope. Dual-channel optical detection
allows wavelength and/or polarization discrimination. The inset illustrates the
principle of surface-speciic excitation where only luorophores close to the aperture
end (red dots) are eficiently excited, in contrast to those outside the near-ield region
(gray dots). The optical near-ield generated at the aperture has a signiicant intensity
at distances < 100 nm away from the aperture, selectively exciting luorophores that
are in close proximity to the cell surface. (b) Tapered NSOM ibre (above) together
with a SEM image of the probe end (below). Schematic adapted with permission from
Ref. 67. © (2009) National Academy of Sciences, USA.
9.2.1 Different NSOM Configuraons
The most commonly used NSOM coniguration for biological applications
is based on aperture-type ibre probes as described earlier, although other
types of approaches have also been implemented. For instance, instead of
using the probe to illuminate the sample, one can employ far-ield optics to
illuminate the sample and use the probe to collect the evanescent ield in
close proximity to the sample surface. Although perfectly suitable for some
photonic applications, its use in luorescence imaging is less appropriate
since far-ield illumination translates in unnecessary sample photobleaching.
A different experimental strategy to NSOM is based on the use of metallic
tips, known in the literature as apertureless NSOM
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when the tip is used as
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