Spatially offset Raman spectroscopy is a common approach to testing substances held in containers or behind non-transparent layers, while Stimulated Raman Spectroscopy enables the imaging of cells and living tissues at faster speeds, with greater resolution and higher sensitivity than other conventional methods.
Spatially Offset Raman Spectroscopy (SORS)
SORS can be used for a wide range of applications, like the determination of raw materials in pharmaceuticals, detection of explosives in airport screening checkpoints, the detection of narcotics at points of entry and hazardous material detection in remote locations.
Standard, or "spontaneous", Raman spectroscopy is restricted to analyzing surfaces of materials, or samples behind transparent containers or coatings. When it comes to hazardous materials like high-potency pharmaceutical raw materials, SORS allows screeners to avoid the opening containers that can result in dangerous exposure.
SORS is able to analyze through several millimeters of ‘barrier’ material, enabling precise chemical testing beyond a layer of paper, plastic, fabric or skin. Specific knowledge about the composition of a container or barrier is not necessary to perform SORS, and the technique does not require direct contact.
SORS uses more than one spectroscopy measurement to test a sample. A “standard” Raman measurement, involving a laser and collection of resultant spectra, results in a signal that is generally dominated by the surface of the material. A spatially-offset measurement, involving the separation of the laser excitation and spectra collection, produces a signal that is more representative of the target material in a container or behind a thin barrier. A calculation involving two spectroscopy measurements is used to generate the desired result.
Stimulated Raman Spectroscopy (SRS)
The scientific phenomenon behind SRS was first identified in the early 1960s; however, it has only recently been identified as the key to an alternative Raman-imaging technique.
The investigation of a cell, or tissue, in its natural environment still presents considerable challenges for researchers. Raman spectroscopy methods offer some possibilities for the visualization of living tissues without the use of labels, as they produce imaging contrasts rooted in different chemical compositions. Importantly, water has a weak Raman scattering cross-section, meaning molecules and cells can be imaged in aqueous environments. SRS tactics have been found to overcome the low signals associated with standard Raman imaging, and therefore SRS methods offer much stronger real-time imaging of living cells and tissues.
SRS is based on the paired-alignment of two incident laser beams, called the pump and Stokes beams. By adjusting the frequency difference of these beams to fit a particular molecular vibration, it causes a stimulated excitation of the target. This sequence triggers a loss of intensity in the pump beam and a gain in the Stokes beam. By modulating one of the beams, normally the Stokes beam, the shift in pump beam can be assessed, delivering a contrast that can be used to produce an image or establish the spectral fingerprint of the sample.
SRS is commonly used to analyze the composition of a material, as well as perform ultrafast microscopy and imaging. Through the identification of various SRS signals associated with particular materials in a sample, the chemical composition of a sample can be determined. Three-dimensional imaging via SRS involves the laser scanning of a sample in a raster pattern while the focus depth is adjusted. Splitting up the frequency of the two lasers by the vibrational frequency of a molecular transition in the sample causes only a single, particular vibrational level in the sample, which is used to create an image.
Ultrafast microscopy enabled by SRS is much more rapid than what is possible with traditional microscopy techniques, as it avoids time-consuming frequencies scanning.
Sources and Further Reading
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