36 research outputs found

    Engineered nanofluidic platforms for single molecule detection, analysis and manipulation

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    Since the pioneering studies on single ion-channel recordings in 1976, single molecule methods have evolved into powerful tools capable of probing biological systems with unprecedented detail. In this work, we build on the versatility of a type of nanofluidic devices, called nanopipettes, to explore novel modes of single molecule detection and manipulation with the aim of improving spatial and temporal control of biomolecules. In particular, a novel nanopore configuration is presented, where biomolecules were individually confined into a zeptoliter volume bridging two adjacent nanopores at the tip of a nanopipette. As a result of this confinement, the transport of biomolecules such as DNA and proteins was slow down by nearly three orders of magnitude, leading to an improved sensitivity and superior signal-to-noise performances compared to conventional nanopore sensing. Active ways of controlling the transport of biomolecule by combining the advantages of nanopore single-molecule sensing and Field-Effect Transistors are also presented. These hybrid platforms were fabricated in a simple two step process which integrates a gold electrode at the apex of a nanopipette. We show that these devices were effective in modulating the charge density of the nanopore and in actively switching "on" and "off" the transport of DNA through the nanopore. Finally, a nanoscale dielectrophoretic nanotweezer device has been developed for high resolution manipulation and interrogation of individual entities. Two closely spaced carbon nanoelectrodes were embedded at the apex of a nanopipette. Voltage and frequency applied to the electrodes generated a highly localized force capable of trapping and manipulating a broad range of biomolecules. These dielectrophoretic nanotweezers were suitable for probing complex biological environments and a new technique for minimally invasive single-cell nanobiopsy was established. Such study provides encouraging results on how nanopipettebased platforms can be integrated as a future tool for routinely interrogating molecules at the nanoscale.Open Acces

    Méthodes optiques d’attribution d’identifiants moléculaires à des cellules uniques pour assurer leur traçabilité

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    Bien qu’apparues récemment, les technologies de séquençage de cellules uniques ont déjà largement illustré l’immense variabilité observable entre les cellules d’un tissu. Dès lors, nombre de techniques permettant de caractériser un échantillon biologique évoluent rapidement vers l’analyse individuelle de chaque cellule constituant l’échantillon. Cette variabilité est en effet porteuse d’informations déterminantes qui sont perdues lorsqu’une étude est réalisée en moyenne sur des millions de cellules. Or, si l’on exclut la microscopie, la majorité des protocoles applicables aux cellules uniques requiert une homogénéisation de l’échantillon en une suspension de cellules. Dès que la structure de l’échantillon est brisée, toute information concernant des éléments distinctifs facilement observables au microscope tels que la position, la forme, les contacts avec l’environnement, la direction et la vitesse de déplacement de la cellule étudiée est perdue. Ces éléments sont pourtant des descripteurs clefs du développement d’un embryon ou d’une pathologie, des réponses immunitaires, du fonctionnement du système nerveux, de la croissance et de la différenciation cellulaire. L’objectif de ce travail est de développer une approche qui permette d’associer une identité aux cellules d’intérêt afin d’en assurer la traçabilité tout au long des protocoles expérimentaux auxquels elles seront soumises par la suite. Les informations obtenues sur chacune de ces cellules par observation au microscope pourront ainsi être corrélées à celles obtenues lors d’analyses subséquentes. Dans un premier temps, notre but a été d’attacher un marqueur fluorescent à des cellules arbitrairement choisies une à une dans une image de microscope. Nous avons pour cela développé « Cell labeling via photobleaching » (CLaP). Cette méthode repose sur l’utilisation d’un laser pour photoblanchir un fluorophore, ce qui génère un radical libre et permet la liaison d’une biotine à la membrane plasmique des cellules. Cette procédure est non toxique, n’affecte pas le transcriptome des cellules visées et le marquage peut être détecté plusieurs jours après avoir été placé. Nous faisons la démonstration de principe de l’utilisation de CLaP en conjonction avec la plateforme Fluidigm C1TM pour obtenir la séquence de quelques cellules choisies individuellement dans un échantillon. Nous étendons ensuite l’utilisation de cet outil à des échantillons tridimensionnels, ainsi qu’à l’ancrage simultané de plusieurs étiquettes de couleur, et à la génération de liaisons entre les cellules et leur substrat afin de permettre leur isolation. Nous changeons ensuite de paradigme et, plutôt que d’essayer de reconnaître les cellules d’intérêt après avoir séquencé toutes les cellules de l’échantillon, nous cherchons à les isoler. Ces cellules purifiées restent viables et peuvent ensuite être séquencées, réinjectées, cultivées… Pour cela, nous proposons « Single-Cell Magneto-Optical Capture », scMOCA, une adaptation du précédent protocole qui permet de coller des billes magnétiques à la surface de cellules d’intérêt pour les extraire à l’aide d’un simple aimant. Ces manipulations nous ont permis de purifier sans dommages des cellules reconnues très sensibles tels des neurones primaires et des cellules souches embryonnaires. Nous démontrons les capacités de cette procédure en générant des lignées de cellules choisies pour leur capacité exceptionnelle à réparer les dommages induits à l’ADN et ainsi qu’en purifiant des cellules multinucléées jouant un rôle déterminant dans l’apparition des résistances aux médicaments et les récidives de cancer et finalement en extrayant les premières cellules qui se différencient en adipocytes à partir d’une culture de cellules 3T3.Even though they only recently appeared, single cell sequencing techniques have already highlighted huge variability among cells. Since then, numerous techniques arose that allow the characterization of each individual cell from a sample. This variability indeed holds crucial information that is lost when studies average observations across millions of cells. Outside of microscopy, most single cell protocols require the creation of a homogenized cell suspension from the sample. Because the spatial structure of the sample is broken, any information easily obtained with a microscope about shape, position, cell-cell contacts, migration direction and speed is lost. These descriptors are nevertheless key to understanding both embryo and pathology development, immune responses, nervous system functioning and cell growth and differentiation. The objective of this project is to develop a technique that allows giving an identity to cells of interest to trace them across any protocol they might later undergo. This will allow pairing microscopy generated information with that obtained with any downstream analysis. Our first goal was to tether fluorescent markers to cells that were individually chosen in a microcopy image. We developed Cell labeling via photobleaching (CLAP), in which a fluorophore is bleached using a laser to generate a free radical that allows binding a biotin to plasma membranes. This procedure is non-toxic, leaves transcriptomes untouched, and the tag can be found for several days. We show a proof of principle of the use of this technique with the Fluidigm C1TM platform to sequence individually chosen single cells. We then extended this new tool for 3-dimentional samples, for the simultaneous use of multiple color stamps, and for sorting cells by binding them to their substrate. We then considered the problem through a different angle: instead of trying to recognize data originating from single cells of interest after sequencing all cells from a sample, one can try to first isolate these few cells prior to sequencing, reinjecting or further culturing them. To this aim, we propose Single-Cell Magneto-Optical Capture (scMOCA), in which the above protocol is adapted to attach magnetic beads on cell surfaces. Viable cells can then be extracted with high efficiency and purity with a simple magnet; even from populations of very sensitive cells such as primary neurons or embryonic stem cells. Using this procedure, we generated cell lines selected for their outstanding ability to quickly repair induced DNA damage. We then purified multinucleated cells which are involved in the appearance of drug resistance and in cancer relapse and extracted cells that differentiated into adipocytes faster than the rest of the culture

