Confocal Raman Microscope: Comprehensive Guide
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Confocal Raman Microscopy: What Is It?
For high-resolution chemical imaging of materials, confocal Raman microscopy combines the spectrum data from Raman spectroscopy with the spatial filtering of an optical microscope.
While the confocal optics of the microscope allow the analysis volume within the sample to be spatially filtered with high resolution in both the lateral (XY) and axial (Z) axes, the spectral Raman information is sensitive to the vibrational modes of the sample and provides extensive chemical, physical, and structural insight.
Chemical analysis of individual particles, discrete sample features, or layers down to less than 1 mm in size is made possible by the interaction of spectral and spatial information.
Raman Confocal Microscope
Figure 1 depicts a schematic of a confocal Raman microscope's optical design. The sample to be examined is set up on the microscope stage, where the laser's excitation light is reflected by a beam splitter and directed downward onto the sample by the microscope's objective lens.
The objective lens gathers the sample's Rayleigh and Raman scatter, which is then sent through the beam splitter and rejected filter to eliminate Rayleigh scatter. Through a confocal pinhole, the residual Raman scatter is then concentrated and brought into the spectrograph, where it is separated by wavelength and the spectrum is measured by an array detector.
The sample is moved on the microscope stage, providing spatial information. An array of Raman spectra are then collected, and these spectra are utilized to create three-dimensional Raman spectral maps of the material.
(Figure 1, Optical Layout, taken from Edinburgh Instruments)
The best excitation source for Raman microscopy is a laser because it can be focused to a tiny area on the sample for high spatial resolution, produces high intensity light that boosts Raman scattering and sensitivity, is monochromatic and provides great spectrum resolution.
The laser's wavelength has an impact on the background fluorescence, spatial resolution, and Raman scattering intensity. High-end Raman microscopes, such the NMI mK-CFM/Raman, may hold many lasers concurrently as a result, enabling rapid software switching to the laser wavelength that is most suited for each sample.
Both the laser excitation and the Raman scatter are collected from the sample using the objective lens. The laser spot size on the sample and the spatial resolution of the Raman mapping are both influenced by the numerical aperture (NA) of the objective lens and the laser wavelength. The Rayleigh criteria determines the theoretically achievable lateral resolution, which is diffraction-limited:
The spatial resolution is enhanced by increasing the NA (increased magnification) of the objective lens.
Raman scattering only makes up around 1 in 10 million of the scattered photons; the remainder are dispersed by Rayleigh. By filtering the scattered light via an edge or notch rejection filter, the strong Rayleigh scatter, which does not carry any valuable sample information, is removed before detection.
Sharp long-pass optical filters called edge filters absorb all wavelengths up to their "edge" and then transmit all remaining wavelengths. The edge wavelength is selected to allow the Stokes Raman scattered light to be transmitted while the Rayleigh scattered light is heavily absorbed. Compared to notch filters, they allow for the observation of lower Raman shift wavenumbers but only of the Stokes lines.
Holographic filters known as "notch filters" transmit all other wavelengths while having a pronounced absorption peak at a selected wavelength that is chosen to correspond with the laser wavelength.
The advantage of notch filters is that the Stokes and anti-Stokes Raman scatter can be observed simultaneously. The drawback is that they have a wider absorption profile that makes it difficult to see low wavenumber peaks and have a limited lifespan under laser irradiation, necessitating regular replacement.
The confocal pinhole, which is the distinguishing characteristic of a confocal microscope, is utilized to boost contrast, eliminate fluorescent background, and improve spatial resolution while doing Raman mapping. As seen in Figure 2, the confocal pinhole prevents out of focus Raman scatter from entering the spectrograph and being observed. Both in-plane and out-of-plane Raman scatter penetrate the spectrograph and would be seen in the absence of a confocal pinhole (left). The out of plane Raman scatter, on the other hand, is prevented from accessing the spectrograph by the confocal pinhole (right).
(Figure 1, Confocal Pinhole, taken from Edinburgh Instruments)
For 3D mapping, the confocal pinhole is crucial because it acts as a spatial filter in the axial (Z) direction by preventing Raman scatter from above and below the focus plane (extrafocal). Without the pinhole, there would be no axial spatial filtering and the Raman scatter would be captured with no axial resolution from the full axial volume of the sample. The pinhole is also helpful for lateral 2D mapping because it increases contrast by reducing background fluorescence and extrafocal scatter while only slightly enhancing lateral (XY) resolution.
When researching Raman microscopes, the words really confocal and pseudo confocal are widely used. Pseudo-confocal microscopes simulate the effect of a pinhole using the orthogonal arrangement of the entrance slit of the spectrograph and the pixels of the CCD camera, in contrast to truly confocal microscopes, such as NMI mK-CFM/Raman which have a physical pinhole in the confocal plane of the microscope and provide superior confocal performance.
The spectrograph spatially separates the various Raman scatter wavelengths and projects them onto an array detector for detection using mirrors and a diffraction grating. The spectral resolution of the Raman microscope, which determines how well the microscope is able to resolve closely spaced Raman peaks, is determined by the focal length of the spectrograph, the width of the entry slit, and the density of the grooves on the diffraction grating. The diffraction grating's groove density has the most impact on spectral resolution, and spectral resolution rises as groove density does as well. There is a trade-off though, since the spectral range that the grating is effective over is constrained by how dense the grooves are.
An array detector that captures the spectrum in a single acquisition is used to detect the Raman scatter. A CCD camera, a high sensitivity camera with a rectangular array of pixels onto which the Raman spectrum is captured and transformed into an electronic signal, is the most popular type of array detector. The recommended detector for quick Raman mapping with short acquisition times is an EMCCD (Electron Multiplying CCD), which is identical to a standard CCD but has an extra on-chip electron-multiplying amplification stage that permits quicker data readouts while maintaining the high sensitivity.
Advantages Of Raman Spectroscopy
Raman spectroscopy has a variety of benefits, some of which are given below.
1. Sample direct scanning can reduce the amount of time and chemical required for analysis.
2. Raman spectroscopy decreases the number of analysts needed. (Analysis may be completed by one person.)
3. Material real-time diagnostics.
4. There is no need to prepare the sample.
5. Samples may be examined more than once without being ruined, and the same may be used for additional test analyses.
6. It can distinguish chemical structures even when they have the same atoms but are arranged differently.
7. Sample analysis can be done through a clear polybag or container. No need to open the container to scan it.
8. Analyses of almost all materials are possible.
Disadvantages Of Raman Spectroscopy
1. Raman spectroscopy is very sensitive
2. Considered as costly equipment.
3. You cannot use metal or an alloy.
4. The samples' low concentration is difficult to measure
5. Sample destruction is possible due to sample heating from laser radiation.
What type of image does a confocal microscope produce?
Confocal microscopy is a powerful instrument that creates sharp images of fixed or living cells and tissues and can greatly increase optical resolution and contrast over that of a conventional microscope.
Raman Spectroscopy Application Areas
Raman spectroscopy has several uses in numerous sectors. It is a tool for analytical investigation in identification.
Applications for Raman spectroscopy are found in the following sectors;
1. Pharmaceutical industry
2. Forensic science Lab
4. Planetary science
Where can I buy a Confocal Raman Microscope?
If you are looking for a high quality Confocal Raman Microscope, you can check NanoMagnetics Instruments' mK-Confocal/Raman Microscope (mK-CFM/Raman).
Contact us to get a quote for Raman Microscope.