Beyond imaging with coherent anti-Stokes Raman scattering microscopy

La microscopie optique permet de visualiser des échantillons biologiques avec une bonne sensibilité et une résolution spatiale élevée tout en interférant peu avec les échantillons. La microscopie par diffusion Raman cohérente (CARS) est une technique de microscopie non linéaire basée sur l’effet Raman qui a comme avantage de fournir un mécanisme de contraste endogène sensible aux vibrations moléculaires. La microscopie CARS est maintenant une modalité d’imagerie reconnue, en particulier pour les expériences in vivo, car elle élimine la nécessité d’utiliser des agents de contraste exogènes, et donc les problèmes liés à leur distribution, spécificité et caractère invasif. Cependant, il existe encore plusieurs obstacles à l’adoption à grande échelle de la microscopie CARS en biologie et en médecine : le coût et la complexité des systèmes actuels, les difficultés d’utilisation et d’entretient, la rigidité du mécanisme de contraste, la vitesse de syntonisation limitée et le faible nombre de méthodes d’analyse d’image adaptées. Cette thèse de doctorat vise à aller au-delà de certaines des limites actuelles de l’imagerie CARS dans l’espoir que cela encourage son adoption par un public plus large. Tout d’abord, nous avons introduit un nouveau système d’imagerie spectrale CARS ayant une vitesse de syntonisation de longueur d’onde beaucoup plus rapide que les autres techniques similaires. Ce système est basé sur un laser à fibre picoseconde synchronisé qui est à la fois robuste et portable. Il peut accéder à des lignes de vibration Raman sur une plage importante (2700–2950 cm-1) à des taux allant jusqu’à 10 000 points spectrales par seconde. Il est parfaitement adapté pour l’acquisition d’images spectrales dans les tissus épais. En second lieu, nous avons proposé une nouvelle méthode d’analyse d’images pour l’évaluation de la structure de la myéline dans des images de sections longitudinales de moelle épinière. Nous avons introduit un indicateur quantitatif sensible à l’organisation de la myéline et démontré comment il pourrait être utilisé pour étudier certaines pathologies. Enfin, nous avons développé une méthode automatisé pour la segmentation d’axones myélinisés dans des images CARS de coupes transversales de tissu nerveux. Cette méthode a été utilisée pour extraire des informations morphologique des fibres nerveuses dans des images CARS de grande échelle.Optical-based microscopy techniques can sample biological specimens using many contrast mechanisms providing good sensitivity and high spatial resolution while minimally interfering with the samples. Coherent anti-Stokes Raman scattering (CARS) microscopy is a nonlinear microscopy technique based on the Raman effect. It shares common characteristics of other optical microscopy modalities with the added benefit of providing an endogenous contrast mechanism sensitive to molecular vibrations. CARS is now recognized as a great imaging modality, especially for in vivo experiments since it eliminates the need for exogenous contrast agents, and hence problems related to the delivery, specificity, and invasiveness of those markers. However, there are still several obstacles preventing the wide-scale adoption of CARS in biology and medicine: cost and complexity of current systems as well as difficulty to operate and maintain them, lack of flexibility of the contrast mechanism, low tuning speed and finally, poor accessibility to adapted image analysis methods. This doctoral thesis strives to move beyond some of the current limitations of CARS imaging in the hope that it might encourage a wider adoption of CARS as a microscopy technique. First, we introduced a new CARS spectral imaging system with vibrational tuning speed many orders of magnitude faster than other narrowband techniques. The system presented in this original contribution is based on a synchronized picosecond fibre laser that is both robust and portable. It can access Raman lines over a significant portion of the highwavenumber region (2700–2950 cm-1) at rates of up to 10,000 spectral points per second and is perfectly suitable for the acquisition of CARS spectral images in thick tissue. Secondly, we proposed a new image analysis method for the assessment of myelin health in images of longitudinal sections of spinal cord. We introduced a metric sensitive to the organization/disorganization of the myelin structure and showed how it could be used to study pathologies such as multiple sclerosis. Finally, we have developped a fully automated segmentation method specifically designed for CARS images of transverse cross sections of nerve tissue.We used our method to extract nerve fibre morphology information from large scale CARS images

