Demixing fluorescence time traces transmitted by multimode fibers

Fiber photometry is a significantly less invasive method compared to other deep brain imaging microendoscopy approaches due to the use of thin multimode fibers (MMF diameter $<$ 500 $\mu$m). Nevertheless, the transmitted signals get scrambled upon propagation within the MMF, thus limiting the technique's potential in resolving temporal readouts with cellular resolution. Here, we demonstrate how to separate the time trace signals of several fluorescent sources probed by a thin ($\approx$ 200 $\mu$m) MMF with typical implantable length in a mouse brain. We disentangled several spatio-temporal fluorescence signals by using a general unconstrained non-negative matrix factorization (NMF) algorithm directly on the raw video data. Furthermore, we show that commercial and low-cost open-source miniscopes display enough sensitivity to image the same fluorescence patterns seen in our proof of principle experiment, suggesting that a whole new avenue for novel minimally invasive deep brain studies with multimode fibers in freely-behaving mice is possible.Comment: Main text: 13 pages, 4 Figures. Supp info: 9 pages, 8 Figure

Mechanisms of molecular interaction between antimicrobial polyelectrolytes and membrane models by nonlinear vibrational spectroscopy

Pesquisa em novas moléculas e estratégias antimicrobianas é crucial devido ao aumento de resistência a antibióticos pelos microrganismos. Polímeros antimicrobianos tem várias vantagens quando comparados a outros biocidas pequenos: maiores tempo de vida, potência, especificidade e baixa toxicidade residual. Logo, outras aplicações tecnológicas como recobrimentos, embalagens ou produtos têxteis antimicrobianos poderem ser exploradas. Em particular, derivados hidrossolúveis de quitosana, como os oligômeros de quitosana (OQ), são biopolímeros catiônicos extraídos de fontes renováveis que são candidatos promissores a serem agentes antimicrobianos de amplo espectro (fungos, bactérias gram-positivas e bactérias gram-negativas). Diferentemente da quitosana, que é sobretudo bioativa em pHs ácidos, OQ permanece catiônico &ndash; e portanto ativo &ndash; em pH fisiológico. Não obstante, o mecanismo exato pelo qual o polímero age nas membranas celulares permanece desconhecido em nível molecular. Este trabalho visa investigar o mecanismo de interação entre os OQ e modelos de membrana biomiméticos (Filmes de Langmuir). Para comparação, outro polieletrólito catiônico sintético com propriedades antibacterianas, o PAH &ndash; poli(hidrocloreto de alilamina) &ndash; foi investigado. Nós realizamos a Espectroscopia por Geração de Soma de Frequência (SFG) em Filmes de Langmuir de fosfolipídeos em água pura e em subfases contendo antimicrobianos. A Espectroscopia SFG nos permite obter o espectro vibracional de moléculas interfaciais (filme lipídico e moléculas que estão interagindo com ele: água e antimicrobianos) sem nenhuma contribuição de moléculas do interior do volume e é muito sensível às conformações lipídicas da membrana. Um fosfolipídeo zwitteriônico (DPPC) foi usado para modelar membranas tipo-humana, enquanto outro carregado negativamente (DPPG) modelava a tipo-bacteriana. Isotermas em subfases contendo antimicrobianos mostraram que ambos PAH e OQ causam uma pequena expansão das monocamadas de DPPC. Entretanto, para as monocamadas de DPPG ambos os polieletrólitos geraram uma expansão significativa. Entre eles, os OQ causaram um efeito mais drástico. Espectros SFG dos estiramentos CH mostraram que a conformação lipídica permaneceu bem empacotada em todos os casos (ligeiramente menos ordenada com PAH) apesar das expansões da membrana. Isto indica que os OQ foram inseridos formaram ilhas de OQ dentro do filme lipídico. Mudanças na forma de linha dos estiramentos da água interfacial indicaram que a adsorção de PAH em ambos os filmes foram capazes de compensar as cargas negativas, gerando uma inversão de cargas na superfície. Os espectros SFG dos grupos fosfato também indicaram que, em água pura, as cabeças polares de DPPC estão com uma orientação mais ordenada do que no caso do DPPG. Contudo, quando interagindo com os polieletrólitos catiônicos, as cabeças dos DPPGs se