Cellular level nanomanipulation using atomic force microscope aided with superresolution imaging

Atomic force microscopes (AFM) provide topographical and mechanical information of the sample with very good axial resolution, but are limited in terms of chemical specificity and operation time-scale. An optical microscope coupled to an AFM can recognize and target an area of interest using specific identification markers like fluorescence tags. A high resolution fluorescence microscope can visualize fluorescence structures or molecules below the classical optical diffraction limit and reach nanometer scale resolution. A stimulated emission depletion (STED) microscopy superresolution (SR) microscope coupled to an AFM is an example in which the AFM tip gains nanoscale manipulation capabilities. The SR targeting and visualization ability help in fast and specific identification of subdiffraction-sized cellular structures and manoeuvring the AFM tip onto the target. We demonstrate how to build a STED AFM and use it for biological nanomanipulation aided with fast visualization. The STED AFM based bionanomanipulation is presented for the first time in this article. This study points to future nanosurgeries performable at single-cell level and a physical targeted manipulation of cellular features as it is currently used in research domains like nanomedicine and nanorobotics

Imaging amyloid fibers at the nanoscale: Method development and applications for hybrid materials and biomedicine

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 13-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 13-06-2021In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the nanoscale, enabling the observation of structural and molecular components. Different imaging tools are specialized in the characterization of a specific aspect of the sample and, when they are combined, complementary information is obtained providing a more comprehensive understanding of the system. This thesis focuses on the application of (super-resolution) fluorescence microscopy in combination with atomic force microscopy (AFM) for revealing specific chemical information in a high-resolution topography map. Particularly, correlative microscopy is applied to the characterization of amyloid fibers, which are misfolded protein aggregates with interest in nanomaterials research and biomedicine. This manuscript is organized in seven chapters. Chapter 1 introduces the imaging techniques used in the thesis. It also gives a general overview on amyloid fibers, their application as hybrid materials, their importance in biomedicine for being involved in different diseases, and the phototherapeutic approaches available to treat them. In Chapter 2, the general materials and methods used during the thesis are explained. Chapter 3 provides a detailed discussion about technical aspects of correlative super-resolution fluorescence microscopy and AFM such as sample preparation, data analysis and image alignment. Furthermore, the advantage of using AFM as a “ground truth” to evaluate different aspects of super-resolution techniques, such as labeling or image reconstruction, is highlighted. In Chapter 4, the methodology developed in Chapter 3 is applied to evaluate the functionalization of amyloid fibers with quantum dots or organic fluorophores. Thus, correlative microscopy is presented as a useful technique for characterizing luminescent hybrid materials at the nanoscale. In the context of biomedicine, amyloid aggregates are important for being involved in different diseases (e.g. Alzheimer or Parkinson). Photochemical strategies to degrade amyloid structures are becoming an interesting alternative. In this thesis, a thioflavin T (ThT) derivative (ROS-ThT), which is able to target pathogenic aggregates in the presence of functional proteins, is used to study photodamage effects on amyloid fibers. In addition to fluorescence, this photocatalyst or photosensitizer produces singlet oxygen Abstract upon blue light exposure, affecting amyloid structures through oxidation. The purpose of Chapter 5 is to select a useful amyloid model to evaluate photodamage at the nanoscale, and therefore different fibers were produced, fibrillated and characterized. In Chapter 6, the selected amyloid model is used to study photodamage induced by ROS-ThT at the single-fiber level through imaging techniques, and complemented by classical biochemical assays. These experiments highlight that the combination of fluorescence microscopy and AFM is useful to probe the heterogeneity of amyloid material and to disentangle the complex dependence between photocatalyst binding/activity and fiber morphology and/or composition. The aim of Chapter 7 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods may impact the study of amyloids in different fields of researchQuiero agradecer también al Ministerio de Economía y Competitividad por financiar mi trabajo con la beca FPI BES-2016-076293 dentro del proyecto MAT2015-66605-P y al Ministerio de Ciencia, Innovación y Universidades por financiar el proyecto PCI2018‐093064

Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences

Correlating data from different microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted by the Abbe diffraction limit. We detail system integration and demonstrate system performance using sub-resolution fluorescent beads and applied to a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1) tagged with an enhanced green fluorescent protein (EGFP) at the N-terminal.Ana I. Gomez Varela wishes to acknowledge support from the Xunta de Galicia, Conselleria de Cultura, Educacion e Ordenacion Universitaria e da Conselleria de Economia, Emprego e Industria (Programa de axudas de apoio a etapa de formacion posdoutoral 2017). Adelaide Miranda and Pieter De Beule acknowledge financial support from Norte's Regional Operational Programme 2014-2020-Norte2020 (NORTE-01-0145-FEDER-000019). Sandra Paiva and Rosana Alves thank Fulbright Commission Portugal and Luso-American Development Foundation (FLAD) for their financial support to perform research work at UC Berkeley, California, USA. We thank the U.S. Embassy in Portugal for supporting David Drubin's visit to Portugal. We thank Ann Fisher of the UC Berkeley Cell Culture Facility for help with cell culture. We thank Dr. Kartoosh Heydari of the Cancer Research Lab Flow Cytometry Core Facility of UC Berkeley. Rosana Alves and Claudia Barata-Antunes are recipients of PhD fellowships from the Portuguese Foundation for Science and Technology (PD/BD/113813/2015 and PD/BD/135208/2017, respectively). The authors want to thank Nikon and Izasa Scientific for their support to the experiment by providing a N SIM-E microscope set-up on loan. We also gratefully acknowledge Dr. Kees van der Oord from Nikon Instruments Europe B.V. for his assistance with the SIM microscope as well as Paulo Madureira and Carlos Pitaes from IZASA Portugal and Jordi Recasens from IZASA Spain for their assistance with the integration of the SIM and AFM microscopes. Finally, we want to thank Benjamin Holmes for fruitful discussions and relentless support

Optical nanoscopy

AbstractThis article deals with the developments of optical microscopy towards nanoscopy. Basic concepts of the methods implemented to obtain spatial super-resolution are described, along with concepts related to the study of biological systems at the molecular level. Fluorescence as a mechanism of contrast and spatial resolution will be the starting point to developing a multi-messenger optical microscope tunable down to the nanoscale in living systems. Moreover, the integration of optical nanoscopy with scanning probe microscopy and the charming possibility of using artificial intelligence approaches will be shortly outlined

Dissection of DNA double-strand-break repair using novel single-molecule forceps.

Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity

Bioimage informatics in STED super-resolution microscopy

Optical microscopy is living its renaissance. The diffraction limit, although still physically true, plays a minor role in the achievable resolution in far-field fluorescence microscopy. Super-resolution techniques enable fluorescence microscopy at nearly molecular resolution. Modern (super-resolution) microscopy methods rely strongly on software. Software tools are needed all the way from data acquisition, data storage, image reconstruction, restoration and alignment, to quantitative image analysis and image visualization. These tools play a key role in all aspects of microscopy today – and their importance in the coming years is certainly going to increase, when microscopy little-by-little transitions from single cells into more complex and even living model systems. In this thesis, a series of bioimage informatics software tools are introduced for STED super-resolution microscopy. Tomographic reconstruction software, coupled with a novel image acquisition method STED< is shown to enable axial (3D) super-resolution imaging in a standard 2D-STED microscope. Software tools are introduced for STED super-resolution correlative imaging with transmission electron microscopes or atomic force microscopes. A novel method for automatically ranking image quality within microscope image datasets is introduced, and it is utilized to for example select the best images in a STED microscope image dataset.Siirretty Doriast

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies

AFM-STED correlative nanoscopy provides a new view on the formation process of misfolded protein aggregates

The main part of my PhD work focused on the application of an advanced integrated technique, based on the coupling of an atomic force microscope (AFM) and a stimulated emission depletion (STED) microscope in the study of amyloid fibrils formation. This coupled system allows the acquisition of super-resolution fluorescence images, perfectly overlapped with AFM topography. Exploiting the extended capability offered by this technique, I highlighted some important features on the mechanisms followed by the labeled and unlabeled proteins through their aggregation pathway. The results demonstrates that labeled molecules are involved only in selected pathways of aggregation, among the multiple that are present in the aggregation reaction. In a second part of my work, I investigated the process of interaction between Alpha-synuclein (\u3b1-Syn), the pathological peptide associated to the Parkinson\u2019s disease, and model lipid membranes. The aim of this study was to identify molecular mechanisms that are indicated as the base of neurodegeneration, not only in Parkinson\u2019s disease, but also in a large class of disorders, indicated as protein misfolding diseases

