Biomedical Engineering Reference
In-Depth Information
microscopy. The former is limited to low spatial resolution due to the longer
wavelength involved and by the vibrational absorption of the common solvent
water. Spontaneous Raman scattering microscopy with laser excitation in the
visible and near-infrared avoids this problem. However, it is often limited by
the weak Raman scattering cross section that necessitates high laser powers
and by the presence of auto-fluorescence background.
The signal-to-background limitations inherent to spontaneous Raman de-
tection schemes can be circumvented by the use of a nonlinear process that
significantly enhances the Raman scattering signal. As such, surface-enhanced
Raman scattering has been demonstrated to provide single-molecule detection
sensitivity [9, 10], but requires the molecular sample in the vicinity of surfaces
of metal colloidal particles or substrates and often lacks the reproducibility
of enhancement eciency. In an alternative attempt, coherent Raman scat-
tering (CRS) has been combined with optical microscopy. Because of its co-
herent nature, in which the molecular bonds oscillate in phase and interfere
constructively, the CRS signal can be orders of magnitude more sensitive than
spontaneous Raman scattering. Although it is not possible to detect a single
vibrational mode at room temperature, it is feasible to detect a macromolecule
with thousands of identical vibrational modes that interfere coherently. CRS
microscopy has been shown to provide advantages for the vibrational imaging
of biological samples for the following reasons [11-13]: (i) It does not require
fluorescent probes. (ii) Since there is no population of electronically excited
molecular states, photobleaching and damage to biological samples are sup-
pressed. (iii) It is much more sensitive than spontaneous Raman microscopy,
requiring only a moderate average power for excitation, which is tolerable by
most biological samples. (iv) Being a nonlinear microscopy with signal gen-
eration confined to the focal volume, it exhibits three-dimensional sectioning
capability similar to multi-photon-induced fluorescence microscopy. (v) The
use of near-infrared excitation minimizes sample heating due to the lack of
water absorption and provides a deep penetration depth for imaging through
thick tissues or cells.
The most predominant realization of CRS microscopy to date is coherent
anti-Stokes Raman scattering (CARS) microscopy [14, 11], which has emerged
as a highly sensitive tool for label-free vibrational imaging and microspec-
troscopy in the life and material sciences [15-19]. Only recently, stimulated
Raman scattering (SRS) has been used as a vibrational contrast for optical
microscopy [20, 21, 12, 13, 22]. CARS and SRS microscopy can be readily
combined with other nonlinear coherent optical processes that provide im-
age contrast, such as second-harmonic generation (SHG) [23], sum-frequency
generation (SFG) [24], and third-harmonic generation (THG) [25]. Among
them, SFG generation provides vibrational contrast, but this technique is
surface sensitive instead of bulk sensitive. Common to all techniques, ultra-
short pulses of high peak powers and moderate average power are required
for ecient signal generation. In combination with laser beam scanning, CRS
microscopy has been demonstrated to provide high image acquisition rates
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