ordenam, ficando preferencialmente perpendicular à interface. Experimentos com antimicrobianos injetados na subfase enquanto os filmes de Langmuir já estavam condensados indicaram que os OQ foram capazes de penetrar na monocamada, embora causando uma expansão no filme menor. Esta comparação evidencia que a escolha da metodologia experimental afeta o resultado, mas ambas podem ser complementares, visto que podem representar diferentes fases do ciclo celular das biomembranas. A visão detalhada provida aqui para as interações moleculares desses polieletrólitos com filmes lipídicos podem os elucidar mecanismos de atividade biocida deles e auxiliar no planejamento racional de novos polímeros antimicrobianos.Research on new antimicrobial molecules and strategies is crucial due to the increasing microorganism resistance to antibiotics. Antimicrobial Polymers have many advantages when compared to other small biocides: increased lifetimes, potency, specificity and lower residual toxicity. Therefore, they have great potential for technological applications, such as antimicrobial coatings, packages, or textile products. In particular, water-soluble derivatives of chitosan, such as chitosan oligomers (CO), are cationic biopolymers obtained from renewable sources that are promising candidates to a wide-spectrum antimicrobial agent (fungi, gram positive and gram negative bacteria). Unlike chitosan, which is mainly bioactive at acidic pH, CO remain cationic - and therefore active - at physiological pH. Nevertheless, the exact mechanism by which this polymer acts on the cell membranes remains unknown at the molecular level. This work aims at investigating the molecular interaction between CO and a biomimetic cell membrane model (Langmuir Film). For comparison, another synthetic cationic polylelectrolyte with antibacterial properties, PAH &ndash; poly(alylamine hydrochloride), has been investigated. We have carried out Sum-Frequency Generation (SFG) Spectroscopy on Langmuir Films of phospholipids on pure water and on antimicrobial containing subphases. SFG Spectroscopy allows obtaining the vibrational spectrum of interfacial molecules (lipid Langmuir Film and molecules interacting with it &ndash; water and antimicrobials), without any contribution from the bulk molecules, and is quite sensitive to the conformation of membrane lipids. A zwitterionic phospholipid (DPPC) was used to model human-like membranes, while a negatively charged phospholipid (DPPG) modeled bacterial-like membranes. Surface pressure-area isotherms on antimicrobial-containing subphases showed that both PAH and CO led to a small expansion of DPPC monolayers. However, for DPPG monolayers both polyelectrolytes led to significant expansion, with CO causing a more dramatic effect. SFG spectra in the CH stretch range showed that the lipid chain conformation remained always well ordered in all cases (slightly less ordered upon interacting with PAH), despite membrane expansion. This indicates that CO were inserted in the monolayer, forming islands of CO within the lipid film. Changes in the SFG spectral lineshape of OH stretches for the interfacial water molecules indicated that PAH adsorption on both DPPC and DPPG films was able to overcompensate the lipid negative charge and led to an overall surface charge reversal. The SFG spectra of the phosphate groups also indicated that in pure water the DPPC headgroups had a more ordered orientation than in the case of DPPG. Nevertheless, upon interaction with the cationic polyelectrolytes, the DPPG headgroups also become ordered, with a preferential orientation towards the subphase. Experiments with the antimicrobials injected in the subphase under a condensed Langmuir film indicated that CO were also capable of monolayer penetration, albeit causing a reduced film expansion. This comparison indicates that the choice of experimental methodology affects the outcome, but both may be complementary, as they may represent different phases of a biomembrane lifecycle. The detailed view provided here for the molecular interaction of these polyelectrolytes with lipid films may shed light on the mechanism of their biocidal activity and aid on a rational design of new antimicrobial polymers

