Optically sectioned Raman images can be obtained from a sample of interest by either using a confocal back-scattering or off-axis geometry. In the confocal Raman spectroscopy/imaging system, a pin-hole is utilized to spatially filter away light emanating from outside the desired excitation volume (Fig.1), while in an off-axis Raman system (Fig.2), the excitation and collection arms are inclined at an angle. In the off-axis system, excitation of Raman light can be achieved using a now numerical aperture (NA) optics, and it prevents sample damage by excessively focused light, while the collection of Raman light can be achieved using high NA optics. Thus the spurious contributions by portions of a sample that are outside the region of interest can be avoided. Another important advantage of the off-axis system is the excitation of low background from the system optics
Fig.1: Schematic of a confocal Raman imaging/spectroscopy set-up
To obtain 3D images in a confocal Raman system, the sample is positioned in a plane perpendicular to the direction of excitation using the translational stages. Thus Raman spectra is acquired point by point and finally information is stitched together to obtain Raman image from desired region of interest. For biological tissues, where the Raman cross section is low, this step can be time consuming.
Wide field Raman imaging is employed to obtain Raman images of larger regions of interest in shorter time. The slit of the spectrometer can be used as a spatial filter, thus avoiding the use of small pin-holes and increasing throughput. But, spectral resolution may be compromised and hence a balance of aperture size to the Raman signal desired is required.
Fig.2: Schematic of an off-axis excitation Raman imaging/spectroscopy set-up
These two imaging methods can be used to effectively perform optical sectioning and extract information from a small sampling volume from within various heterogenous layers. Further, through planar and depth scanning of each small sampling volume, a 3D image of the sample under investigation can be developed.
The lateral and axial resolutions can be qualitatively defined according to the following equations:
Lateral resolution ~ (2*λ)/(π*NA) (1)
Axial resolution ~ (n*λ)/(NA2) (2)
where λ is the wavelength of the light in air, n is the refractive index, and NA is the numerical aperture of the excitation/collection optics respectively.