Oldenbourg Rudolf

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Oldenbourg

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Rudolf

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0000-0003-1055-8692

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Time lapse movie of meiosis II in a living spermatocyte from the crane fly, Nephrotoma suturalis, viewed with polarized light microscopy

( 2002-11) LaFountain, James R. ; Oldenbourg, Rudolf

The events of meiosis II in two living spermatocytes obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at prophase II through telophase II to the near completion of cytokinesis following meiosis II.
Image simulation for biological microscopy : microlith

(Optical Society, 2014-05-13) Mehta, Shalin B. ; Oldenbourg, Rudolf

Image simulation remains under-exploited for the most widely used biological phase microscopy methods, because of difficulties in simulating partially coherent illumination. We describe an open-source toolbox, microlith (https://code.google.com/p/microlith), which accurately predicts three-dimensional images of a thin specimen observed with any partially coherent imaging system, as well as images of coherently illuminated and self-luminous incoherent specimens. Its accuracy is demonstrated by comparing simulated and experimental bright-field and dark-field images of well-characterized amplitude and phase targets, respectively. The comparison provides new insights about the sensitivity of the dark-field microscope to mass distributions in isolated or periodic specimens at the length-scale of 10nm. Based on predictions using microlith, we propose a novel approach for detecting nanoscale structural changes in a beating axoneme using a dark-field microscope.
Quantification of collagen organization in histopathology samples using liquid crystal based polarization microscopy

(The Optical Society, 2017-08-29) Keikhosravi, Adib ; Liu, Yuming ; Drifka, Cole ; Woo, Kaitlin M. ; Verma, Amitabh ; Oldenbourg, Rudolf ; Eliceiri, Kevin

A number of histopathology studies have utilized the label free microscopy method of Second Harmonic Generation (SHG) to investigate collagen organization in disease onset and progression. Here we explored an alternative label free imaging approach, LC-PolScope that is based on liquid crystal based polarized light imaging. We demonstrated that this more accessible technology has the ability to visualize all fibers of interest and has a good to excellent correlation between SHG and LC-PolScope measurements in fibrillar collagen orientation and alignment. This study supports that LC-PolScope is a viable alternative to SHG for label free collagen organization measurements in thin histology sections.
Massive bioaccumulation and self-assembly of phenazine compounds in live cells

(John Wiley & Sons, 2015-06-05) Min, Kyoung Ah ; Rajeswaran, Walajapet G. ; Oldenbourg, Rudolf ; Harris, Grant ; Keswani, Rahul K. ; Chiang, Mason ; Rzeczycki, Phillip ; Talattof, Arjang ; Hafeez, Mahwish ; Horobin, Richard W. ; Larsen, Scott D. ; Stringer, Kathleen A. ; Rosania, Gus R.

Clofazimine is an orally administered drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine is synthesized and tested for ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation is consistently greater in macrophages than in epithelial cells. Microscopically, some self-assembled structures exhibit a pronounced, diattenuation anisotropy signal, evident by the differential absorption of linearly polarized light, at the peak absorbance wavelength of the phenazine core. The measured anisotropy is well above the background anisotropy of endogenous cellular components, reflecting the self-assembly of condensed, insoluble complexes of ordered phenazine molecules. Chemical variations introduced at the R-imino position of the phenazine core lead to idiosyncratic effects on the compounds' bioaccumulation behavior as well as on the morphology and organization of the resulting intracellular structures. Beyond clofazimine, these results demonstrate how the self-assembly of membrane permeant, orally bioavailable small molecule building blocks can endow cells with unnatural structural elements possessing chemical, physical, and functional characteristics unlike those of other natural cellular components.
Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

(American Society for Cell Biology, 2014-02-26) LaFountain, James R. ; Oldenbourg, Rudolf

We use liquid crystal polarized light imaging to record the life histories of single kinetochore (K-) fibers in living crane-fly spermatocytes, from their origins as nascent K-fibers in early prometaphase to their fully matured form at metaphase, just before anaphase onset. Increased image brightness due to increased retardance reveals where microtubules are added during K-fiber formation. Analysis of experimentally generated bipolar spindles with only one centrosome, as well as of regular, bicentrosomal spindles, reveals that microtubule addition occurs at the kinetochore-proximal ends of K-fibers, and added polymer expands poleward, giving rise to the robust K-fibers of metaphase cells. These results are not compatible with a model for K-fiber formation in which microtubules are added to nascent fibers solely by repetitive “search and capture” of centrosomal microtubule plus ends. Our interpretation is that capture of centrosomal microtubules—when deployed—is limited to early stages in establishment of nascent K-fibers, which then mature through kinetochore-driven outgrowth. When kinetochore capture of centrosomal microtubules is not used, the polar ends of K-fibers grow outward from their kinetochores and usually converge to make a centrosome-free pole.
Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments

(eLife, 2017-11-06) Spira, Felix ; Cuylen-Haering, Sara ; Mehta, Shalin B. ; Samwer, Matthias ; Reversat, Anne ; Verma, Amitabh ; Oldenbourg, Rudolf ; Sixt, Michael ; Gerlich, Daniel W.

The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.
Mapping birefringence in three dimensions using polarized light field microscopy : the case of the juvenile clamshell

( 2018-05) Tran, Mai Thi ; Oldenbourg, Rudolf

We report methods to generate three dimensional maps of birefringence, its position and orientation in juvenile shells of the Atlantic hard clamshell (Mercenaria mercenaria). For measuring the retardance and optic axis orientation of curved shell surfaces in three dimensions, we developed enhanced acquisition and processing algorithms and combined results from conventional and light field imaging approaches to reconstruct the three dimensional shell shape and its anisotropic optical properties. Our work represents the first successful attempt to generate such maps at a spatial resolution of about 2 m and angular steps of about 9° in terms of the inclination angles of the optic axis. The maps of clamshell birefringence provide structural insights into the early mineralization during juvenile clamshell development.
Time lapse movie of meiosis I in a living spermatocyte from the crane fly, Nephrotoma suturalis, viewed with polarized light microscopy

( 2002-11) LaFountain, James R. ; Oldenbourg, Rudolf

The events of meiosis I in a living spermatocyte obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at diakinesis through telophase to the near completion of cytokinesis following meiosis I.
Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy

(American Society of Cell Biology, 2011-10-26) LaFountain, James R. ; Cohan, Christopher S. ; Oldenbourg, Rudolf

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is “park,” in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.
Reconfigurable self-assembly through chiral control of interfacial tension

( 2011-12-01) Gibaud, Thomas ; Barry, Edward ; Zakhary, Mark J. ; Henglin, Mir ; Ward, Andrew ; Yang, Yasheng ; Berciu, Cristina ; Oldenbourg, Rudolf ; Hagan, Michael F. ; Nicastro, Daniela ; Meyer, Robert B. ; Dogic, Zvonimir

From determining optical properties of simple molecular crystals to establishing preferred handedness in highly complex vertebrates, molecular chirality profoundly influences the structural, mechanical, and optical properties of both synthetic and biological matter at macroscopic lengthscales1,2. In soft materials such as amphiphilic lipids and liquid crystals, the competition between local chiral interactions and global constraints imposed by the geometry of the self-assembled structures leads to frustration and the assembly of unique materials3-6. An example of particular interest is smectic liquid crystals, where the 2D layered geometry cannot support twist, expelling chirality to the edges in a manner analogous to the expulsion of a magnetic field from superconductors7-10. Here, we demonstrate a previously unexplored consequence of this geometric frustration which leads to a new design principle for the assembly of chiral molecules. Using a model system of colloidal membranes11, we show that molecular chirality can control the interfacial tension, an important property of multi-component mixtures. This finding suggests an analogy between chiral twist which is expelled to the edge of 2D membranes, and amphiphilic surfactants which are expelled to oil-water interfaces12. Similar to surfactants, chiral control of interfacial tension drives the assembly of myriad polymorphic assemblages such as twisted ribbons with linear and circular topologies, starfish membranes, and double and triple helices. Tuning molecular chirality in situ enables dynamical control of line tension that powers polymorphic transitions between various chiral structures. These findings outline a general strategy for the assembly of reconfigurable chiral materials which can easily be moved, stretched, attached to one another, and transformed between multiple conformational states, thus enabling precise assembly and nano-sculpting of highly dynamical and designable materials with complex topologies.
pH dependent isotropic to nematic phase transitions in graphene oxide dispersions reveal droplet liquid crystalline phases

( 2014-05) Tkacz, Rachel ; Oldenbourg, Rudolf ; Mehta, Shalin B. ; Miansari, Morteza ; Verma, Amitabh ; Majumder, Mainak

Size fractionation, amplified by the surface charge density of graphene oxide (GO) sheets, broadens the pH dependent isotropic (I) to nematic (N) phase transition in aqueous dispersions of graphene oxide (GO). In this biphasic region, a highly organized droplet nematic phase of uniform size (20 ± 2.8 μm diameter) with an isotropic interior is observed.
Chromosome malorientations after meiosis II arrest cause nondisjunction

(American Society for Cell Biology, 2007-02-21) Janicke, Marie A. ; Lasko, Loren ; Oldenbourg, Rudolf ; LaFountain, James R.