    Label-Free Biosensors for Cell Biology

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    Recent advances in single-cell subcellular sampling

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    Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine

    Detecting uterine cervical cancer cells using molecular biomarkers

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    Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire.Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection

    STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

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    Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

    Nanoimprint Lithography Technology and Applications

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    Nanoimprint Lithography (NIL) has been an interesting and growing field in recent years since its beginnings in the mid-1990s. During that time, nanoimprinting has undergone significant changes and developments and nowadays is a technology used in R&D labs and industrial production processes around the world. One of the exciting things about nanoimprinting process is its remarkable versatility and the broad range of applications. This reprint includes ten articles, which represent a small glimpse of the challenges and possibilities of this technology. Six contributions deal with nanoimprint processes aiming at specific applications, while the other four papers focus on more general aspects of nanoimprint processes or present novel materials. Several different types of nanoimprint processes are used: plate-to-plate, roll-to-plate, and roll-to-roll. Plate-to-plate NIL here also includes the use of soft and flexible stamps. The application fields in this reprint are broad and can be identified as plasmonics, superhydrophibicity, biomimetics, optics/datacom, and life sciences, showing the broad applicability of nanoimprinting. The sections on the nanoimprint process discuss filling and wetting aspects during nanoimprinting as well as materials for stamps and imprinting

    Multi-functional Fluorescence Microscopy via PSF Engineering for High-throughput Super-resolution Imaging

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    Image-based single cell analysis is essential to study gene expression levels and subcellular functions with preserving the native spatial locations of biomolecules. However, its low throughput has prevented its wide use to fundamental biology and biomedical applications which require large cellular populations in a rapid and efficient fashion. Here, we report a 2.5D microcopy (2.5DM) that significantly improves the image acquisition rate while maintaining high-resolution and single molecule sensitivity. Unlike serial z-scanning in conventional approaches, volumetric information is simultaneously projected onto a 2D image plane in a single shot by engineering the fluorescence light using a novel phase pattern. The imaging depth can be flexibly adjusted and multiple fluorescent markers can be readily visualized. We further enhance the transmission efficiency of 2.5DM by ~2-fold via configuring the spatial light modulator used for the phase modulation in a polarization-insensitive manner. Our approach provides a uniform focal response within a specific imaging depth, allowing to perform quantitative high-throughput single-molecule RNA measurements for mammalian cells over a 2 x 2 mm2 region within an imaging depth of ~5 µm in less than 10 min and immunofluorescence imaging at a volumetric imaging rate of \u3e 30 Hz with significantly reduced light exposure. With implementation of an adaptive element, our microscope provides an extra degree of freedom in correcting aberrations induced by specimens and optical components, showing its capability of imaging thick specimens with high-fidelity of preserving volumetric information with fast imaging speed. We also demonstrate multimodal imaging that can be switched from 2.5DM to a 3D single-molecule localization imaging platform by encoding the depth information of each emitter into the shape of point spread function, which enables us to obtain a resolution of \u3c 50 nm. Our microscope offers multi-functional capability from fast volumetric high-throughput imaging, multi-color imaging to super-resolution imaging
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