Assembly and optimization of a super-resolution STORM microscope for nanoscopic imaging of biological structures

Fluorescence microscopy is a widely used technique for imaging of biological structures due to its noninvasiveness although resolution of conventional fluorescence microscopes is limited to about 200-300 nm due to the diffraction limit of light. Super-resolution fluorescence microscopy offers an extension of the original method that allows optical imaging below the diffraction limit. In this thesis, a microscope for localization-based super-resolution fluorescence microscopy techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) was established. An epifluorescence microscope was built for this purpose that provides both widefield and Total Internal Reflection Fluorescence (TIRF) excitation modalities and focus was put on the special requirements of localization-based super-resolution methods. This included a high mechanical and optical stability realized by a compact design and implementation of a home-built perfect focus system. The setup was further designed to allow both two- and three-dimensional imaging. The work also included both the development of a setup control software and a software for the analysis of the required data. Different analysis methods and parameters were tested on simulated data before the performance of the microscope was demonstrated in two and three dimensions at appropriate test samples such as the cellular microtubule network. These experiments showed the capability of super-resolution microscopy to reveal underlying structures that cannot be resolved by conventional fluorescence microscopy. Resolutions could be achieved down to approximately 30 nm in the lateral and 115 nm in the axial dimension. Subsequently, the established method was applied to two biological systems. The first is a study of the budding of the human immunodeficiency virus type 1 (HIV-1) from the host cell. In this step of the viral reproduction cycle, the virus hijacks the cellular endosomal sorting complex required for transport (ESCRT) machinery to achieve membrane fission. ESCRT consists of the subcomplexes ESCRT-0, -I, -II and -III and additional related proteins, from which HIV-1 recruits certain components. The fission process is initiated by the HIV-1 Gag protein, which recruits the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX to the virus assembly site. Subsequently, ESCRT-III proteins CHMP4 and CHMP2 form transient lattices at the membrane, which are actively involved in membrane fission. However, the actual geometry of the ESCRT machinery assembling at the HIV-1 budding site that is driving the fission process is still not fully understood. Different models proposed either constriction of the budding neck by lattices surrounding the neck, by ESCRT structures within the neck or within the bud itself. A problematic aspect in previous studies was the usage of modified, tagged versions of the involved proteins for visualization. In this study, super-resolution microscopy was therefore applied to endogenous Tsg101, ALIX and CHMP2 isoform CHMP2A and to a version of CHMP4 isoform CHMP4B with a small HA-tag to elucidate the size and the distribution of the structure relative to the HIV-1 assembly sites. ESCRT structures colocalizing with HIV-1 exhibited closed, circular structures with an average size restricted to 45 and 60 nm in diameter. This size was significantly smaller than found for HIV-1 assembly sites and the constriction of the size, which was not observed for non-colocalizing ESCRT structures at the cell membrane, ruled out an external restriction model. Super-resolution imaging of ALIX often revealed an additional cloud-like structure of individual molecules surrounding the central clusters. This was attributed to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Together with experiments that confirmed the non-physiological behavior of tagged Tsg101 and a relative orientation of ESCRT clusters towards the edge of the assembly site, the results strongly point toward a within-neck model. A second project focused on the influence of external constriction on cell migration. The latter plays an important role in various processes in the human body ranging from wound healing to metastasis formation by cancer cells. Migration is driven by the lamellipodium, which is a meshwork of fine actin filaments that drive membrane protrusion. Endothelial cells were grown on micropatterns that confined the freedom of movement of the cells. Three-dimensional super-resolution imaging revealed that the lamellipodia of these cells showed a much broader axial extension than was the case for control cells that grew without confinement of migration. The different organization of the actin filament network showed a clear effect of environmental conditions on cellular migration. Overall, it was possible to build a super-resolution fluorescence microscope over the course of this study and establish the required analysis methods to allow STORM and PALM imaging below the diffraction limit of light. Two applications further showed that these tools are capable of answering currently discussed questions in the biological sciences

Richtlinien zur Umsetzung taktiler Grafiken: Richtlinien für Bildbeschreibungen und zur Erstellung taktiler Grafiken

Die vorliegenden Richtlinien sollen dabei helfen, aus visuellen Grafikvorlagen brauchbare textuelle sowie taktile Umsetzungen für blinde Menschen zu erstellen. Sie sollen somit insbesondere unerfahrenen Bearbeitern als Anleitung und Unterstützung dienen