Data-driven microscopy: placing high-fidelity data in a population-wide context

Mikroskopi är idag ett fundamentalt verktyg inom forskning, där det tillåter oss att skåda in och utforska våra prover i hög detalj. Mycket utav utvecklingen av nya mikroskopimetoder har strävat efter att öka den detaljnivå vi kan uppnå. Samtidigt har utvecklingen inom hårdvara, med tillgång till bättre och mer kraftfulla instrument, lett till utveckligen av metoder där fokuset är att studera en hel population av celler. Till skillnad från när vi studerar ett fåtal celler i hög detalj, tillåter det oss att sätta perspektiv på det vi ser. Det ger oss en förmåga att säga vad det normala beteendet som man kan förvänta sig är, och vilka celler som sticker ut i en population. Med andra ord, vad som är intressant.Samtidigt finns det ett stort intresse av att veta hur varje individuell cell beter sig. Varje cell är, precis som oss människor, unik. De har olika historia, olika ålder och befinner sig i olika tillstånd. Precis som våra celler i kroppen är unika, är även de cellerna som kan orsaka sjukdom unika. För att förstå varför vissa personer är mer känsliga mot sjukdom, och hur en infektion svarar på våra behandlingar behövs en förståelse och an förmåga att studera celler på individuell nivå, samtidigt som vi bibehåller ett perspektiv utifrån populations-nivå.Denna brist på perspektiv har länge varit ett problem inom mikroskopi. Den vanliga lösningen på detta problem är att vi, som människor, kan tolka en bild och peka på vad det är som är intressant eller inte. Vi är, trots allt, extremt duktiga på att tolka visuell information. Men detta är inte en helt felfri lösning. Som människor kan vi vara relativt okonsekventa, vi tolkar oftast utifrån hur vi vill att datan ser ut. Med andra ord, vi saknar förmågan att vara objektiva i vår metodik för att samla in bilder i hög detalj.Min avhandling har till stor del handlat om att utveckla ett verktyg som tillåter oss att sätta perspektiv på det vi studerar med mikroskopi. Detta har lett till Arbete 1, där vi presenterar en allmän strategi (data-styrd mikroskopi) för hur vi kan arbeta med mikroskopi för att samla in data på en hel population, samtidigt som vi kan samla in data med hög detalj på relevanta fynd i populationen. Vi presenterar även här en teknisk lösning, och utför metoden i tre olika scenarion: ett för att studera en population av celler mer allmänt, ett för att fånga det ögonblick som bakterier infekterar mänskliga celler, och ett där vi studerar och fångar in data på relevanta (från ett populations-kontext) cancerceller och följer dem över tid. Denna metod tillåter oss att samla in data i hög detalj på ett objektivt sätt, och att sätta perspektiv på det vi studerar.I Arbete 2 har vi vidare utvecklat på vår metod, där vi försöker lösa problemet att hitta en och samma cell i flera olika mikroskop. Eftersom vi, genom mikroskopi, jobbar på en så ofantligt liten skala, är det oftast väldigt svårt att orientera sig och hitta rätt inom ett prov. Det är lite som att spela På spåret och gissa vart man är, fast utan alla ledtrådar man får på varje nivå. Eftersom vi har tillgång till data på en hel population, så utgick vi från att det borde finnas samband mellan celler och deras grannar i ett prov som är unika för just dem. Genom att använda sig av dessa unika samband kom vi fram med en lösning där vi snabbt kan kalibrera ett prov på ett nytt mikroskop. Det öppnar dörrarna för oss forskare att återanvända prov, att lättare justera provet med nya markörer (för det vi vill visualisera inom cellerna), och att kunna tolka ett prov med data insamlat från flera system.COVID-19 pandemin var en stor omställning för samhället och vården. Likväl var det en stor omställning för många forskningslabb, där en kapplöpning startade för att så snabbt som möjligt förstå sig på hur viruset fungerar och hur vårt immunförsvar svarar på dess infektion. Det var i detta kontext som mitt tredje arbete utfördes. Genom den erfarenhet jag samlat på mig inom mikroskopi och att analysera bilder på stora dataset, bidrog jag med hjälp för att studera hur framtagna antikroppar kan förhindra bindningen av virus-lika partiklar till celler. Antikroppar är ett protein som immunförsvaret producerar i respons mot en patogen. En bättre förståelse kring hur antikroppar verkar, och vad skillnaden mellan en bra och en dålig antikropp är kan leda till framtagningen av bättre vaccin-program och behandlingar inom sjukvården.I Arbete 4 medverkade jag i ett arbete där bakterien Streptococcus pyogenes var i fokus. S. pyogenes enda värd är människor, och ansvarar för över 600 miljoner infektionsfall per år globalt. På bakteriens yta dominerar ett protein, M-proteinet, ett multi-funktionellt protein som bakterien (bland annat) använder sig för att binda till ytor och förhindra immunförsvarets förmåga att göra sig av med bakterien. I arbetet upptäckte vi att fibronektin binder till bakterien (specifikt M-proteinet) olika mycket beroende på mängden antikroppar som finns i miljön. Fibronektin är ett protein som vi människor producerar, och bidrar (bland annat) till att skapa den miljön som celler befinner sig i. Mängden fibronektin varierar beroende på var i kroppen man kollar. Till exempel, i saliv har du en relativt låg mängd fibronektin jämfört med i blodet. Detta ledde till hypotesen att bakterien är special-anpassad för olika miljöer i dess förmåga att undkomma immunförsvaret. En bättre förståelse kring hur bakterien är anpassad till våra olika miljöer och dess infektionsförlopp kan leda till bättre och mer anpassade behandlingar inom sjukvården

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo