Surface-Enhanced Raman Scattering-Based Bioimaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 321

where λ is the wavelength of incident light and na (= n sin θ ) is the numerical aperture
of an optical system, n is the refractive index of the medium, and θ is the angle
subtended by the optics. The depth of the field or vertical resolution of μRS is
approximated by conrady expression as given in Equation 11.2 [24]:

λ

=

=

λ

∆ Z (11.2)

2

sin 2

NA

n θ

under normal conditions ( n = 1 for air, λ = 632.8 nm, na = 0.95), the typical lateral
and depth resolutions would be about 400 and 700 nm, respectively. using the small-
est visible wavelength (~400 nm) and a high na ( n = 1.515, na ~ 1.4), one can
estimate the highest lateral resolution as 200 nm (based on abbe criterion) and the
smallest field depth as 400 nm. However, if a series of spectra are recorded at very
close equidistant locations, a reduced spot size (considering the gaussian profile of
the beam) is obtained through a convolution of the spot profile.

11.3.3 raman instrumentation tailored for Bioimaging

To date, very few of instrumentation developments are solely dedicated to Raman-
based in vivo bioimaging applications. Most of in vivo demonstrations so far have
relied on either handheld Raman spectrometers or confocal Raman microscopes
(discussed earlier), which are suboptimal for bioimaging applications. often,
commercial Raman microspectrometers that are designed for probing relatively flat
substrates are customized for bioimaging applications [20]. for example, for
performing Raman mapping on small animals using a confocal Raman microscope,
a low magnification objective (12×) with long working distance, intentional defocus-
ing of the laser beam, and high sensitivity settings by sacrificing spectra resolution
are necessary to attain satisfactory results. a high na, commonly used in Raman
microscope setup, results in a small laser spot illumination, which limits the permis-
sible laser power that can be used before tissue damage. Moreover, only a very small
amount of scattered photons emanate from the small laser spot due to a highly
diffusive propagation of light photons through tissue so that high na objectives can
only efficiently collect light from this small spot size. a 12× open-field lens with a
defocused beam provides a spot size of 20 × 200 µm, which is much larger than that
using higher na. While this approach of customizing suboptimal instrumentation
suffices proof-of-concept demonstrations, advanced Raman instrumentation tailored
for bioimaging applications is critical for enabling rapid progress of this technique in
preclinical and clinical settings.

11.3.3.1 Portable and Ergonomic Devices for Live Subjects and Surgery
intraoperative handheld device is an excellent tool for a surgeon to identify tumor
margins and satellite nodules during surgical procedures. We would like to quickly
point out a recent report, which made significant progress along this direction using
portable Raman. nie and coworkers have developed a handheld spectroscopic device