The combination of linear and nonlinear Raman microspectroscopy has been established to be a powerful tool for
biomedical diagnostics. In this contribution we discuss our recent approaches towards CARS (coherent anti-Stokes
Raman scattering) based quantification of analytes, which is generally complicated by the CARS-signal strength
dependence on the square of the molecular concentration and on the interplay between a molecular-specific vibrational
signal and a nonresonant contribution in the signal generation. Due to these complications the quantification of analytes
presents a major challenge in CARS microscopy.
Here we discuss two recently developed approaches, i.e. on the one hand a simplified setup for coherent anti-Stokes
Raman scattering (CARS) microscopy, which allows for recording CARS images with 30 cm-1 excitation bandwidth for
probing Raman bands between 500 and 900 cm-1 with minimal requirements for alignment. This experimental
arrangement is based on electronic switching between CARS images recorded at different Raman resonances by
combining a photonic crystal fiber (PCF) as broad-band light source and an acoustooptical programmable dispersive
filter (AOPDF) as tunable wavelength filter.
On the other hand, we discuss how the introduction of carbon-deuterium (C-D) bonds into drug compounds constitutes a
non-invasive labeling approach that allows for higher intrinsic CARS contrast to be obtained. The quantitative detection
of C-deuterated drugs by Raman microspectroscopy and CARS microscopy is examined. Concentration-dependent
studies on drugs with aliphatic and aromatic C-D moieties were performed in a two-channel microfluidic chip, using the
corresponding non-deuterated (C-H) isotopomers as an internal reference.
Nanoparticle probes for use in targeted detection schemes and readout by surface-enhanced Raman scattering (SERS)
comprise a metal core, Raman reporter molecules and a protective shell. One design of SERS labels specifically
optimized for biomedical applications in conjunction with red laser excitation is based on tunable gold/silver nanoshells,
which are completely covered by a self-assembled monolayer (SAM) of Raman reporters. A shell around the SAM-coated
metal core stabilizes the colloid and prevents particle aggregation. The optical properties and SERS efficiencies
of these plasmonic nanostructures are characterized both experimentally and theoretically. Subsequent bioconjugation of
SERS probes to ligands such as antibodies is a prerequisite for the selective detection of the corresponding target
molecule via the characteristic Raman signature of the label. Biomedical imaging applications of SERS-labeled
antibodies for tumor diagnostics by SERS microscopy are presented, using the localization of the tumor suppressor p63
in prostate tissue sections as an example.
We discuss the combination of a CARS-imaging system with microfluidics. Such system is a versatile tool to quantify
the relative contributions of resonant and non-resonant scattering at the CARS frequency. We will show that the twochannel
microfluidic chip employed in combination with deuterated isotopomers as an internal standard allows for fast
and quantitative detection of organic molecules by CARS microscopy. The experimental design enables the
simultaneous measurement of both the chemically relevant Raman-resonant signal and the non-Raman-resonant
We propose a methodology for enhancing the diffraction limited
spatial resolution attained in Raman and Fourier transform infrared microspectroscopic imaging techniques. Near-field scanning optical microscopy (SNOM) and spectroscopy employ apertureless and aperture approaches to provide ultra-high spatially resolved images at the nanometer level. In contrast, we employ conventional spectral acquisition schemes modified by spatial oversampling with the subsequent application of deconvolution techniques. As an example, this methodology is applied to flat samples using point illumination.
Simulated data, assuming idealized sample concentration profiles, are presented together with experimental Raman microspectroscopic data from chemically and morphologically well-defined test samples. Intensity profiles determined using conventional mapping and imaging techniques are compared to those obtained by the probe/deconvolution