In this work, we present two approaches for Broadband coherent anti-Stokes Raman spectroscopy (BCARS) imaging for biomedical research applications. The first approach is optimized for fast microscopy. The setup combines a chirped pulse amplification laser with the generation of supercontinuum generation in YAG crystal and PCF fibers. The second approach uses a fiber-based optical parametric amplifier to enable fast-tuned narrow-band pulses for fast hyperspectral multi-modal image acquisition in endoscopy settings.
Stimulated Raman Histology (SRH) can be used for label-free histological tissue analysis. However the current approaches are limited to two Raman peaks, thus restricting chemically-specific insights. We present a broadband coherent Raman platform (CRP), which overcomes these limitations combining an all-fiber dual output laser and a multichannel lock-in amplifier. This enables the simultaneous detection of 38 channels across the entire CH spectrum (2800-3100 cm-1) in parallel, facilitating chemometric and multimodal tissue analysis. By incorporating AI-driven virtual H&E staining and tissue segmentation for diagnostic purposes, it advances SRH towards clinical-diagnostic.
The intraoperative assessment of tumor margins of head and neck cancer is crucial for complete tumor resection and patient outcome. The current standard is to take intraoperatively tumor biopsies for frozen section analysis or to wait for the postoperative final histopathology report. The intraoperative evaluation is time-consuming, subjective, and methodologically limited. Optical methods like hyperspectral imaging (HSI) are of high interest to overcome these limitations. We present a framework to connect in-vivo HSI measurements to ex-vivo histopathological assessments with results as first steps towards the use of HSI as a tool for in-vivo tumor evaluation during head and neck surgery.
Modern medicine faces a major challenge in preventing, diagnosing, and treating cancer, especially in its early stages. Current tools for early detection and minimally invasive treatment are insufficient. Laser-based nonlinear endoscopy combining CARS, SHG, and TPEF emerges as a promising solution for cancer diagnostics by detecting molecular changes in tissue for differentiating cancer from healthy tissue. We developed an endomicroscopic system for head and neck cancer diagnosis and femtosecond laser ablation of tissue. The system has been validated through ex-vivo measurements on patient tissue slices and machine learning methods are being developed for quantitative comparison with standard H&E histopathology.
Acknowledgements:
Funding from the European Community’s Horizon 2020 Programme under the grant agreement No. 860185 (PHAST), No 101016923 (CRIMSON) and from the German Federal Ministry of Education and Research (BMBF): within the project TheraOptik (FKZ 13GW0370E) and LPI (Grant Number 13N15467) is acknowledged.
We combine an all-fiber dual wavelength, self-synchronized laser and a dedicated multi-channel detection unit to perform state-of-the-art multiplex Stimulated Raman Scattering (SRS) microscopy. The system covers the full CH spectrum in 1 μs reaching shot-noise limited performances with 25 μW per detection channel. This all-inone solution is based on a passively synchronized dual-wavelength laser source with shot-noise limited relative intensity noise from 600 kHz and a modular multi-channel lock-in detection unit. The synergistic design between laser source and detection system simplifies multiplex SRS implementation for real-time full-chemical imaging.
SignificanceConventional diagnosis of laryngeal cancer is normally made by a combination of endoscopic examination, a subsequent biopsy, and histopathology, but this requires several days and unnecessary biopsies can increase pathologist workload. Nonlinear imaging implemented through endoscopy can shorten this diagnosis time, and localize the margin of the cancerous area with high resolution.AimDevelop a rigid endomicroscope for the head and neck region, aiming for in-vivo multimodal imaging with a large field of view (FOV) and tissue ablation.ApproachThree nonlinear imaging modalities, which are coherent anti-Stokes Raman scattering, two-photon excitation fluorescence, and second harmonic generation, as well as the single photon fluorescence of indocyanine green, are applied for multimodal endomicroscopic imaging. High-energy femtosecond laser pulses are transmitted for tissue ablation.ResultsThis endomicroscopic system consists of two major parts, one is the rigid endomicroscopic tube 250 mm in length and 6 mm in diameter, and the other is the scan-head (10 × 12 × 6 cm3 in size) for quasi-static scanning imaging. The final multimodal image accomplishes a maximum FOV up to 650 μm, and a resolution of 1 μm is achieved over 560 μm FOV. The optics can easily guide sub-picosecond pulses for ablation.ConclusionsThe system exhibits large potential for helping real-time tissue diagnosis in surgery, by providing histological tissue information with a large FOV and high resolution, label-free. By guiding high-energy fs laser pulses, the system is even able to remove suspicious tissue areas, as has been shown for thin tissue sections in this study.