Structure à l'échelle nanométrique des filaments d'actine sondée par microscopie polarisée à super-résolution

Actin filaments are key structural building blocks that affects cell behavior and function. Although they are extensively studied, open questions still arise on how their organization at the nanoscale influences cell morphology and processes. An important actin assembly is stress fibers, which are supramolecular bundles made of F-actin filaments and crosslinkers that have an important role in cell adhesion and motility. Actin stress fibers bear cellular forces, and can be subdivided into different categories, in particular those which are contractile and those which are not. How actin filaments are organized in different stress fiber categories at the nanoscale is a topic still not well understood. To that end, polarized fluorescence microscopy (PFM) techniques can be very useful once they can provide subdiffraction quantitative information of the molecular organization with high specificity in cells. In this thesis, we build up an optical microscope system capable of performing two different PFMs methods to study the organization of actin filaments at the nanoscale. The first PFM method, is a replica of a previously built system in our group, which is an ensemble averaged polarization resolved technique. The latter uses a fast polarization rotation performed by a Pockels cell placed in the excitation path, and a multi-focus parallel confocal scanning for fast imaging (spinning disk). The second PFM method is a novel polarized super-resolution method that we named 4polar-dSTORM. The method is based on direct Stochastic Optical Reconstruction Microscopy (dSTORM) method that splits single-molecule fluorescence signals into 4 projected polarization channels. With the latter, we were able to measure each single-fluorophore mean orientation (along a bundle) independently of the fluorophore wobbling (angular fluctuations). Thanks to that, we were able to perform the first 2D unbiased quantification of the actin filament static order at the nanoscale with an optical resolution up to approximately 20nm. With these techniques, we were able to conclude that actin filament organization is quite constant (within angular deviations of about 20° to 30°) along all stress fiber categories and they are highly stable structures upon contractility perturbation or stimuli. Interestingly, since 4polar-dSTORM is a single-molecule based method, other actin complex structures such as very dense nanoscale actin filament meshworks (e.g., in lamellipodia) could be studied, illustrating the potential of the method to investigate unknown organizations.Les filaments d'actine sont des éléments structurels clés qui affectent le comportement et la fonction des cellules. Bien qu'ils soient largement étudiés, des questions restent ouvertes sur la manière dont leur organisation à l'échelle nanométrique influence la morphologie et les processus cellulaires. Un assemblage d'actine important est celui des fibres de stress, qui sont des faisceaux supramoléculaires constitués de filaments de F-actine et de réticulants qui jouent un rôle important dans l'adhésion et la motilité des cellules. Les fibres de stress de l'actine supportent des forces cellulaires et peuvent être subdivisées en différentes catégories, en particulier celles qui sont contractiles et celles qui ne le sont pas. La manière dont les filaments d'actine sont organisés en différentes catégories de fibres de stress à l'échelle nanométrique est un sujet encore mal compris. À cette fin, les techniques de microscopie à fluorescence polarisée (PFM) peuvent être très utiles dès lors qu'elles peuvent fournir des informations quantitatives de sous-diffraction de l'organisation moléculaire avec une grande spécificité dans les cellules. Dans cette thèse, nous construisons un système de microscope optique capable de réaliser deux méthodes PFM différentes pour étudier l'organisation des filaments d'actine à l'échelle nanométrique. La première méthode PFM, est une réplique d'un système précédemment construit dans notre groupe, qui est une technique de résolution de polarisation moyenne d'ensemble. Cette dernière utilise une rotation rapide de la polarisation effectuée par une cellule de Pockels placée dans le trajet d'excitation, et un balayage confocal parallèle multi-focal pour une imagerie rapide (disque rotatif). La seconde méthode PFM est une nouvelle méthode de super-résolution polarisée que nous avons appelée 4polar-dSTORM. Cette méthode est basée sur la méthode de microscopie à reconstruction optique stochastique directe (dSTORM) qui divise les signaux de fluorescence monomoléculaire en 4 canaux de polarisation projetés. Avec ce dernier, nous avons pu mesurer chaque orientation moyenne d'un fluorophore unique (le long d'un filament) indépendamment de l'ondulation du fluorophore (fluctuations angulaires). Grâce à cela, nous avons pu effectuer la première quantification 2D non biaisée de l'ordre statique du filament d'actine à l'échelle nanométrique avec une résolution optique allant jusqu'à environ 20 nm. Grâce à ces techniques, nous avons pu conclure que l'organisation des filaments d'actine est assez constante (avec des écarts angulaires d'environ 20° à 30°) le long de toutes les catégories de fibres de stress et qu'il s'agit de structures très stables en cas de perturbation de la contractilité ou de stimuli. Il est intéressant de noter que, puisque la méthode 4polar-dSTORM est basée sur une seule molécule, d'autres structures complexes d'actine telles que des maillages de filaments d'actine très denses à l'échelle nanométrique (par exemple dans les lamellipodes) pourraient être étudiées, ce qui illustre le potentiel de la méthode pour étudier des organisations inconnues

