Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Astigmatic traction force microscopy (aTFM).

Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity.

Extended mechanical force measurements using structured illumination microscopy.

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.

Cytoskeletal actin dynamics are crucial for the activation of T-cells. Immortalised Jurkat T-cells have been the model system of choice to examine and correlate the dynamics of the actin cytoskeleton and the immunological synapse leading to T-cell activation. However, it has remained unclear whether immortalised cellular systems, such as Jurkat T-cells can recapitulate the cytoskeletal behaviour of primary T-cells. Studies delineating the cytoskeletal behaviour of Jurkat T-cells in comparison to primary T-cells are lacking. Here, we employ live-cell super-resolution microscopy to investigate the cytoskeletal actin organisation and dynamics of living primary and immortalised Jurkat T-cells at the appropriate spatiotemporal resolution. Under comparable activation conditions, we found differences in the architectural organisation and dynamics of Jurkat and primary mouse and human T-cells. Although the three main actin network architectures in Jurkat T-cells were reminiscent of primary T-cells, there were differences in the organisation and molecular mechanisms underlying these networks. Our results highlight mechanistic distinctions in the T-cell model system most utilised to study cytoskeletal actin dynamics.

Simultaneous Quantification of the Interplay Between Molecular Turnover and Cell Mechanics by AFM-FRAP.

Quantifying the adaptive mechanical behavior of living cells is essential for the understanding of their inner working and function. Yet, despite the establishment of quantitative methodologies correlating independent measurements of cell mechanics and its underlying molecular kinetics, explicit evidence and knowledge of the sensitivity of the feedback mechanisms of cells controlling their adaptive mechanics behavior remains elusive. Here, a combination of atomic force microscopy and fluorescence recovery after photobleaching is introduced offering simultaneous quantification and direct correlation of molecule kinetics and mechanics in living cells. Systematic application of this optomechanical atomic force microscopy-fluorescence recovery after photobleaching platform reveals changes in the actin turnover and filament lengths of ventral actin stress fibers in response to constant mechanical force at the apical actin cortex with a dynamic range from 0.1 to 10 nN, highlighting a direct relationship of active mechanosensation and adaptation of the cellular actin cytoskeleton. Simultaneous quantification of the relationship between molecule kinetics and cell mechanics may thus open-up unprecedented insights into adaptive mechanobiological mechanisms of cells.

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy.

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells.

Bacterial cell wall nanoimaging by autoblinking microscopy.

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this 'autoblinking' phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.

Parasitosis intestinales y su relación con factores socioeconómicos y condiciones de habitat en niños de Neuquén, Patagonia, Argentina / Intestinal parasitosis in relation to socioeconomic factors and habitat conditions in children of Neuquen, Patagonia, Argentina

Se investigó la prevalencia y distribución de parásitos intestinales (PI) en niños de 2 poblaciones de diferente nivel socioeconómico del área periurbana de la ciudad de Neuquén (Sectores I y II) a fin de evaluar su relación con las condiciones de hábitat y factores socio-económicos. Se procesaron muestras seriadas de materia fecal y de escobillado anal de 126 niños entre 2 y 14 años de edad. Se registraron datos acerca de condiciones de hábitat y factores socioeconómicos mediante visitas domiciliarias y encuestas observaciones estructuradas. Se detectó presencia de PI en el 50,7 por ciento de los niños del Sector I (barrio suburbano con adecuadas condiciones sanitarias y nivel socioeconómico medio o medio-bajo) y en el 92,9 por ciento de los niños del Sector II (asentamiento marginal con deficientes condiciones sanitarias y bajo nivel socioeconómico). Se identificaron 7 especies de protozoos intestinales y 4 especies de helmintos. Blastocystis hominis fue la especie más frecuente encontrada en ambas poblaciones. No se encontraron helmintos diferentes de Enterobius vermicularis en el Sector I y la prevalencia de tales especies fue muy baja en el Sector II. Las condiciones de hábitat deficientes y los bajos parámetros socioeconómicos se relacionaron con una mayor prevalencia de PI de transmisión directa como protozoos y E. vermicularis en las poblaciones estudiadas. Sin embargo, aún en ese contexto favorable a la transmisión, las especies parasitarias que requieren estadíos intermedios de maduración en el suelo no encuentran un hábitat adecuado para su diseminación en esta región patagónica.

