What automation means for Raman microscopy

Damon Strom looks at new advances in self-optimisation, modular optics and remote operation

Raman microscopy, once used primarily by specialists, is being adopted across an ever-increasing variety of scientific disciplines. Automation has been essential to the growth of the technique. Software-driven routines can expedite the measurement process and make advanced experiments much easier to carry out.

Recent advances in automated Raman microscopy include self-alignment and self-calibration, modular optics and remote-operation capability. These technologies will revise expectations of a fully automated Raman imaging system. This overview briefly covers the technique, details the new developments and presents an application that benefits from the advantages that they provide.

The Raman effect and Raman microscopy variations

The Raman effect describes the slight energy shift of scattered light due to interaction with molecular vibrations. These shifts are unique to each material and can be seen in the form of Raman spectra. Raman microscopy is a nondestructive, label-free technique that acquires Raman spectra to identify the contents of samples. Confocal Raman microscopy features a beam path geometry that strongly rejects light from outside the focal plane to maximise sensitivity and allow 3D measurements. Raman imaging acquires a Raman spectrum at every pixel to visualise the physical distribution of sample components.

Automated alignment and calibration aiding Raman imaging

Raman imaging experiments usually include a number of optical elements in their beam paths. Fully automated instruments control every piece with an integrated software suite that also saves their settings along with the measurement.

New opto-mechanical technology makes it possible for automated systems to self-align and self-calibrate. This optimises system performance for individual experimental set-ups and ensures consistent and repeatable results. It also reduces the researcher’s workload by requiring less user input and eliminating potential sources of error.

Modular optics enhancing automated Raman microscopy

Optical modules built to a common hardware standard can enhance the flexibility of automated Raman microscopes by enabling them to incorporate new capabilities as they are introduced. They can also simplify the configuration of a beam path for an experiment and help maintain flexibility as requirements evolve.

Modules necessary for the self-optimisation described above include a calibration source that can validate and calibrate spectrometer gratings, an output coupler that maximises the outgoing signal to spectrometers, and motorised iris diaphragms that adjust the beam path for optimum contrast and homogeneity in white-light imaging.

New multi-wavelength input couplers can optimise the optics for each excitation laser. They can also facilitate measurements with techniques complementary to Raman while remaining at the same sample position. Excitation wavelengths can be chosen to best produce the Raman effect, or to generate or avoid photoluminescence from the sample.

Remotely operated Raman imaging

Automated components enable remotely operated Raman imaging measurements in environmental enclosures such as glove boxes. This can be very useful in semiconductor research and life science, among other fields. Raman microscopes capable of self-optimisation also allow the instrument to be completely controlled from another location. Only the placing of the sample on the stage requires physical interaction with the microscope. This provides the full capability of a laboratory instrument from anywhere, including home offices.

Example of automated Raman microscope application

The measurement shown above was carried out using a WITec alpha300 apyron automated Raman imaging microscope outfitted with WITec UHTS ultra-high throughput spectrometers optimised for different wavelengths. After the sample was fixed on the microscope stage, the entire measurement was performed through WITec’s Suite Five integrated software and EasyLink handheld controller.

The images above (A-E) show the correlative Raman analysis of a tungsten diselenide (WSe2) flake. Different layers are visible in the white-light image (A). In approximately two minutes, a clear and detailed 75 x 75 µm² Raman image of 10,000 spectra was recorded (B). The flake shown consists of single-layer (red), double-layer (green) and multi-layer (blue) areas. The same measurement after smoothing is shown in (C). A measurement aquired in about 17 minutes of more than 100,000 spectra produced an even sharper image (D). The increased signal to noise ratio was achieved by reducing the pixel size from 750 nm (B) to 230 nm (D). The photoluminescence image (E) shows the same structures as the Raman image and even the grain boundary between the larger and the smaller flake is visible. The integration time was 6ms per pixel for all measurements. Measurement by Thomas Diesing - WITec.

Changing perceptions of what “fully automated” means for Raman microscopy

An investigation of a tungsten diselenide flake quickly produced high-resolution images with an instrument that can be controlled remotely and requires minimal user input. White-light, Raman and photoluminescence measurements were acquired from the same sample area with the beam path automatically optimised for each technique.

The advantages that automation initially brought to Raman imaging microscopy, in accessibility and the rate with which samples can be measured, have been augmented by new capabilities in self-alignment and self-calibration, modular optics, and remote operation. These technologies will change perceptions of what “fully automated” means for Raman microscopes.

Damon Strom is with WITec

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