Non-invasive label-free investigation and typing of head and neck cancers by multimodal nonlinear microscopy

Abstract

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.

Transient absorption microscopy: advances in chemical imaging of photoinduced dynamics

“…Several examples of optical‐microspectroscopic tools that have been developed and applied to this extent include parametric generation microspectroscopy (e.g. second harmonic generation (SHG) , third harmonic generation , four‐wave‐mixing (FWM) processes , coherent anti‐Stokes Raman scattering (CARS) ), nonlinear dissipation optical microspectroscopy (e.g. stimulated Raman scattering (SRS) , two‐photon excited fluorescence ) or pump‐probe microspectroscopy (e.g.…”

“…Conceptually, each of the abovementioned nonlinear microscopic tools provides some specific information on the material properties of the samples investigated: For instance, CARS, SRS or FWM deduce information from vibrational modes of the molecules in the sample and even in cells and tissues of living organisms . SHG, on the other hand, obtains its contrast from the sample's ability to generate second‐harmonic light .…”

“…The advancement of arguably the most intensely used technique for optical microscopy ‐ fluorescence microscopy – has led to a multitude of different implementations including wide‐field fluorescence microscopy , confocal laser scanning fluorescence microscopy , nonlinear versions of fluorescent microscopy based on two‐ or multi‐photon absorption and super resolution techniques such as STED, STORM, PALM etc. The main limitation, however, irrespective of the specific implementation of fluorescence microscopy is the necessity to use exogenous (e.g.…”

Raman Imaging: An Impending Approach Towards Cancer Diagnosis

“…Popp et al. investigated unstained samples from the brain of a domestic pig using multimodal nonlinear imaging approach combining CARS, second harmonic generation, and two photons excited fluorescence microscopy [47] . Same group demonstrated that integration of multiple multiphoton modalities can be highly productive in recognizing advanced carcinoma of hypopharynx, larynx, and the left tonsil in a clinical case study and has the potential to replace conventional H&E staining [48] .…”

Pump–probe micro-spectroscopy and 2D materials

“…The spatially resolved micro-spectroscopic tool is used to characterize photo-induced processes in several areas relating to materials with complicated structural. Examples of the many micro-spectroscopic techniques that have been developed and used in this context include FWM (four-wave mixing) processes [3], SHG and THG (second and third harmonic generator) [4][5][6][7], CARS (coherent anti-Stokes Raman scattering) [8][9][10], nonlinear optical micro spectroscopy (e.g. SRS (stimulated Raman scattering)) [11][12][13][14], and pumpprobe micro-spectroscopy [15][16][17] (e.g.…”