Richtlinien zur Umsetzung taktiler Grafiken: Richtlinien für Bildbeschreibungen und zur Erstellung taktiler Grafiken

Die vorliegenden Richtlinien sollen dabei helfen, aus visuellen Grafikvorlagen brauchbare textuelle sowie taktile Umsetzungen für blinde Menschen zu erstellen. Sie sollen somit insbesondere unerfahrenen Bearbeitern als Anleitung und Unterstützung dienen

Richtlinien zur Umsetzung taktiler Grafiken: Richtlinien für Bildbeschreibungen und zur Erstellung taktiler Grafiken

Die vorliegenden Richtlinien sollen dabei helfen, aus visuellen Grafikvorlagen brauchbare textuelle sowie taktile Umsetzungen für blinde Menschen zu erstellen. Sie sollen somit insbesondere unerfahrenen Bearbeitern als Anleitung und Unterstützung dienen

Inclusive Production of Tactile Graphics

International audienceIn this article a collaborative workstation for creating audio-tactile graphics is presented. The system is based on a common open source office suite and supports a transcriber for tactile graphics with several tools. In addition the system allows a blind reviewer to get involved at every stage of the creation process. This is achieved through a refreshable two-dimensional tactile display. The blind participant can independently manipulate graphical objects and make annotations in parallel. As a result, a tandem team of a sighted graphic creator and a blind partner may create a tactile graphic with better quality

Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein

Suturing methods in prolapse surgery: a biomechanical analysis

Introduction!#!Pelvic organ prolapse is a common problem in urogynecological surgery. Abdominal and laparoscopic sacrocolpopexy is currently considered to be the gold standard of treatment. The main problem remains the anatomical point of fixation as well as how sutures are placed. We evaluated the biomechanical difference between an in-line ligament suture versus an orthogonal ligament suture and a single suture versus a continuous suture at the anterior longitudinal ligament in an in-vitro, sacrocolpopexy model.!##!Methods!#!Biomechanical in-vitro testing was performed on human, non-embalmed, female cadaver pelvises. An Instron test frame (tensinometer) was used for load/ displacement analysis. The average patient age was 75 years. Ligament preparation yielded 15 ligaments available for testing. Recorded parameters were the ultimate load, failure displacement, and stiffness.!##!Results!#!This in-vitro analysis of different suturing methods showed the difference between an orthogonal and an in-line approach to be the ultimate load. Orthogonal sutures displayed an ultimate load of 80 N while in-line suturing yielded only 57 N (p < 0.05). For the anterior longitudinal ligament, this study demonstrated that continuous suture is significantly superior to a single suture regarding failure displacement (p < 0.05).!##!Conclusion!#!We established baseline biomechanical parameters for the sacrospinous ligament and anterior longitudinal ligament. An orthogonal suture is superior to an in-line suture in an in-vitro model. A continuous suture is superior to a single suture at the anterior longitudinal ligament. Clinical trials might be able to evaluate whether any clinical significance can be established from these findings

Inframammary Fold Reconstruction: A Biomechanical Analysis

Background: Inframammary fold reconstruction has scarcely been evaluated in literature. No biomechanical analyses have been performed comparing different reconstructive methods. This evaluation compares the gold-standard suture reconstruction with an intrarib anchor system (Micro BioComposite SutureTak, Arthrex). Methods: Three analysis groups were compared including 8 Sawbone blocks, 22 embalmed cadaver, and 27 regular cadaver specimens (N = 57). Transient mechanical analysis was performed at 5 N/s using an Instron 5565 test frame. Results: Ultimate load favored the anchor system (compared with the gold-standard suture) by a factor of 9.8 (P < 0.0001) for the regular cadaver group and a factor of 1.7 (P < 0.038) for the embalmed cadaver group. A similar statistically significant benefit was shown for stiffness and load at 2-mm displacement. Conclusions: This analysis showed an anchor system to be the biomechanically superior fixation method in terms of ultimate load, fixation stiffness, and displacement at failure when compared with the gold-standard suture method in inframammary fold reconstruction. Because of superior stability in every aspect, an anchor system may be considered for inframammary fold reconstruction