About Raman microscopy
Raman microscopy (µ-Raman) is the combination of conventional light microscopy and a unique chemical identification by Raman spectroscopy.
Both techniques are quite powerful on their own, but when combined they offer the possibility to chemically examine smallest objects (> 0.5 µm) and thus, they link spectral with spatial information.
In contrast to infrared microscopy, Raman microscopes are much easier to implode because they use light that is compatible with simple glass optics. Therefore, Raman microscopes are often developed on the basis of a very high quality optical microscope.
About sampling and confocality
Generally and depending on the analytical task, no elaborate sample prerparations are necessary in Raman microscopy.Usually, samples are placed beneath the microscopy as the are. At most, cross sections are prepared or large workpieces are cut to size to fit on the stage.
However, the same sample restrictions as in Raman spectroscopy still apply and the sample may not show strong fluorescence or absorbance of the excitation wavelength.
Some samples require a confocal Raman microscope, which offers spatial resolution in all three dimensions. This way, you can measure inside containers (e.g. glass vials) or characterize samples in 3D.
Calibrating a Raman microscope
For precise and reliable µ-Raman results an accurate calibration of the wavelength axis is essential. Many operational changes of a Raman microscope usually have more or less severe consequences in terms of wavenumber calibration.
A (re)calibration is performed by measuring a silicon standard, but modern microscopes offer continuous calibration for maximum convenience.
If not continuously calibrated, recalibration should be done regularily and even after seemingly minor instrument adjustments like laser, aperture or grating changes, sudden shocks and vibrations as well as temperature shifts and variations, to ensure optimal spectral data.
Spectral resolution describes the ability to resolve spectral features into their individual elements.If it is too small, some spectral signals disappear in wide "bands".
If it is too large, the measurement takes much longer than required without any advantages for the user. It is therefore important to know which spectral resolution is ideal for the particular sample. What makes the resolution "too low" or "too high" depends on the respective application and the analytical task at hand.
Spatial resolution is important as it influences how sharply we see objects. In Raman microscopy, it is vital to distinguish different structures in a sample. Hence, the better the spatial resolution, the more detailed the information obtained.
The lateral and axial resolution is determined by various parameters. To achieve the highest resolution in both areas, a confocal Raman microscope must be used. Typically, spatial resolution is a decisive parameter in Raman imaging.
In optical microscopy, confocality means that an illuminated sample spot and a pinhole aperture within the beam path both share the same focal point. In practical terms, instead of the entire sample, only a small part is illuminated by a point-shaped light source. The pinhole then blocks unfocussed light, thus increasing contrast and depth of field.
What is confocal Raman Microscopy?
This principle can be applied to Raman spectroscopy, thus enhancing spatial resolution along x,y- (lateral) and z-axis (depth) while also enabling depth profiling. Raman microscopes, however, may differ in their confocal design.
True confocal design
The biggest advantage of a true confocal Raman microscope, is the independent control of spatial and spectral resolution. This is achieved by placing a pinhole aperture in front of the spectrometer entrance slit. Variable pinhole apertures control the degree of confocality, while the entrance slit controls the spectral resolution of the spectrometer. The downside of this design are the difficulties encountered when trying to keep both apertures ideally aligned to maintain optimum performance.
In a simplified configuration the spatial resolution can be controlled by a combination of the entrance slit in one direction, and the spatial resolution of the CCD detector in the orthogonal direction. Spectrograph limitations lead to inferior performance when it comes to spatial resolution but by reducing the number of optics in the pseudo-confocal setup, the overall throughput is greatly improved.
Hybrid-confocal design (FlexFocus)
Since both, a high troughput and true confocal design offer obvious advantages, a Raman microscope can be equipped with a hybrid aperture array containing a set of pinholes and slits that can act as the confocal aperture and the spectrograph entranc. This Hybrid design combines the benefits of the two designs and allows on-demand access to a true confocal or high throughput setup.
What are advantages of Raman spectroscopy
Raman has several major advantages in comparison to other vibrational spectroscopy technique such as FTIR and NIR absorption. Opposed to absorption, Raman effect is the inelastic light scattering off a sample. As a result, Raman spectroscopy requires no or little sample preparation in measurement of solids, liquids, and gases. Not only directly, but also through transparent windows such as glass and plastic. Water has very low Raman signal and therefore Raman spectroscopy can easily detect compounds that dissolved in water without strong interference. That makes Raman spectroscopy very suitable for biological samples in native state.
How long does it take to acquire a Raman spectrum?
The exposure time depends on many factors, such as the expectation of spectral quality, laser power, and samples cross-section for Raman scatter. Typically, good-quality Raman spectra can be acquired within a few seconds.
Can Raman spectra be obtained from a mixture of materials?
The Raman spectrum contains information about all molecules that are measured. Therefore, Raman spectra obtained from mixture contain peaks from various molecules. If the spectra of components are known, quantitative information about the composition can be generated.
What else information can Raman detect, other than chemical structure?
Raman spectroscopy can provide, directly or indirectly, various information such as isotopes in molecules, allotropes, crystallinity, polymorphism, doping in crystal lattice, tension, pressure, and temperature.
The intensity of a spectrum is linear to concentration. The relationship between peak intensity and concentration can be calibrated with known samples. In mixtures, Raman peaks provide quantitative information about the concentration of compounds at the same time.
Unfortunately, the best laser wavelength for a specific application is also not always obvious. Many system variables must be considered to optimize the excitation wavelength in a Raman spectroscopic experiment. The scattering efficiency, influence of fluorescence, detector efficiency, as well as the availability of the cost-efficient and easy-to-use system, are the main aspects need to be considered. The resulting most used wavelength is 785 nm and/or 523 nm. The 532 nm is particularly suitable for inorganic materials, e.g. graphene and fullerenes.
The laser power at the sample on a Raman microscope is typically from sub-mW level to a few tens of mW. The Raman intensity is directly proportional to the laser power. However, there is an increased risk of sample damage when using strong laser power. The laser power can be lowered to avoid sample damage but in doing this we need longer exposure time to acquire good-quality spectra.