Confocal Raman Microscopy

Confocal Raman spectroscopy offers a number of significant advantages over other spectroscopic or optical microscopy techniques.

Confocal Raman Microscopy offers a number of significant advantages over other spectroscopic or optical microscopy techniques. It can be used for depth profiling and microscopic area mapping of samples with a spatial resolution of around 1 μm.

Overview

Figure 1 depicts a general layout for Confocal Raman microscopy. Raman utilizes a laser (a) as a source due to weak Raman scattering, about a million times weaker than optical spectroscopy. A beamsplitter (b) splits the light, with the beam path going to the sample (c) through the objective (d).  The beam is then split between the camera (e) and the spectrograph (f) after going through a rejection filter. The light then reflects and wavelength are separated by the grating (g) before reaching the detector (h).

Diagram of a Confocal Raman Microscope

Spectral Resolution

Spectral resolution in a  Confocal Raman microscope system is defined by several factors – focal length of the spectrograph, number of lines on the grating and resolution of the elements in the detector. Focal length is the distance between the focusing mirror and the detector, longer focal length provides greater resolution due to the wider separation of light (Figure 2). The grating is an optical component that consists of periodic grooves at a specific angle (blaze angle) to match the wavelength of the excitation laser. Resolution increases with a greater number of lines (Figure 3), given as lines or groove per millimeter on the surface (a common grating is 900 gr/mm) and the blaze wavelength. Light striking the grating surface is dispersed following the Huygens–Fresnel principle. The number of elements of a detector also determines resolution with increasing elements giving increased resolution but less sensitivity.

Focal Point and Resolution.
Figure 2. Focal Point and Resolution.

 

Grating and Resolution.
Figure 3 Grating and Resolution.

Spatial Resolution

In  Confocal Raman microscopy, spatial resolution is extremely important in being able to discriminate small structural differences in the sample. Optical microscope objective lenses (up to x100 magnification) are used to observe the sample and pass the Raman scatter to the spectrograph. This means that many of the factors and techniques that affect light microscopy can be applied to Raman microscopy. Spatial resolution is constrained by the resolving power of the objective lens. The spot size for a microscope measurement is dependent on three variables:  refractive index of the sample (n), the numerical aperture of the objective (NA) and the wavelength of the incident light (see Figure 4).  The estimation of the beam waist, or focal plane, volume does not include contributions from scattered light to or from the sample.

Confocal Raman Microscope Objectives and Spot Size.
Figure 4. Microscope Objectives and Spot Size.

Lasers

Selection of the laser wavelength is important in obtaining meaningful Raman data; the excitation wavelength can be selected from the UV though visible to near IR, with a range of power options depending on the application. Multiple lasers with a variety of wavelengths can often be installed in a Raman microscope to allow data to be measured for different types of sample (Figure 5). A popular laser wavelength is 532nm because it offers relatively high Raman excitation energy with low fluorescence. However, for greater reduction in fluorescence, longer wavelength lasers such as 785nm and 1064nm are often used. Less frequently required UV lasers offer the additional benefit of “resonance Raman”, an excellent choice for carbonaceous materials.

Confocal Raman Microscopy Laser Options and Applications.
Figure 5. Laser Options and Applications.

Rejection Filters

Rejection filters are used to remove light of the probe laser’s wavelength before it reaches the spectrograph.  There are three major types of filters: edge, notch and low-E filters (Figure 6).  Edge, the most commonly used, rejects both the laser light as well as the anti-Stokes region.  The intensity is >92 % and the cut off is about 100 cm-1. If the anti-Stokes region is also desired, then a notch filter may be used at the cost of intensity (>80 %) and cut off (150 cm-1). The Low-E filter is for studies requiring low wavenumber studies (5-50 cm-1 cut off) and also have a lower intensity (>80 %).

Rejection Filters.
Figure 6. Rejection Filters.

