Confocal Raman microscopy refers to the ability to spatially filter the analysis volume of the sample, in the XY (lateral) and Z (depth) axes.
Raman microscopy couples a Raman spectrometer to a standard optical microscope, allowing high magnification visualization of a sample and Raman analysis with a microscopic laser spot. Raman microscopy is easy: simply place the sample under the microscope, focus, and make a measurement.
Just adding a microscope to a Raman spectrometer does not give a controlled sampling volume - for this a spatial filter is required. Confocal Raman microscopy refers to the ability to spatially filter the analysis volume of the sample, in the XY (lateral) and Z (depth) axes.
There are several methods in use today (for example, true confocal aperture, or pseudo confocal slit-binning techniques) and some are better than others. However, it is well established that by using a true confocal Raman microscope, it is possible to analyze individual particles or layers with dimensions as low as 1 碌m and below.
For a true confocal design, typical spatial resolution is in the order of 0.5-1 碌m.
For a true confocal design, the limits of spatial resolution are defined principally by the laser wavelength and quality of the laser beam that is used, and the type of microscope objective selected and so on. For the highest spatial resolution, a correctly matched high magnification objective and visible laser excitation will often produce optimum results. Typical spatial resolution is in the order of 0.5-1 碌m.
As its name suggests, remote in situ Raman analysis is a method of analyzing a sample in situ and/or in a remote location, rather than having to extract some of the sample and take it to a Raman spectrometer. It is often used in industrial settings, where examples include monitoring of reaction components in a reaction vessel (from a small glass flask through to industrial scale reactors), and analysis of chemicals at multiple positions in pipe lines.
Remote in situ Raman is often carried out using optical fibers, allowing a Raman probe head to be coupled to a spectrometer (which can be many hundreds of meters away from the analysis point). A single cable is used to transmit the laser to the sample, while another fiber is then used to transfer the Raman signal from the sample to a standard spectrometer and detection system. These two cables are connected to a compact, rugged Raman probe head which focuses the laser onto the sample, and collects the Raman signal.
The probes are suitable for use at high temperatures and pressures. They can operate in either an immersion mode (where the analysis head is dipped within the reaction liquid) or in a stand-off mode (where the analysis is made by focusing the laser through a transparent window in the reaction vessel or pipeline).
In situ Raman analysis can be used for:
Transmission Raman Spectroscopy (or TRS) is a form of Raman analysis which is ideally suited for bulk analysis of opaque/turbid materials
Transmission Raman Spectroscopy (or TRS) is a form of Raman analysis which is ideally suited for bulk analysis of opaque/turbid materials. Transmission Raman is based on the collection of Raman light propagating through the sample in the direction of the excitation laser 鈥� in essence, the sample is illuminated with the excitation laser from one side, and the Raman signal is collected from the other.
Despite the sample being opaque, light from the laser can pass through the sample via light scattering processes. Many of these photons contain Raman information, and thus Transmission Raman spectroscopy is possible.
Unlike traditional Raman spectrometers and microscope systems, the transmission geometry allows true bulk analysis from the entire volume of the sample (for example, a pharmaceutical tablet).
Transmission Raman is non-contact, non-invasive and non- destructive. It requires no sample preparation. Importantly, the measurement is insensitive to particle size effects, sample homogeneity and orientation.
Transmission Raman spectroscopy can be used to understand:
Resonance Raman spectroscopy is a variant of 鈥榥ormal鈥� Raman spectroscopy. 鈥楴ormal鈥� Raman spectroscopy uses laser excitation at any wavelength in order to measure the Raman scattering of this laser light.
Notwithstanding the many practical issues caused by the use of different laser wavelengths, the end result will be very similar whatever wavelength is used.
In resonance Raman, the excitation wavelength is carefully chosen to overlap with (or be very close to) an electronic transition 鈥� this typically means in an area of UV-visible absorption. Such overlap can result in scattering intensities which are increased by factors of 102-106 鈥� thus, detection limits and measurement times can be significantly improved. However, since the excitation coincides with UV-visible absorption, fluorescence backgrounds can be significant and more problematic than with 鈥榥ormal鈥� Raman scattering.
An alternative approach is Surface Enhanced Raman Scattering (SERS), which offers similar order of magnitude increases in intensity. The advantage of SERS over resonance Raman is that fluorescence is suppressed while the Raman is enhanced, thus removing the fluorescence background problem of resonance Raman.
For certain specific applications the benefits of resonance Raman can be powerful. One such example is the use of resonance Raman for the analysis of environmental pollutants, where concentrations in the parts per billion (ppb) and parts per million (ppm) range can be detected.
Practically, resonance Raman can be explored on any Raman system, and the actual measurement is made in the standard way. The obvious requirement is to have suitable laser excitation in order to meet resonance conditions.
The transmission configuration allows the use of the highest numerical aperture (NA) objectives (including immersion objectives). The reflection configuration can be used for any kind of samples (opaque and transparent) but is limited to lower NA objectives.
TERS (Tip Enhanced Raman Spectroscopy) brings Raman spectroscopy into nanoscale resolution Imaging. TERS is a super-resolution chemical technique. Better yet, it is a label- free super-resolution imaging technique which has been extended by our novel technology into an important new imaging technology.
TERS imaging is performed with an AFM/Raman system, where a Scanning Probe microscope (SPM that can be used in atomic force, scanning tunneling, or normal/shear force mode) is integrated with a confocal Raman spectrometer through an opto-mechanical coupling. The scanning probe microscope allows for nanoscale imaging, the optical coupling brings the excitation laser to the functionalized tip (or probe), and the spectrometer analyzes the Raman (or otherwise scattered) light providing a hyperspectral image with nanometer scale chemical contrast.
A TERS system is based on a metallic tip (generally made of gold or silver) employed to concentrate the incident light field at the apex. The tip acts as a nano-source of light and local field enhancer, greatly improving the Raman sensitivity (by a factor of 103鈥�107) and reducing the probed volume to the 鈥渘ano鈥� region immediately below the tip. The optical coupling that combines the two instruments uses a confocal scheme. Two different configurations exist for this coupling: one in transmission and one in reflection (Fig. 28), having their own advantages and drawbacks.
The transmission configuration allows the use of the highest numerical aperture (NA) objectives (including immersion objectives) giving high power density at the focus point and enabling the collection of high signal level, but it can only be used for transparent samples. The reflection configuration can be used for any kind of samples (opaque and transparent) but is limited to lower NA objectives.
By combining point-by-point scanning with simultaneous spectrum acquisition, near-field Raman mappings can be performed with lateral resolution down to ten nanometers or less.
Transmission Raman devices for pharmaceutical applications are available in locations other than the United States of America, Europe, and India.
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