Browsing by Author "Gustavsson, Anna-Karin"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    A Flexible Multimodal 3D Single-Molecule Super-Resolution Microscope for Whole Cell Imaging
    (2023-07-20) Nelson, Tyler Evan; Gustavsson, Anna-Karin
    Single-molecule super resolution techniques can be used to resolve subcellular structures in nanoscale detail, but they are sensitive to background signal which is common in fluorescently labeled cells. Most microscopes are limited to standard epi- illumination, which generates high background fluorescence by illuminating the entire sample at once. Specialized illumination schemes like light sheets or Total Internal Reflection Fluorescence (TIRF) are useful to improve the resolution, but the usefulness of these methods can be limited in certain regions of the cell. In this thesis, we demonstrate a flexible, multimodal super resolution imaging system which combines the optical sectioning capacity of a tilted light sheet with the excellent contrast and homogeneous illumination of a flat-field epi- and TIRF setup. This imaging platform also includes a two-channel 4f point spread function (PSF) engineering system combined with long axial range phase masks for 3D imaging. We show that our microscope greatly reduces background fluorescence throughout thick mammalian cells and improves the performance of single-molecule super-resolution imaging in cells in both 2D and 3D and has the potential to image in 3D throughout an entire cell.
  • Thumbnail Image
    Item
    Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics
    (The American Society for Cell Biology, 2022) Gustavsson, Anna-Karin; Ghosh, Rajarshi P.; Petrov, Petar N.; Liphardt, Jan T.; Moerner, W. E.; Smalley–Curl Institute; Institute of Biosciences and Bioengineering
    Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.
  • Thumbnail Image
    Item
    Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses
    (Frontiers Media S.A., 2021) Gagliano, Gabriella; Nelson, Tyler; Saliba, Nahima; Vargas-Hernández, Sofía; Gustavsson, Anna-Karin; Smalley-Curl Institute; Laboratory for Nanophotonics; Institute of Biosciences & Bioengineering
    The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.
  • Thumbnail Image
    Item
    Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells
    (Optica Publishing Group, 2024) Nelson, Tyler; Vargas-Hernández, Sofía; Freire, Margareth; Cheng, Siyang; Gustavsson, Anna-Karin; Smalley-Curl Institute;Institute of Biosciences & Bioengineering;Center for Nanoscale Imaging Sciences
    Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.
  • Thumbnail Image
    Item
    Single-Objective Tilted Light Sheet Illumination with Exchange-PAINT and Deep Learning for Fast, Accurate, and Precise 3D Single-Molecule Super-Resolution Imaging in Mammalian Cells
    (2023-05-03) Gagliano, Gabi; Gustavsson, Anna-Karin
    Single-molecule super-resolution fluorescence microscopy is a powerful method for imaging detailed biological structures at the nanoscale. However, imaging capabilities in thick samples such as mammalian cells remain limited due to increased fluorescence background which degrades the achievable localization precision of fluorescent emitters. Light sheet fluorescence microscopy is a simple solution in which a thin plane of light is used to optically section the sample, resulting in an increased signal-to-background ratio and thus improving the achievable localization precision of single molecules. However, light sheet illumination is sensitive to shadowing artifacts from imperfections in the optical path and scattering throughout the sample which may impact the homogeneity of the illumination. Additionally, most light sheet systems employ two objectives, which may suffer from steric hindrance and drift between the sample and illumination objective. In this work, I present a single-objective light sheet microscopy setup which has been combined with the additional innovations of (i) dithering of the light sheet for artifact reduction, (ii) a 3D-printed microfluidic chip for control of the extracellular environment and reflection of the light sheet into the sample, (iii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iv) engineered point spread functions (PSFs) for 3D imaging, and (v) deep learning for high-density localization of single molecules. Altogether, this approach improves the localization precision, imaging speeds, and multi-target accuracy and enable fast, accurate, and precise multi-target 3D single-molecule super-resolution cellular imaging. First, I introduce single-molecule localization microscopy, light sheet illumination, and the experimental methods of Exchange-PAINT and point spread function engineering which are used in this work. I then describe the design of the light sheet which has been devised with width, thickness, and confocal parameter specifically suited for mammalian cell imaging, and outline the construction and calibration of the optical setup. Finally, I validate the optical system in terms of background reduction, localization precision improvement, and imaging speed by performing multi-target single-molecule super-resolution imaging of nuclear lamina proteins lamin B1, lamin A/C, and emerin, and the microtubule protein alpha-tubulin. The single-objective dithered light sheet achieves a localization precision below ten nanometers in xy and below 12 nanometers in z, enabling a range of applications from nuclear protein investigation to whole cell imaging. I plan to use this system to investigate the relationship between nuclear lamina protein organization and chromatin dynamics in Hutchinson-Gilford Progeria Syndrome (HGPS), a disorder caused by a mutation in the LMNA gene.
  • Thumbnail Image
    Item
    Ubiquitination in and around the peroxisome
    (2023-04-19) Traver, Melissa Sue; Bartel, Bonnie; Braam, Janet; Gustavsson, Anna-Karin
    Seedlings store neutral lipids such as triacylglycerol (TAG) in cytosolic lipid droplets to fuel seedling growth before the onset of photosynthesis. Fatty acids released from lipid droplet TAG are catabolized in peroxisomes—membrane-bound organelles that sequester reactive oxygen species-generating reactions including fatty acid beta-oxidation. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Peroxisomes in plants and yeast have a dedicated ubiquitin-conjugating enzyme, PEX4, that ubiquitinates proteins associated with the peroxisomal membrane. PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. Several lipid droplet proteins and peroxisomal proteins are ubiquitinated as a signal for subsequent membrane extraction and/or degradation. For example, oleosins, which serve as lipid droplet coat proteins, are ubiquitinated, extracted, and degraded in parallel with lipid droplet TAG mobilization. In this thesis, I probed the dynamic interactions between lipid droplets and peroxisomes, including the mechanism of oleosin ubiquitination and the role of oleosin in TAG mobilization. Using forward genetics, I found that the ubiquitin-ligase MIEL1 (MYB30-interacting E3 ligase) is needed for timely oleosin degradation during seedling lipid mobilization. I further demonstrated that MIEL1 associates with peroxisomes and impacts oleosin levels post-transcriptionally, supporting the hypothesis that MIEL1 targets peroxisome-proximal seed oleosins for degradation during seedling lipid mobilization. I expanded my study of the mechanisms and biological consequences of ubiquitination in and around the peroxisome by characterizing the Arabidopsis PEX4-PEX22 complex using structural biology, enzymology, and genetic approaches. The work in this thesis expands our understanding of the complex and interwoven relationships between peroxisomes and lipid droplets.
  • Thumbnail Image
    Item
    Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet
    (Springer Nature, 2024) Saliba, Nahima; Gagliano, Gabriella; Gustavsson, Anna-Karin; Smalley-Curl Institute;Center for Nanoscale Imaging Sciences
    Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. We combine these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets. We then demonstrate that this platform, termed soTILT3D, enables whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.