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.

Do You Get What You See? Understanding Molecular Self-Healing.

The self-healing ability of self-healing materials is often analyzed using morphologic microscopy images. Here it was possible to show that morphologic information alone is not sufficient to judge the status of a self-healing process and molecular information is required as well. When comparing molecular coherent anti-Stokes Raman scattering (CARS) and morphological laser reflection images during a standard scratch healing test of an intrinsic self-healing polymer network, it was found that the morphologic closing of the scratch and the molecular crosslinking of the material do not take place simultaneously. This important observation can be explained by the fact that the self-healing process of the thiol-ene based polymer network is limited by the mobility of alkene-containing compounds, which can only be monitored by molecular CARS microscopy and not by standard morphological imaging. Additionally, the recorded CARS images indicate a mechanochemical activation of the self-healing material by the scratching/damaging process, which leads to an enhanced self-healing behavior in the vicinity of the scratch.

Investigation of Microalgal Carotenoid Content Using Coherent Anti-Stokes Raman Scattering (CARS) Microscopy and Spontaneous Raman Spectroscopy.

The yield of high-value products, such as pigments that could be extracted from microalgae, is affected by various nutritional and physical factors. Consequently, there is a need for fast visualization techniques that investigate the responses of individual microalgal cells to changing environmental conditions without introducing perturbations. Here, we apply CARS microscopy to map the distribution of pigments in the diatoms Ditylum brightwellii and Stephanopyxis turris and report their relative change in response to varying light cycles using a marker-based watershed analysis of the acquired images. Simultaneously, the underlying specific pigment composition alterations are revealed using Raman microspectroscopy at 785 nm excitation. In regards to assessing the chemical content of microalgae, these methods present themselves as viable alternatives to the standard techniques currently in use because of their non-disruptive nature and the wealth of complementary information that could be obtained from them.

Dual-focus coherent anti-Stokes Raman scattering microscopy using a compact two-beam fiber laser source.

We have developed a dual-focus coherent anti-Stokes Raman scattering (CARS) microscope based on a dual output, compact fiber laser source. The underlying concepts of time-multiplexed, two-beam scanning and demultiplexed detection that we already employed for second-harmonic generation are here naturally extended for CARS microscopy. The layout of a robust, all-fiber laser source was reconfigured to provide two outputs, each containing the two colors necessary for the CARS process. The utilization of the design for simultaneously imaging two laterally or axially separated fields of view and, thus, inherently speeding up the image acquisition process, is demonstrated on human artery tissue samples.

Hepatic Vitamin A Content Investigation Using Coherent Anti-Stokes Raman Scattering Microscopy.

Standard techniques for examining the distribution of vitamin A in liver either require staining or lead to rapid photobleaching of the molecule. A potentially better alternative approach is to use coherent anti-Stokes Raman scattering (CARS) microscopy; a fast, label-free, non-disruptive imaging method that provides contrast based on molecular vibrations. This contribution evaluates the viability of CARS microscopy for imaging vitamin A within thick hepatic tissue under physiological conditions by tuning into its characteristic vibrational band in the fingerprint region. Additional information about the morphology and architecture of the tissue was acquired using second harmonic generation (SHG) and multi-photon excited fluorescence (MPEF) to help mapping the intra-lobular positions of the vitamin A droplets. We demonstrate the capability of our multimodal imaging framework to selectively image lipid-soluble vitamin A droplets deep in bulk liver tissue with a high contrast while co-registering a complementary morphological background that clearly visualizes hepatic lobules. The results obtained envisage the good prospect of the technique for in vivo studies assessing vitamin A distribution heterogeneity and how it is affected by the progression of hepatic diseases.

Fiber-based dual-focus time-demultiplexed second harmonic generation microscopy.

We present a dual-focus second harmonic generation (SHG) microscopy approach based on stable, compact, and inexpensive fiber technology. One-tenth of the fiber laser output is coupled into a 100 m (≙500 ns) long single-mode fiber and further amplified to achieve two separately guided beams with time-alternating pulse trains. SHG detection is performed sequentially, generating two individual images in one scan. Thus, the configuration allows for imaging of distinct areas within the field of view at twice the repetition rate of the fiber laser but is readily extended to a multiple of the repetition rate with tens of foci.

Multimodal nonlinear microscopy of head and neck carcinoma - toward surgery assisting frozen section analysis.

BACKGROUND: Treatment of early cancer stages is deeply connected to a good prognosis, a moderate reduction of the quality of life, and comparably low treatment costs. METHODS: Head and neck squamous cell carcinomas were investigated using the multimodal combination of coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) microscopy. RESULTS: An increased median TPEF to CARS contrast was found comparing cancerous and healthy squamous epithelium with a p value of 1.8·10(-10) . A following comprehensive image analysis was able to predict the diagnosis of imaged tissue sections with an overall accuracy of 90% for a 4-class model. CONCLUSION: Nonlinear multimodal imaging is verified objectively as a valuable diagnostic tool that complements conventional staining protocols and can serve as filter in future clinical routine reducing the pathologist's workload. © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1552, 2016.

Texture analysis and classification in coherent anti-Stokes Raman scattering (CARS) microscopy images for automated detection of skin cancer.

Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful tool for fast label-free tissue imaging, which is promising for early medical diagnostics. To facilitate the diagnostic process, automatic image analysis algorithms, which are capable of extracting relevant features from the image content, are needed. In this contribution we perform an automated classification of healthy and tumor areas in CARS images of basal cell carcinoma (BCC) skin samples. The classification is based on extraction of texture features from image regions and subsequent classification of these regions into healthy and cancerous with a perceptron algorithm. The developed approach is capable of an accurate classification of texture types with high sensitivity and specificity, which is an important step towards an automated tumor detection procedure.