We have developed a coherent Raman scattering microscope that combines total internal reflection illumination with surface plasmon resonance. The excitation geometry is based on an objective-type Kretschmann configuration, which allows widefield excitation of surface plasmon polariton modes in a thin gold film on a glass substrate. The surface plasmon fields enhance the excitation efficiency, enabling image acquisition at 10 frames/s. Since the evanescent field extends only over a length scale of ~100 nm, structures close the substrate surface are observed while bulk contributions are suppressed. We discuss the operational principles of this microscope in detail and point out its applications in cell biology.
SERS spectroscopy is currently gaining wider acceptance in biological research due to its ability to obtain signals from
very low quantities of material, and to obtain information from within live cells. SERS spectroscopy yields very narrow
bands (10-100 times narrower than typical fluorescence bands) and spectra suffer from minimal interference from
aqueous media, making SERS spectroscopy ideal for multiplex detection of intracellular components. Typically for
sensing, nanoparticles are labelled with suitable sensing molecules such as a dye or thiol. Nanoparticle labelling involves
two different types of interaction between the label and the enhancing surface, chemisorption and physisorption. The
former is considerably stronger and more stable than the latter and hence chemisorbed labels are more appropriate for
intracellular nanosensor design. In this paper, we demonstrate the difference in stability of both types of Raman label
inside live cells over periods of time. Chinese hamster ovary (CHO) cells were infused with a mixture of differently
labelled stable nanosensors and were imaged using SERS microspectroscopy. We also demonstrate the applicability of
SERS mapping for high-throughput multiplex detection using micropatterned cell arrays.
Raman spectroscopy and its various derivatives continue to offer the analyst fast, powerful, non-invasive and nondestructive
means by which to identify multiple analytes simultaneously and in real time. By virtue of the huge
enhancements possible in Raman scattering, generated by both surface enhancement and the resonance Raman effect, or
when coupled with other techniques such as confocal microscopy, Raman spectroscopy is becoming more and more
applicable to the types of assay being conducted in lab-on-a-chip applications, such as flow cytometry, cell patterning
and trapping, and microarrays, all of which often involve the detection of extremely low quantities of analyte. Surface
enhanced Raman scattering (SERS, or when coupled with the resonance Raman phenomenon, SERRS) spectroscopy has
proven to be of particular use as a robust optical detection method in microfluidic environments. In this paper, we
demonstrate the use of SERRS multiplex detection to quantitatively characterize individual microdroplets in a
continuous stream whose contents are gradually varied using a bespoke pump control algorithm.