Surface-Enhanced Raman Scattering-Based Bioimaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 320

is used to illuminate the surface under investigation. The light source can be built into
the spectrometer or an external source can be used for illumination. Modern Raman
spectrometers generally include holographic gratings for improved excitation light
rejection, notch filter for reflected and Rayleigh scattered light rejection, and a liquid
nitrogen or Peltier-cooled charge-coupled device (ccD) to reduce variations in dark
current at the detector. a small local area of the sample is probed, where the ccD
detects only photons coming from a narrow spatial domain of the material [22]. The
filtered light is analyzed as a function of the probed position where the signal is read
by the detector and the intensity of each frequency is measured by an individual pixel
on the array. The intensity of the Raman signal is a function of four factors: material
properties (absorptivity, reflectivity, and the intrinsic strength of the Raman modes),
source laser power, the width of the spectrometer admission slit, and the width of the
resolution slit. Based on the intensity and frequency of the Raman signal received,
spatially resolved structure and properties of the material are identified.
owing to the submicron spatial resolution achieved with μRS, precise areas of
interest can be chosen to identify and analyze physical and chemical properties of the
materials under investigation or location of predesigned bright SERS probes, which
form exogenous contrast agents for SERS-based bioimaging. a motorized X , Y , and
Z stage with a nominal resolution as small as approximately 10 nm is used for scanning
the surface for lateral and depth information. Raman spectral mapping complements
the visual image obtained with the microscope by providing information about the
variation in chemical (or physical) properties of a heterogeneous surface.
Modern μRS systems are accompanied with powerful spectral acquisition and
analysis software, which enables the creation of 1D (cross section), 2D, and 3D
maps of various features from the 1D, 2D, or 3D array of spatially resolved Raman
spectra. Various features that can be routinely mapped include intensity variations
of specific peaks (by plotting the user-defined peak intensity or integrated area
under the peak), intensity ratio of two different bands, peak position (by user-
defined peak fitting routines such as gaussian and Lorentzian), and peak widths.
The obtained images can be further processed to highlight the spatial variations of
the acquired spectra. for example, Boolean maps, which present a binary represen-
tation of the desired attribute (such as intensity, peak width range), are obtained by
specifying the threshold values of the parameter. The Boolean maps are extremely
helpful to visualize the distribution of exogenous contrast agents in a living animal
or tissue.

11.3.2 resolution criteria

Dictated by the diffraction limit of light, the lateral resolution of a far-field optical micro-
scope is defined as the smallest distance between two features that can still be resolved
in the image and is given by Rayleigh criterion as shown in Equation 11.1 [23]:

λ
NA

R = 061 . (11.1)