Multimodal-nonlinear-optical-microspectroscopy for biological and diagnostic applications

Multimodal non-linear imaging techniques provide non-invasive and potentially in vivo means to investigate tissue with cellular resolution. A particularly promising approach that has garnered attention as of late is the combination of coherent antiStokes Raman scattering (CARS), second harmonic generation (SHG) and two photon excited autofluorescence (TPEF) microscopy. In the first section of this thesis, the diagnostic potential of multimodal non-linear imaging has been demonstrated in the case of head and neck squamous cell carcinoma. The second part of this thesis investigates the feasibility of CARS microscopy for imaging intense bands in the finger-print region (800-1800 cm 1) wherein the presence of multiple overlapping peaks and interference with non-resonant background present challenges. Specifically, the emphasis is on imaging the prominent peaks arising from conjugated C=C double bonds in retinol, tretinoin, β-carotene, and various microalgal pigments. The first CARS fingerprint imaging application in the thesis is concerned with the vitamin A content of liver tissue. Analogously, in a uni-cellular application, CARS has been employed to image carotenoids in the diatoms D. brightwellii and S. turris. As part of the effort in transferring multimodal microscopic technologies to the enduser, the third part of the thesis examines two beam excitation and demultiplexed detection as a means of doubling the speed of laser scanning microscopes based on compact fiber laser sources. Another area of improvement explored is the resolution of the CARS microscopic setup wherein, based on results from numerical studies, a Bessel like beam was employed as one of the excitation arms in the setup to enhance lateral resolution

Nonlinear Microscopy Techniques: Principles and Biomedical Applications

Nonlinear optical microscopy techniques have emerged as a set of successful tools within the biomedical research field. These techniques have been successfully used to study autofluorescence signals in living tissues, structural protein arrays, and to reveal the presence of lipid bodies inside the tissular volume. In the first section, the nonlinear contrast technique foundations is described, and also, a practical approach about how to build and combine this setup on a single confocal system platform shall be provided. In the next section, examples of the usefulness of these approaches to detect early changes associated with the progression of different epithelial and connective tissular diseases are presented

Clinical cancer diagnosis using optical fiber-delivered coherent anti-stokes ramon scattering microscopy

This thesis describes the development of a combined label-free imaging and analytical strategy for intraoperative characterization of cancer lesions using the coherent anti-Stokes Raman scattering imaging (CARS) technique. A cell morphology-based analytical platform is developed to characterize CARS images and, hence, provide diagnostic information using disease-related pathology features. This strategy is validated for three different applications, including margin detection for radical prostatectomy, differential diagnosis of lung cancer, as well as detection and differentiation of breast cancer subtypes for in situ analysis of margin status during lumpectomy. As the major contribution of this thesis, the developed analytical strategy shows high accuracy and specificity for all three diseases and thus has introduced the CARS imaging technique into the field of human cancer diagnosis, which holds substantial potential for clinical translations. In addition, I have contributed a project aimed at miniaturizing the CARS imaging device into a microendoscope setup through a fiber-delivery strategy. A four-wave-mixing (FWM) background signal, which is caused by simultaneous delivery of the two CARS-generating excitation laser beams, is initially identified. A polarization-based strategy is then introduced and tested for suppression of this FWM noise. The approach shows effective suppression of the FWM signal, both on microscopic and prototype endoscopic setups, indicating the potential of developing a novel microendoscope with a compatible size for clinical use. These positive results show promise for the development of an all-fiber-based, label-free imaging and analytical platform for minimally invasive detection and diagnosis of cancers during surgery or surgical-biopsy, thus improving surgical outcomes and reducing patients' suffering

