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Raman microscopy, also referred to as confocal Raman microscopy, is a technique that combines Raman spectroscopy with optical microscopy.
By combining an optical microscope with a Raman spectrometer, scientists and researchers can generate high-resolution images of microscopic samples. The optical microscope (typically a confocal microscope) provides spatial resolution and filtering, while the spectrometer provides spectral information. For this reason, it is a superior technology of choice for the characterization of various materials and samples.
The optics enable the analysis volume within the sample to be spatially filtered with high-resolution across all axes. The spectrometer—sensitive to the vibrational modes of the sample—provides chemical, physical, and structural insights.
When these spatial filters are added to a Raman microscope, it is called a confocal Raman microscope. In confocal Raman microscopy, the resolution is so fine that individual particles as small as 1 micron or less can be analyzed. The resolution of these microscopes is also affected by the specific wavelengths of the laser and the type of objective lens used.
Raman microscopes are widely used in materials science, cell biology, and medicine and have applications in pharmaceutical development, particulate identification, chemical imaging and analysis, and molecular and polymer analysis. In addition, these devices can be used to map areas of a sample or perform depth profiling without sample preparation.
One of the reasons these microscopes are considered so helpful is that, by taking Raman spectrums at all points on an object and filtering for specific spectral bands, an image can be reconstructed from the spectral information. This ability to analyze and produce an image of an object from its Raman spectrum distinguishes Raman microscopy from Raman spectroscopy.
We’ll briefly explain the fundamentals, components, and various imaging techniques of confocal Raman microscopy, a technique made possible through the combination of confocal microscopy and Raman spectroscopy.
It’s important to note that Raman spectrometers are generally similar to Raman microscopes when they are equipped with optical components. (This makes distinguishing between the two a little confusing. Just remember that when a device can both analyze a sample and produce an image of the sample, it’s often considered a Raman Microscope.)
Raman spectroscopy (the basis for Raman analysis in this type of microscopy) is based on the Raman effect, an inelastic scattering phenomenon. It’s a type of vibrational spectroscopy used to analyze a sample chemically. An analysis is performed using a monochromatic light (i.e., a laser), which acts as an excitation source and creates a molecular vibration.
When the light interacts with the sample, a small part of it changes wavelengths. This change is referred to as the Raman effect, and the altered light is collected to gain chemical insights about the sample.
In contrast to optical techniques like infrared microscopy, Raman microscopes use light that is compatible with simple glass optics. This means that Raman microscopes are often developed based on a very high-quality optical microscope.
Generally speaking, no elaborate sample preparations are necessary when using Raman microscopy. Instead, the samples are placed beneath the microscopy as they are. When sample preparation is, in fact, critical, simple cross sections are prepared, or large workpieces are created to fit on the stage.
However, the same sample restrictions required in Raman spectroscopy still apply here, and the sample may not show strong fluorescence or absorbance of the excitation wavelength.
For accurate and reproducible results, calibration of the wavelength axis is necessary. Whether minor or major, many consequences usually happen in terms of wavenumber calibration. For this reason, calibration and recalibration is performed regularly.
To calibrate or recalibrate a device, a silicon standard is measured. However, modern microscopes typically offer continuous calibration for ease of use and reduction of manual recalibration.
Spectral resolution describes the ability to resolve spectral features into their individual elements. If it is too small, spectral signals can disappear in wide “bands.”
On the other hand, if the resolution is too large, the measurements take much longer than required, without any advantages for the end-user. This highlights the importance of knowing which spectral resolution is ideal for a 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 because it influences how sharply we see objects. In Raman microscopy, it is vital to distinguish different structures in a sample. The better the spatial resolution, the more detailed the information obtained.
Various parameters determine the lateral and axial resolution. A confocal Raman microscope must be used to achieve the highest resolution in both areas. Typically, spatial resolution is a decisive parameter in Raman imaging.
Despite the variations, Raman microscopes and spectrometers typically comprise the following components:
When selecting a Raman device, it’s essential to consider all the various components that make up the microscope and spectrometer and their optimal configuration for a particular application. These factors will affect your ability to perform research accurately and efficiently.
Generating images of chemicals pose interesting problems due to the size of the sample that is being imaged and what scientists wish to display.
Raman imaging is a technique that uses Raman spectroscopy to develop chemical images by producing false colors representing the material’s chemical makeup. After full Raman spectra of the target, microscopic regions are obtained, specific colors are used to represent the material’s composition, phase, crystallinity, and strain at that pixel.
Other imaging techniques used include direct imaging, hyper-spectral imaging, and correlative imaging.
This type of imaging refers to a group of techniques that combine Raman with other imaging methods. Popular correlative techniques include Raman-SEM, Raman-AFM, and Raman-SNOM.
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