The prevalence and distribution of intestinal parasites (IP) were investigated in children from two populations of different socioeconomic level, located in the same area of the city of Neuquén, in order to evaluate their relationship with habitat conditions and socioeconomic factors. Serial samples of faeces and anal scraping of 126 children between 2 and 14 years from two sectors of the suburban area of Neuquen (Sector I and Sector II) were analyzed. Data concerning habitat conditions and socioeconomic parameters were obtained by home visits and an observational structured survey. Presence of IP was detected in 50.7% of children from Sector I (suburban neighborhood with adequate sanitary conditions and middle or middle low socioeconomic level) and in 92.9% from children of Sector II (marginal settlement with poor sanitary conditions and low socioeconomic status). Seven intestinal protozoan and 4 helminth species were identified. Blastocystis hominis was the most frequent species found in both populations. No helminths different from Enterobius vermicularis were found in Sector I and the prevalence of such species was very low in Sector II. Deficient habitat conditions and low socioeconomic parameters showed relation with a higher prevalence of IP of direct transmission as protozoan and E.vermicularis in the studied populations. Nevertheless, even in this context favourable to transmission, the parasitic species which require intermediate stages of development in soil, don't find an adequate habitat for dissemination in this region

[Estacionalidad de parásitos intestinales en suelos periurbanos de la ciudad de Neuquén, Patagonia, Argentina]. / Estacionalidad de parásitos intestinales en suelos periurbanos de la ciudad de Neuquén, Patagonia, Argentina.

OBJECTIVE: To determine the presence of eggs, larva, cysts and oocysts of intestinal parasites in the soil of a suburb of Neuquén city during 1 year in order to evaluate their seasonal fluctuations in relation to climatic data and soil characteristics in the studied area. METHODS: A total of 107 soil samples were processed for parasite isolation by sedimentation and flotation methods during the four seasons of the year. Meteorological data were registered and physical, chemical and structural characteristics of the soil were analysed. RESULTS: About 28.9% of the soil samples were positive for at least one parasite form. Six protozoa species (cysts of Entamoeba sp., Enteromonas sp., Endolimax sp., Giardia sp., Iodamoeba sp. and coccidia oocysts) were recovered, but neither larvae nor eggs of human or animal helminths parasites were detected. The percentage of contaminated soil samples and the diversity of species showed a marked decrease in the warm and dry months of the summer. The soil was sandy, without vegetable cover, well drained, and with scarce organic matter content. CONCLUSION: The frequencies of parasite recovery and the number of species show seasonal fluctuations related to the rainfall. The importance of soil as a risk factor for the transmission of intestinal parasites in the studied area is conditioned by its structural characteristics, which prevent retaining the humidity, and by climatic variables. The interrelation of both factors determines unfavourable conditions that could explain the low level of contamination observed in soil as well as the absence of eggs and helminth larvae.

Contaminación ambiental generada por la coexistencia de actividades agrícolas y petroleras en una área del periurbano neuquino. Análisis critico de la gestión ambiental

El presente trabajo es un estudio realizado con el objeto de determinar el grado de compromiso ambiental operado en la Colonia Agrícola (Neuquén), por efecto de la actividad petrolera que en este momento desarrolla una empresa del ramo. Los autores pertenecen a la UN del Comahue, Laboratorio de Investigaciones y Servicios en Control de Calidad Ambiental. Se tuvieron en cuenta aspectos legales, institucionales, económicos e informativos educativos, finalmente la incidencia en la gestión ambiental