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Summary on Coherent Raman Scattering (CRS) Microscopy Workshop in Konstanz, Germany

A vast and growing range of applications revealed

The 1st Workshop on Coherent Raman Scattering (CRS) Microscopy by Leica Microsystems attracted 30 enthusiastic participants from all over the world in February 2019 to beautiful Konstanz, Germany. The group enjoyed a number of great presentations, posters, and intense hands-on sessions on the latest Leica technology, including Coherent Anti-Stokes and Stimulated Raman Scattering microscopy (CARS/SRS), the FALCON lifetime imaging platform, and the THUNDER imagers.

It was amazing to see how the Leica CARS and SRS systems have found their way into principal-investigator (PI), government, and pharma-analytics-core labs, as well as imaging facilities, where they are being put to good use for a wide range of applications in biology, medicine, pharma, and much more. Some of these are described below.

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Neurodegenerative disease

In addition to imaging lipid-rich brain structures, such as myelin, and its degeneration due to diseases, such as multiple sclerosis, SRS has now been shown to enable label-free biophysical / biochemical characterizations of pathological Amyloid-β deposits for Alzheimer’s research. More importantly, SRS is sensitive to protein secondary structure and misfolding, one of the key mechanisms underlying several neurodegenerative diseases.

Pharmaceutical R&D

CARS and SRS microscopy is increasingly being used to follow the uptake and distribution of pharmaceuticals in cells and tissues. Even the metabolism of administered compounds can be studied, opening up entirely new perspectives for the study of drug modes of action. Coherent Raman scattering is also emerging as a versatile tool for quality control of pharmaceutical formulations, allowing for high-resolution, label-free imaging of active pharmaceutical ingredients (APIs) and excipients to determine their spatial distribution on tablet surfaces or in powder formulations. Interestingly, even different API polymorphs can be distinguished spectrally. Some of these new label-free procedures are already replacing previous cumbersome electron microscopy procedures.

Label-free histopathology

SRS and CARS, often in conjunction with two-photon-excited autofluorescence and second-harmonic generation, provide a versatile toolbox for studying tissue physiology and pathology. Early and sensitive detection for a wide spectrum of cancers, neurodegenerative diseases, and fibrosis, among many others, has been demonstrated. First studies are beginning to show how deep learning algorithms can aid in SRS-based tissue classification and diagnostics.

Liquid biopsies

Label-free cytopathology of urine samples for bladder cancer and of pap smears for cervical cancer has been demonstrated. Cancerous cells were identified with high accuracy using deep-learning analyses.

Cell & tissue biology

Label-free fingerprinting of various cell and tissue phenotypes has been demonstrated. Exciting applications for cell and gene therapy are expected in the near future. Even label-free high-content screening applications are on the horizon.

Model organisms

Two-color in vivo SRS imaging of the beating Zebrafish heart was shown. Metabolic imaging of organisms ranging from bacteria to C. elegans is enabling the development of applications from biofuels research to toxicity sensing. 

Food science

Label-free imaging of lipids, proteins, and water to determine distribution in emulsions goes a long way for analyzing food samples. Furthermore, CARS was shown to be sensitive to NaCl concentrations below the 1-percent level in solution. Interestingly, this sensitivity arises from ion-induced changes in the intermolecular coordination of the solvent molecules. The coordination changes leave a fingerprint in the CARS spectra, suggesting it can be used as a more generally applicable detection mechanism.

Vibrational Tags

Prof. Wei Min (Columbia University, New York, USA) delivered a fantastic keynote presentation on the emerging applications of “vibrational tags”. Based on alkyne or nitrile bonds or the substitution of hydrogen by deuterium, these tags produce a unique spectral peak in the “silent region” of SRS spectra. Because these tags are so small (just a single chemical bond added to the molecule of interest, compared to a ‘giant’ antibody or fluorescent protein), non-perturbative imaging of small molecules is now becoming a possibility. Some of the coolest applications included: 

  • Following the distribution of tagged compounds (drugs, fatty acids, nutrients, metabolites, …) in cells and tissues. An example in lipid biology that could not be studied previously by any other means demonstrated the aberrant subcellular localization of unhealthy saturated fatty acids, and the restoring effects of healthy unsaturated ones found, for example, in fish oil. Even the metabolism (cleavage, etc.) of tagged compounds can be followed via changes in the tag’s SRS spectra.
  • Observing local protein synthesis in the dendritic spines of neurons, one of the proposed key steps in memory formation.
  • Pushing this concept even further, Stimulated Raman Excited Fluorescence (SREF) was introduced, a new technique (Letter, Nature Photonics, 01 April 2019) that combines single-molecule sensitivity with the chemical specificity of coherent Raman imaging.
  • Super-multiplexed optical imaging: 10-color imaging in brain tissues was shown using a newly developed palette of vibrational tags, termed “Carbow”, which are opening up new avenues for future studies of complex biological processes with highly-multiplexed optical readouts.

Thanks a lot to Prof. Andreas Zumbusch and his team at the University of Konstanz for hosting us in late February 2019. The lovely Lake Constance was definitely only the second-best reason to be there at that time!