Super resolution polarized imaging of the organization of protein assemblies in cells

International audienc

Super resolution polarized imaging of the organization of protein assemblies in cells

International audienc

Demixing fluorescence time traces transmitted by multimode fibers

Abstract Optical methods based on thin multimode fibers (MMFs) are promising tools for measuring neuronal activity in deep brain regions of freely moving mice thanks to their small diameter. However, current methods are limited: while fiber photometry provides only ensemble activity, imaging techniques using of long multimode fibers are very sensitive to bending and have not been applied to unrestrained rodents yet. Here, we demonstrate the fundamentals of a new approach using a short MMF coupled to a miniscope. In proof-of-principle in vitro experiments, we disentangled spatio-temporal fluorescence signals from multiple fluorescent sources transmitted by a thin (200 µm) and short (8 mm) MMF, using a general unconstrained non-negative matrix factorization algorithm directly on the raw video data. Furthermore, we show that low-cost open-source miniscopes have sufficient sensitivity to image the same fluorescence patterns seen in our proof-of-principle experiment, suggesting a new avenue for novel minimally invasive deep brain studies using multimode fibers in freely behaving mice

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

International audienceSingle-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules’ orientation in addition totheir localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules’ localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility

Semifluorinated thiols in Langmuir monolayers : a study by nonlinear and linear vibrational spectroscopies

A series of semifluorinated thiols of the general formula CmF2m+1CnH2nSH (abbr. FmHnSH) have been synthesized and characterized in Langmuir monolayers with surface pressure-area isotherms, complemented with polarization-modulated reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation (SFG) techniques. A comparative analysis was performed for compounds having the same length of fluorinated segment (F10) and variable length of the hydrogenated part (H6, H10, H12), and having identical hydrogenated segment (H12) connected to a fluorinated moiety of different lengths (F6, F8, F10). For the sake of comparison, an alkanethiol (H18SH) was also examined, and F10H10COOH and F10H10OH molecules were used for helping the assignment of SFG spectra of CH stretches. SFG was applied to investigate the hydrocarbon chain and the terminal CF3 group, while PM-IRRAS was used to probe CF2 groups. The number of gauche defects in the hydrocarbon chain increased with the increasing length of the molecule, either by elongation of the hydrogenated or perfluorinated part. SFG measurements recorded at three polarization combinations (ppp, ssp, sps) enabled us to estimate the tilt angle of the terminal CF3 group in semifluorinated thiol molecules as ranging from 35° to 45°, which is consistent with nearly vertical fluorinated segments. Upon increasing the surface pressure, the fluorinated segment gets slightly more upright, but the hydrocarbon chain tilt increases while keeping the same average number of gauche defects. The extent of disorder in the hydrogenated segment may be controlled by varying the size of the fluorinated segment, and this could be exploited for designing functionalized surfaces with insertion of other molecules in the defect region