IMPROVING LONG-TERM, LIVE-CELL FLUORESCENCE MICROSCOPY

Fluorescence microscopy is one of the most powerful tools for studying sub-cellular dynamics with molecular specificity at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of a detection objective using an orthogonal excitation path with a laterally restricted illumination pattern. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both the detection efficiency and native spatial resolution of the detection path. To address this limitation, we present a novel live-cell LSFM method: Lateral Interference Tilted Excitation (LITE) microscopy, in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, efficient, coverslip-based detection objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. Additionally, we formulate and test an optical model to explain the photobleaching improvement that we observe when imaging thin (~5 μm) fluorescent organisms.In addition to developing microscopy technology that improves photobleaching over conventional fluorescence microscopy, we also have proposed theoretical optics to further remove imaging limitations for live-cell biology. Conventional optical microscopy relies on the refraction of light with lenses to magnify and resolve specimens; however, comparatively little work has been done on using reflection as a means of microscopy. We therefore propose using paraboloidal mirrors as primary illumination and detection optics to improve illumination consistency and detection efficiency in fluorescence microscopy.Doctor of Philosoph

Anillin localization suggests distinct mechanisms of division plane specification in mouse oogenic meiosis I and II

Anillin is a conserved cytokinetic ring protein implicated in actomyosin cytoskeletal organization and cytoskeletal-membrane linkage. Here we explored anillin localization in the highly asymmetric divisions of the mouse oocyte that lead to the extrusion of two polar bodies. The purposes of polar body extrusion are to reduce the chromosome complement within the egg to haploid, and to retain the majority of the egg cytoplasm for embryonic development. Anillin's proposed roles in cytokinetic ring organization suggest that it plays important roles in achieving this asymmetric division. We report that during meiotic maturation, anillin mRNA is expressed and protein levels steadily rise. In meiosis I, anillin localizes to a cortical cap overlying metaphase I spindles, and a broad ring over anaphase spindles that are perpendicular to the cortex. Anillin is excluded from the cortex of the prospective first polar body, and highly enriched in the cytokinetic ring that severs the polar body from the oocyte. In meiosis II, anillin is enriched in a cortical stripe precisely coincident with and overlying the meiotic spindle midzone. These results suggest a model in which this cortical structure contributes to spindle re-alignment in meiosis II. Thus, localization of anillin as a conserved cytokinetic ring marker illustrates that the geometry of the cytokinetic ring is distinct between the two oogenic meiotic cytokineses in mammals

CENP-A and topoisomerase-II antagonistically affect chromosome length

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening

Microfluidics Chamber

mock-up of microfluidics chambe

SPIM parts

OpenSPIM part

Laser collimator parts

Cylindrical spacer for laser collimato

C. Elegans Embryo

sample c. elegans embry

Characterization of DTACC Structure and Function

Microtubules (MTs) are essential for numerous cellular processes. MTs impact cell shape, serve as intra-cellular transport tracks, and form the mitotic spindle. Microtubule-associated proteins (MAPs) modulate MTs to afford differing MT dynamics and ultra-structure. The TACC proteins, a highly conserved MAP family, localize to the MT plus-end and to centrosomes where they affect MT dynamics and interact with other MAPs to modulate their MT-regulating potency. We focus on the interplay between TACC and the MT polymerase, XMAP215. To elucidate TACC’s mechanisms of centrosome-localization and XMAP215- recuitment in cells, we used Drosophila S2 cells as a model cell system. We used truncational analysis of Drosophila TACC (DTACC) in order to identify regions of DTACC that localize to the centrosome and to MT plus-ends using fluorescence microscopy. These results suggest the determinants of DTACC that confer in vivo Msps (Drosophila XMAP215) binding and are consistent with our in vitro biochemical binding assays.Bachelor of Scienc