Confocal Raman Microscopy Sampling

Fluorescing Samples

Fluorescence can be a side effect in Raman measurement that can occur in either the target molecule or

the surrounding matrix. There are three physical methods for reducing fluorescence. The first is to select a laser wavelength at which fluorescence does not occur, this is typically at longer wavelengths (e.g. 785 or 1064 nm). The second method is to use aperture size and shape to mask as much of the sample matrix as possible to eliminate matrix fluorescence. The third method is to expose the sample to high wattage until matrix no longer fluoresces (photobleaching).  A software option is the use of a fluorescence rejection algorithm, which is highly effective at removing the broader fluorescence spectrum, leaving the sharper Raman peaks in a clean baseline, as shown in Figure 7. This feature can be used either automatically during measurement for fast imaging, or post run for more additional data processing. This often works well for samples with strong fluorescence when using shorter wavelength lasers (e.g. 405 or 457nm), which give a more intense Raman signal.

Fluorescence Removal by Software.
Figure 7. Fluorescence Removal by Software.

Minerals

Confocal Raman microscopy enjoys many practical benefits over FTIR and XRD with respect to mineral analysis.  The metal oxide bonds present in many mineral samples are highly absorbing in Raman.  These bonds appear in the 200-1000 cm-1 spectral region for Raman.  While XRD can identify polymorphs), it cannot do so on a microscopic level.  Raman bands of polymorphs are easy to distinguish, each giving its own representative spectra. Figure 8 shows a Raman image of rutile and anatase TiO2 fibers on a substrate.

Polymorphs of TiO2 Determined by Raman.
Figure 8. Polymorphs of TiO2 Determined by Raman.

Multilayer Samples

Confocal Raman microscopy can be used to determine layers in a multi-layer sample.  Firstly, a cross-section of a sample can be made and the sample mapped across the sections (Figure 9).  For very thin layers (<3 μm), confocal microscopy is effective due to its spatial resolution in the xy directions.  Thin layers (<30 μm) can be probed without sample preparation with depth profiling by confocal microscopy.  Since scattered light in the z dimension is filtered out, a map in the z-direction can be obtained simply by moving the stage in the z direction.

Cross section of Multi-layered Material.
Figure 9. Cross section of Multi-layered Material.

Particulates

Another application for Confocal Raman Microscopy is analysis of small particles.  These particles may be inclusions embedded in a matrix, filtrates on a filter, or particles on a substrate.  The size range can go from ~1 μm and up, depending on the laser and objective used.  Modern software/hardware can now find particles on a surface and label them via a library search automatically.  Figure 10 shows this process for particles on a substrate.

Automatic Mapping and Identifying of Particles on a Substrate.
Figure 10. Automatic Mapping and Identifying of Particles on a Substrate.

Chemical Mapping

Another application for confocal raman imaging is chemical mapping.  A scan is taken at each point on a sample’s surface and matched against a library.  The data then can be further analyzed using chemometric analysis to give compositional data for the sample.  This can be further enhanced by using a fast mapping stage and fast detector for rapid analysis (32,000 measurement points in around 16 minutes). Figure 11 shows a Raman image showing constituents in a pharmaceutical tablet.

Chemical Analysis of Raman Map of a Tablet.
Figure 11. Chemical Analysis of Raman Map of a Tablet.

Differential Interference Contrast Observation (DIC)

Differential Interference Contrast Observation (DIC) uses polarized light and a Nomarski-modified Wollaston prism to enhance the observation of images with low contrast. DIC uses phase difference in light to stereographically view very small step differences in the submicron order. Nomarski prisms are used to create bright and dark contrast from the differences in the two beams directly reflected at the sample’s surface. This technique can be applied equally to low contrast biological and non-biological samples that have small unevenness in the surface, an example may be seen in Figure 12.

DIC Image of Biological Sample.
Figure 12. DIC Image of Biological Sample.

Polarized Light Observation (PLO)

Polarized Light Observation (PLO) exploits the differences in anisotropic properties to enhance the observation of materials with low contrast. PLO uses two polarized elements located in the optical path on each side of the sample being observed. It is particularly useful for samples such as biomolecules and biostructures, minerals, ceramics, mineral fibers, extended polymers, liquid crystals, etc.  DIC of a mineral sample may be seen in Figure 13.

PLO of Mineral Sample Showing Domains.
Figure 13. PLO of Mineral Sample Showing Domains.