Looking for a perfect match: multimodal combinations of Raman spectroscopy for biomedical applications

Raman spectroscopy has shown very promising results in medical diagnostics by providing label-free and highly specific molecular information of pathological tissue ex vivo and in vivo. Nevertheless, the high specificity of Raman spectroscopy comes at a price, i.e., low acquisition rate, no direct access to depth information, and limited sampling areas. However, a similar case regarding advantages and disadvantages can also be made for other highly regarded optical modalities, such as optical coherence tomography, autofluorescence imaging and fluorescence spectroscopy, fluorescence lifetime microscopy, second-harmonic generation, and others. While in these modalities the acquisition speed is significantly higher, they have no or only limited molecular specificity and are only sensitive to a small group of molecules. It can be safely stated that a single modality provides only a limited view on a specific aspect of a biological specimen and cannot assess the entire complexity of a sample. To solve this issue, multimodal optical systems, which combine different optical modalities tailored to a particular need, become more and more common in translational research and will be indispensable diagnostic tools in clinical pathology in the near future. These systems can assess different and partially complementary aspects of a sample and provide a distinct set of independent biomarkers. Here, we want to give an overview on the development of multimodal systems that use RS in combination with other optical modalities to improve the diagnostic performance

Development of Coherent Raman Scattering Microscopy for Monitoring Drug Delivery

Topical pharmaceuticals are a vitally important part of modern medicine. Currently, characterising the dermatopharmacokinetics of these drugs is very difficult, and not possible in either real-time, or with a high level of accuracy. This thesis applies three coherent Raman scattering microscopy techniques to the challenge of video-rate monitoring of a porcine skin model undergoing penetration by two different, widely used, pharmaceuticals. It was found that the data taken during these time-course experiments could be used in conjunction with a Beer-Lambert expression, and Fick’s second law, to extract valuable permeation data – namely the skin-solute partition coefficient, and diffusion coefficient – of these pharmaceuticals

Viewing life without labels under optical microscopes

Optical microscopes today have pushed the limits of speed, quality, and observable space in biological specimens revolutionizing how we view life today. Further, specific labeling of samples for imaging has provided insight into how life functions. This enabled label-based microscopy to percolate and integrate into mainstream life science research. However, the use of labelfree microscopy has been mostly limited, resulting in testing for bio-application but not bio-integration. To enable bio-integration, such microscopes need to be evaluated for their timeliness to answer biological questions uniquely and establish a long-term growth prospect. The article presents key label-free optical microscopes and discusses their integrative potential in life science research for the unperturbed analysis of biological samples

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Selective-sampling Raman imaging techniques for ex vivo assessment of surgical margins in cancer surgery

One of the main challenges in cancer surgery is to ensure the complete excision of the tumour while sparing as much healthy tissue as possible. Histopathology, the gold-standard technique used to assess the surgical margins on the excised tissue, is often impractical for intra-operative use because of the time-consuming tissue cryo-sectioning and staining, and availability of histopathologists to assess stained tissue sections. Raman micro-spectroscopy is a powerful technique that can detect microscopic residual tumours on ex vivo tissue samples with accuracy, based entirely on intrinsic chemical differences. However, raster-scanning Raman micro-spectroscopy is a slow imaging technique that typically requires long data acquisition times wich are impractical for intra-operative use. Selective-sampling Raman imaging overcomes these limitations by using information regarding the spatial properties of the tissue to reduce the number of Raman spectra. This paper reviews the latest advances in selective-sampling Raman techniques and applications, mainly based on multimodal optical imaging. We also highlight the latest results of clinical integration of a prototype device for non-melanoma skin cancer. These promising results indicate the potential impact of Raman spectroscopy for providing fast and objective assessment of surgical margins, helping surgeons ensure the complete removal of tumour cells while sparing as much healthy tissue as possible