Endomicroscopic AI-driven morphochemical imaging and fs-laser ablation for selective tumor identification and selective tissue removal.

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.

Feasibility studies of multimodal nonlinear endoscopy using multicore fiber bundles for remote scanning from tissue sections to bulk organs.

Here, we report on the development and application of a compact multi-core fiber optical probe for multimodal non-linear imaging, combining the label-free modalities of Coherent Anti-Stokes Raman Scattering, Second Harmonic Generation, and Two-Photon Excited Fluorescence. Probes of this multi-core fiber design avoid moving and voltage-carrying parts at the distal end, thus providing promising improved compatibility with clinical requirements over competing implementations. The performance characteristics of the probe are established using thin cryo-sections and artificial targets before the applicability to clinically relevant samples is evaluated using ex vivo bulk human and porcine intestine tissues.

Design and test of a rigid endomicroscopic system for multimodal imaging and femtosecond laser ablation.

Significance: Conventional 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.

Aim: Develop a rigid endomicroscope for the head and neck region, aiming for multimodal imaging with a large field of view (FOV) and tissue ablation.

Multimodal nonlinear endomicroscopic imaging probe using a double-core double-clad fiber and focus-combining micro-optical concept.

Multimodal non-linear microscopy combining coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence has proved to be a versatile and powerful tool enabling the label-free investigation of tissue structure, molecular composition, and correlation with function and disease status. For a routine medical application, the implementation of this approach into an in vivo imaging endoscope is required. However, this is a difficult task due to the requirements of a multicolour ultrashort laser delivery from a compact and robust laser source through a fiber with low losses and temporal synchronization, the efficient signal collection in epi-direction, the need for small-diameter but highly corrected endomicroobjectives of high numerical aperture and compact scanners.

In vivo coherent anti-Stokes Raman scattering microscopy reveals vitamin A distribution in the liver.

Here we present a microscope setup for coherent anti-Stokes Raman scattering (CARS) imaging, devised to specifically address the challenges of in vivo experiments. We exemplify its capabilities by demonstrating how CARS microscopy can be used to identify vitamin A (VA) accumulations in the liver of a living mouse, marking the positions of hepatic stellate cells (HSCs). HSCs are the main source of extracellular matrix protein after hepatic injury and are therefore the main target of novel nanomedical strategies in the development of a treatment for liver fibrosis.

FLIM data analysis based on Laguerre polynomial decomposition and machine-learning.

Significance: The potential of fluorescence lifetime imaging microscopy (FLIM) is recently being recognized, especially in biological studies. However, FLIM does not directly measure the lifetimes, rather it records the fluorescence decay traces. The lifetimes and/or abundances have to be estimated from these traces during the phase of data processing.

Non-invasive Imaging Techniques: From Histology to In Vivo Imaging : Chapter of Imaging in Oncology.

In this chapter, we will introduce and review molecular-sensitive imaging techniques, which close the gap between ex vivo and in vivo analysis. In detail, we will introduce spontaneous Raman spectral imaging, coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), second-harmonic generation (SHG) and third-harmonic generation (THG), two-photon excited fluorescence (TPEF), and fluorescence lifetime imaging (FLIM). After reviewing these imaging techniques, we shortly introduce chemometric methods and machine learning techniques, which are needed to use these imaging techniques in diagnostic applications.

Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer.

Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection.

Differential response of liver sinusoidal endothelial cells and hepatocytes to oleic and palmitic acid revealed by Raman and CARS imaging.

Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid.

Multimodal Nonlinear Microscopy for Therapy Monitoring of Cold Atmospheric Plasma Treatment.

Here we report on a non-linear spectroscopic method for visualization of cold atmospheric plasma (CAP)-induced changes in tissue for reaching a new quality level of CAP application in medicine via online monitoring of wound or cancer treatment. A combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence lifetime imaging (2P-FLIM) and second harmonic generation (SHG) microscopy has been used for non-invasive and label-free detection of CAP-induced changes on human skin and mucosa samples. By correlation with histochemical staining, the observed local increase in fluorescence could be assigned to melanin.

Nonlinear Multimodal Imaging Characteristics of Early Septic Liver Injury in a Mouse Model of Peritonitis.

Sepsis constitutes a life-threatening organ failure caused by a deregulated host response to infection. Identifying early biomolecular indicators of organ dysfunction may improve clinical decision-making and outcome of patients. Herein we utilized label-free nonlinear multimodal imaging, combining coherent anti-Stokes Raman scattering (CARS), two-photon excited autofluorescence (TPEF), and second-harmonic generation (SHG) to investigate the consequences of early septic liver injury in a murine model of polymicrobial abdominal infection.