This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n – 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.
Achiral symmetry breaking and positive Gaussian modulus lead to scalloped colloidal membranes

( 2016-11) Gibaud, Thomas ; Kaplan, C. Nadir ; Sharma, Prerna ; Ward, Andrew ; Zakhary, Mark J. ; Oldenbourg, Rudolf ; Meyer, Robert B. ; Kamien, Randall D. ; Powers, Thomas R. ; Dogic, Zvonimir

In the presence of a non-adsorbing polymer, monodisperse rod-like particles assemble into colloidal membranes, which are one rod-length thick liquid-like monolayers of aligned rods. Unlike 3D edgeless bilayer vesicles, colloidal monolayer membranes form open structures with an exposed edge, thus presenting an opportunity to study physics of thin elastic sheets. Membranes assembled from single-component chiral rods form flat disks with uniform edge twist. In comparison, membranes comprised of mixture of rods with opposite chiralities can have the edge twist of either handedness. In this limit disk-shaped membranes become unstable, instead forming structures with scalloped edges, where two adjacent lobes with opposite handedness are separated by a cusp-shaped point defect. Such membranes adopt a 3D configuration, with cusp defects alternatively located above and below the membrane plane. In the achiral regime the cusp defects have repulsive interactions, but away from this limit we measure effective long-ranged attractive binding. A phenomenological model shows that the increase in the edge energy of scalloped membranes is compensated by concomitant decrease in the deformation energy due to Gaussian curvature associated with scalloped edges, demonstrating that colloidal membranes have positive Gaussian modulus. A simple excluded volume argument predicts the sign and magnitude of the Gaussian curvature modulus that is in agreement with experimental measurements. Our results provide insight into how the interplay between membrane elasticity, geometrical frustration and achiral symmetry breaking can be used to fold colloidal membranes into 3D shapes.
Polarized light microscopy in reproductive and developmental biology

( 2013-05) Koike-Tani, Maki ; Tani, Tomomi ; Mehta, Shalin B. ; Verma, Amitabh ; Oldenbourg, Rudolf

The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. Therefore, it is a powerful tool to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on chromosome packing in sperm head, first zygote division of the sea urchin, and differentiation initiated by the first uneven cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end by reporting first results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12 through 20.
Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other

(American Society for Cell Biology, 2004-09-22) LaFountain, James R. ; Oldenbourg, Rudolf

To test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.
Entropy-driven formation of a chiral liquid-crystalline phase of helical filaments

(American Physical Society, 2006-01-11) Barry, Edward ; Hensel, Zach ; Dogic, Zvonimir ; Shribak, Michael ; Oldenbourg, Rudolf

We study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.
Multiplexed spectral imaging of 120 different fluorescent labels

(Public Library of Science, 2016-07-08) Valm, Alex M. ; Oldenbourg, Rudolf ; Borisy, Gary G.

The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image.
Single-fluorophore orientation determination with multiview polarized illumination : modeling and microscope design

(Optical Society, 2017-12-01) Chandler, Talon ; Mehta, Shalin B. ; Shroff, Hari ; Oldenbourg, Rudolf ; La Riviere, Patrick J.

We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric—the solid-angle uncertainty—to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric—single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.
Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process

(American Society for Cell Biology, 2015-01-07) Henson, John H. ; Yeterian, Mesrob ; Weeks, Richard M. ; Medrano, Angela E. ; Brown, Briana L. ; Geist, Heather L. ; Pais, Mollyann D. ; Oldenbourg, Rudolf ; Shuster, Charles B.

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.
Polarized light imaging of birefringence and diattenuation at high resolution and high sensitivity

( 2013-07) Mehta, Shalin B. ; Shribak, Michael ; Oldenbourg, Rudolf

Polarized light microscopy provides unique opportunities for analyzing the molecular order in manmade and natural materials, including biological structures inside living cells, tissues, and whole organisms. 20 years ago, the LC-PolScope was introduced as a modern version of the traditional polarizing microscope enhanced by liquid crystal devices for the control of polarization, and by electronic imaging and digital image processing for fast and comprehensive image acquisition and analysis. The LCPolScope is commonly used for birefringence imaging, analyzing the spatial and temporal variations of the differential phase delay in ordered and transparent materials. Here we describe an alternative use of the LC-PolScope for imaging the polarization dependent transmittance of dichroic materials. We explain the minor changes needed to convert the instrument between the two imaging modes, discuss the relationship between the quantities measured with either instrument, and touch on the physical connection between refractive index, birefringence, transmittance, diattenuation, and dichroism.

Oldenbourg Rudolf

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