The rising incidence of head and neck cancer represents a serious global health challenge, requiring more accurate diagnosis and innovative surgical approaches. Multimodal nonlinear optical microscopy, combining coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) with deep learning–based analysis routines, offers label-free assessment of the tissue’s morphochemical composition and allows early-stage and automatic detection of disease. For clinical intraoperative application, compact devices are required. In this preclinical study, a cohort of 15 patients was examined with a newly developed rigid CARS/TPEF/SHG endomicroscope. To detect head and neck tumor from the multimodal data, deep learning–based semantic segmentation models were used. This preclinical study yields in a diagnostic sensitivity of 88% and a specificity of 96%. To combine diagnostics with therapy, machine learning–inspired image-guided selective tissue removal was used by integrating femtosecond laser ablation into the endomicroscope. This enables a powerful approach of intraoperative “seek and treat,” paving the way to advanced surgical treatment.

Leukocytes are a part of the immune system that plays an important role in the host’s defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells’ types. To prove this hypothesis, UV−Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.

As the vital organ for breathing, speaking, and swallowing, the healthy condition of the larynx can influence our quality of life significantly. Due to high consumption of tobacco, laryngeal cancer is a common tumor of the respiratory tract.¹ A rapid intraoperative diagnosis of it can be established by a frozen section procedure on the suspicious mass, but its results can be inaccurate. Accurate diagnosis relies on histopathological analysis of the formalin-fixed paraffin-embedded process biopsy, which requires several days. “Optical biopsy” as an emerging technique, aiming to provide diagnostic signatures in-situ, noninvasively and in real-time, can assist surgeons in decision-making and reduce pathologist workload.² For example, multimodal nonlinear imaging, including coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG), is capable of providing morphochemical contrast, without the need of exogenous markers.^3,4 A prediction accuracy of 90% can be achieved for diagnosing imaged tissue sections.⁵ Apart from that, indocyanine green (ICG) is one of two U.S. Food and Drug Administration approved exogenous contrast agents for clinical use. It has peak absorption and emission wavelengths in the near-infrared spectrum and offers contrast for vascular structures. Moreover, combining the optical biopsy with fs laser ablation can be potentially useful for immediate and high precision ablation of cancerous tissue.⁶

Headgroup-labelled alkyne-tagged cholesterol mimetics with imidazolium cores were designed and probed for their use as Raman probes in mammalian cells. The analogs feature high membrane integration and tunability of their vibrational signature.

Due to ageing populations the number of tumors is increasing worldwide. Successful surgical treatment requires complete resection of tumors to reduce recurrence rates. To reach this goal, novel methods combining in vivo tumor and tumor margin detection with low invasive precision surgical tools are needed. Coherent anti-Stokes Raman scattering (CARS) imaging is a highly promising optical tool for visualizing tumors based on characteristic changes in tissue morphology and molecular composition, while fs-laser ablation is to date the most precise surgical tool established in ophthalmology. In this contribution, CARS imaging has been combined with fs-laser ablation as a new approach for image-guided precision surgery for the first time. CARS guided fs-ablation has been applied to ablate brain, liver, skin, muscular and vascular tissues with μm-precision using sub-100 fs pulses of μJ level. We demonstrate superior imaging performance and contrast as well as detection of tissue margins by coherent Raman microscopy in comparison to laser reflectance imaging. The combination of CARS-image-guided tissue ablation is a promising tool for minimally invasive surgeries particularly in the vicinity of functional structures in the future.