Here, we present a new handheld multiphoton endomicroscopic system designed for tumor diagnosis in the head and neck region. It consists of an approximate 25-cm-long rigid endomicroscopic probe with two variants (0° and 45° bended tip), connected to a handheld scan-head. The system can achieve a field of view ⪆600 µm for Coherent Anti-stokes Raman Scattering (CARS) and other nonlinear imaging modalities by a non-de-scanned detection and using a de-scanned confocal imaging channel to detect light from tissue labeled with Indocyanine Green (ICG). Furthermore, high-power femtosecond laser pulses can be transmitted through the system for precise tissue ablation which was considered in the optical design of the probe.
The intraoperative assessment of tumor margins of head and neck cancer is crucial for complete tumor resection and patient outcome. The current standard is to take tumor biopsies during surgery for frozen section analysis by a pathologist after H&E staining. This evaluation is time-consuming, subjective, methodologically limited. Optical methods like hyperspectral imaging (HSI) are therefore of high interest to overcome these limitations. We present an approach, that enables delineation of tumor margins with label-free HSI-based histopathological information during surgery using deep learning. We show accuracy on par with traditional intraoperative tumor margin assessment on a data set of seven patients.
Here, we present a new handheld multiphoton endomicroscopic system for tumor diagnosis in the head and neck region. It consists of an approx. 25 cm long rigid endomicroscopic probe with two variants (0° and 45° bended tip), connected to a handheld scan-head. The system can achieve a field of view > 600 μm for coherent anti-Stokes Raman scattering (CARS) and other nonlinear imaging techniques by a non-descanned detection channel, and laser confocal imaging with indocyanine green (ICG) by a descanned detection channel. Furthermore, high-power femtosecond laser pulses can be transmitted through the system for precise tissue ablation without the risk of damaging the optical components.
Here, we report a new handheld endoscopic system for nonlinear multimodal imaging of the head and neck region. It has a long rigid endomicroscopic probe with two versions (0° and 45° bended tip), connecting with a compact scan-head of approx. 10×12×6 cm3 size. The rigid probe is 6 mm in diameter and 24 cm long and allows diffraction-limited multiphoton imaging of tissue with at least 430 μm field of view and sub-micron resolution. The signals of Coherent anti-Stokes Raman Scattering (CARS), second harmonic generation (SHG), and two-photon excited fluorescence (TPEF) are collected by a non-descanned detection path in the scan-head, and the fluorescence of Indocyanine green (ICG) labeled lesions is detected by a confocal descanned configuration. Furthermore, this system is capable of guiding high-power femtosecond laser pulses for tissue ablation without the risk of damaging optical glass components.
Early detection and typing of tumors is pressing matter in clinical research with important impacts for prognosis and
successful treatment. Currently, staining is the golden standard in histopathology but requires surgical removal of tissue.
In order to avoid resection of non-diseased tissue a non-invasive real-time imaging method is required which can be
applied ideally intrasurgically. In this proceeding a combination of second harmonic generation (SHG), two photon
excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) imaging has been employed to investigate tissue
sections of head and neck carcinomas focussing on laryngeal carcinoma. Primary laryngeal and other head and neck
carcinomas consist to 99% of squamous cell carcinoma. By fusing the various imaging methods it is possible to measure
the thickness of the epithelial cell layer as a marker for dysplastic or cancerous tissue degradation and to differentiate
keratinizing and nonkeratininzing squamous cell carcinomas (SCC). As nonkeratinizing SCCs of the oropharynx
correlate with a human papillomavirus (HPV) infection as a subentity of head and neck cancer, and HPV related tumors
are associated with a better clinical prognosis, the differentiation between keratinizing and non-keratinizing forms of
SCCs is of high diagnostic value. TPEF is capable of displaying cell nuclei, therefore, morphologic information as cell
density, cell to cytoplasm ratio, size and shape of cell nuclei can be obtained. SHG - on the other hand - selectively
reveals the collagen matrix of the connective tissue, which is useful for determination of tumor-islets boundaries within
epithelial tissue - a prerequisite for precise resection. Finally CARS in the CH-stretching region visualizes the lipid
content of the tissue, which can be correlated with the dysplastic grade of the tissue.
Multimodal nonlinear imaging constitutes a contemporary approach to investigate the morphochemistry of complex
samples noninvasively and without administration of external labels. Here we discuss our recent success in jointly using
various nonlinear microspectroscopic approaches such as coherent anti-Stokes Raman scattering (CARS), two-photon
fluorescence (TPF) and second-harmonic generation (SHG) to study the chemical composition of surgically removed
tissue sections from laryngeal carcinoma. In particular it will be shown how multimodal nonlinear imaging can be
employed to study the structural and chemical development of disease formation as well as to monitor the clinically
important aspect of tumor boundary detection.
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