Raman Microscopy

Raman microscopy is differentiated from Probe based Raman in that the spot is designed to be focused to very small areas. In this market segment, it is common for customers to want to focus to spots ~1 micron in diameter. Therefore, it is essential to have a single spatial mode laser with good beam quality (M2).

Regardless of the system resolution, the laser requirements are the same in this category: The laser needs to be single spatial mode and very narrow spectral linewidth (<<1 cm-1) in order to focus properly in the system (offering high spatial resolution) and to offer high spectral resolution since Raman microscopes almost always have high performance spectrometers capable of discerning 1 cm-1 Raman peaks.

Optical Isolators

Raman microscopes are nearly perfectly aligned, so retro-reflections from highly polished surfaces like silicon wafers can cause the beam to “fold” and retro-reflect back into the laser causing Catastrophic Optical Damage (COD) of the laser. It is common for researchers to use silicon wafers to align their systems since the wafer is atomically flat and has a strong Raman signal – allowing for easy calibration and alignment of a confocal systems. For this reason, optical isolators are a necessity for this application.

Laser Line Filters

IPS_RamanMicroscopy-web
IPS Teamed with BioMark Diagnostics to develop a novel Surface Enhanced Raman Scattering (SERS) plate reader for cancer screening. The system’s modular design incorporates our high throughput integrated Raman probe with a compact spectrometer and computer interface and will be used to rapidly validate the efficacy of cancer treatments.

Circularized output beam

Focus to a nice small round spot is highly desired in a Raman microscope because it improves the spatial resolution of the system. Specifically, the customer wants to pin-point a specific spot in the microscopic field of view and capture insight into its chemical composition. If the focused spot of the laser is distorted or oblong the customer may capture Raman spectra from areas adjacent to the area of interest.

14IPS_CS3_Eye

Low divergence output beam

Divergence of the beam along with the beam diameter at the entrance to the microscope objective are 2 features that impact the “depth of focus” of the microscope system. This is known as “Confocality”. In high performance microscopes, it is desirable to have a very sharp focus (so a very small depth of focus). This allows the microscope to filter out Raman signal that comes from above or below the surface of the sample. The longer the focal length – the lower the confocality ratio and the less Z-Axis resolution is attainable. Beam Divergence is a critical specification in this area because the lens focal point is dependent upon having a collimated beam entering the back surface of the objective. Divergent beams don’t focus to the same location as collimated beam, so if the user focuses on an object in the microscope and then uses a excitation laser with a divergent beam, the laser will actually focus to a point above or below the surface that is optically in focus. See figure 1a vs 1b for image of collimated vs. divergent focus.

Beam diameter filling aperture of microscope objective

As with the beam divergence issue detailed in d). above, under-filling the objective lens also has an impact on the depth of focus. As can be seen in Figure 1a below, the paraxial (underfilled beam diameter) focusses to a different point than the marginal ray focus. It also has a much longer “depth of focus” since the angle is smaller. This leads to lower confocality ratio

Figure 1: Image depicts issues with the location of the actual vs ideal focal point when divergent vs collimated beams are used AND when the lens is underfilled.

IPS lasers have integral optical isolators, dual laser line filter packs and an adjustable beam expander – so they are ideal sources for Raman microscopy.

Raman Imaging

Raman imaging is a technique that involves creating a multi-dimensional “map” of hyperspectral data by combining both optical imaging and Raman spectroscopy. In its simplest form, it involves plotting the Raman spectra for every point in a 2-dimensional space. This is commonly done by step-and-repeat scans with a moveable sample stage.
IPS_RamanImaging-web

Another approach is to use a line scan/push-broom approach. In this embodiment, a line of excitation light is generated, and a spectrometer is oriented with its slit positioned orthogonally to the laser line.  

The spectrometer uses a 2-D imaging array (rather than a 1D array or a vertically integrated pixel array) to capture the Raman spectra from each section of the line and the laser line is scanned across the entire sample surface by translating the sample under the camera. The resolution of the line-scan is generally limited to the thickness of the line and the number of pixels on the camera. 

Related Products