Spatial Resolution in Raman Spectroscopy
In Raman microscopy, spatial resolution is vital for discriminating different structures in a sample. The better the spatial resolution the more detailed information can be gained. For example, differentiating different components in a single cell or detecting defects in graphene materials. Lateral and axial resolution are governed by different parameters, however to achieve the highest resolution for both a confocal Raman microscope needs to be used.
Two factors contribute to the lateral (XY plane) resolution: the excitation wavelength and the microscope objective used. Theoretically, the spatial resolution can be calculated:
Where λ is the wavelength of the laser and NA the numerical aperture of the objective. For example, using a 405 nm laser and an objective with a NA of 0.9 the theoretical spatial resolution achievable is 275 nm. However, in reality Raman spatial resolution is commonly quoted in the order of 1 µm for ideal samples. This is largely due to sample effects, and how the sample interacts with the Raman photons.
Since the spatial resolution is proportional to laser excitation wavelength, shorter wavelengths give higher spatial resolution Instruments with multiple lasers are common practice to find the best compromise between required spatial resolution and sample constraints, e.g. fluorescence. The other factor contributing to the spatial resolution is the NA, as the objectives NA gets larger the spatial resolution improves. This is because the NA represents the ability of the objective to gather light from the sample area. A typically used high NA value is 0.9 on a 100X objective.
Figure 1 highlights the spatial resolution gained from the truly confocal nature of Edinburgh Instruments RM5 Raman Microscope, closing the pinhole from 2 mm to 25 µm has a massive effect on the resolution of polystyrene beads. Only a truly confocal Raman microscope offers the user such control over the pinhole and therefore over the resolution.
Figure 1: polystyrene beads mapped on the RM5 Raman Microscope with varying pinhole sizes
A) 2 mm B) 100 µm C) 50 µm D) 25 µm
For applications where spatial resolution is critical, immersion objectives can be used to obtain a higher NA. Here an immersion fluid is placed between the front lens of the objective and a coverslip/sample where in normal objectives air would be. The immersion liquid increases the amount of light reaching the objective by reducing the amount of reflection and refraction of light from the sample. This is achieved by using immersion fluids with refractive indices higher than that of air, which is 1. The two most commonly used fluids are water with a refractive index of 1.3, and oil which has a refractive index of approximately 1.5 (dependent on oil type). Water immersion objective have particular use in confocal Raman microscopy for the study of live cells in cell media, whilst oil objectives can be useful for depth studies.
Axial resolution (Z axis) is more complicated, in terms of simply optical microscopy it is proportional to λ/NA2. However, for Raman microscopes both the detection and focusing optics need to be considered. Crucially resolution relies on the confocal design of the instrument and the pinhole diameter, depth resolution in the order of 1 µm can be achieved under ideal conditions. To find out more about the relationship between pinhole diameter and resolution read our post on the role of the pinhole in Raman microscopy. As well as the confocality of the system, the laser wavelength, microscope objective, and sample will affect the achievable depth resolution. The video below shows a 3D Raman map revealing the layers of a transdermal patch. The resulting volume render clearly shows the separate layers of the patch coloured to represent PET (red), PET/PIB (pink), PE (blue) and the active ingredient (green).
The RM5 Confocal Raman Microscope
The RM5 is a compact and fully automated Raman microscope for analytical and research purposes.
The truly confocal design of the RM5 is unique to the market and offers uncompromised spectral
resolution, spatial resolution, and sensitivity. To find out how it can help with your research, please contact us.
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