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Author Posting. © The Optical Society, 2017. This article is posted here by permission of The Optical Society for personal use, not for redistribution. The definitive version was published in Biomedical Optics Express 8 (2017): 4243-4256, doi:10.1364/BOE.8.004243., 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., Morgridge Institute for Research and National Institutes of Health (NIH) (U54DK104310 and R01GM114274); The University of Wisconsin Carbone Cancer Center Cancer Center Biocore and Histology core (UWCCC) (P30 CA014520).

Author Posting. © Optical Society of America, 2017. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Optics Express 25 (2017): 31309-31325, doi:10.1364/OE.25.031309., 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., National Institute of Health (NIH) (R01GM114274, R01EB017293).

This article is distributed under the terms of the Creative Commons public domain dedication. The definitive version was published in PLoS One 11 (2016): e0158495, doi:10.1371/journal.pone.0158495., 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., This work was supported by Grant 2007-3- 13 from the Alfred P. Sloan Foundation (to GGB), National Institutes of Health Grant 1RC1-DE020630 from the National Institute of Dental and Craniofacial Research (NIDCR) (to GGB) and by National Institutes of Health Fellowship 1F31-DE019576 from NIDCR (to AMV).

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 26 (2015): 887-900, doi:10.1091/mbc.E14-07-1244., 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., This research was supported by National Science Foundation STEP grant 0856704 to Dickinson College, student/faculty summer research grants from the Dickinson College Research and Development Committee, Laura and Arthur Colwin Summer Research Fellowships from the Marine Biological Laboratory to J.H.H. and C.B.S., National Institutes of Health Grant EB002583 to R.O., and National Science Foundation collaborative research grants to J.H.H. (MCB-1412688) and C.B.S. (MCB-1412734).

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Advanced Science 2 (2015): 1500025, doi:10.1002/advs.201500025., 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., The authors acknowledge financial support from NIH (Grant R01GM078200 to G.R.R. and R01EB002583 to R.O.), a Rackham Predoctoral Fellowship Award to K.A.M., and University of Michigan M-Cubed funds awarded to K.A.S. and G.R.R.

Author Posting. © Optical Society, 2014. This article is posted here by permission of Optical Society for personal use, not for redistribution. The definitive version was published in Biomedical Optics Express 5 (2014): 1822-1838, doi:10.1364/BOE.5.001822., 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., This study was funded by National Institutes of Health grant RO1 EB002583

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 25 (2014): 1437-1445, doi:10.1091/mbc.E14-01-0008., 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., This work was supported by Grant EB002045 from the National Institute of Biomedical Imaging and Bioengineering awarded to R.O.

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of IOP Publishing for personal use, not for redistribution. The definitive version was published in Journal of Optics 15 (2013): 094007, doi:10.1088/2040-8978/15/9/094007., 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., This work was supported by a fellowship of the Human Frontiers Science Program awarded to SM and a grant from the National Institute of Biomedical Imaging and Bioengineering (grant Nr. R01EB002045) awarded to RO. MS acknowledges support from the National Institute of General Medical Sciences/NIH (grant R01-GM101701)., 2014-09-10

Author Posting. © The Author(s), 2003. This is the author's version of the work. It is posted here by permission of Cold Spring Harbor Laboratory Press for personal use, not for redistribution. The definitive version was published in Live Cell Imaging : A Laboratory Manual, edited by R. D. Goldman and D. L. Spector, :205-237. Cold Spring Harbor Laboratory Press, 2005. ISBN: 9780879696825., In the current chapter we describe the use of a new type of polarized light microscope which we started to develop at the Marine Biological Laboratory about ten years ago. The new “PolScope” is based on the traditional polarized light microscope and enhances it with the use of liquid- crystal devices and special image processing algorithms. The LC-PolScope measures polarization optical parameters in many specimen points simultaneously, in fast time intervals, and at the highest resolution of the light microscope. It rapidly generates a birefringence map whose pixel brightness is directly proportional to the local optical anisotropy, unaffected by the specimen orientation in the plane of view, as well as a map depicting the slow axis orientation of the birefringent regions. The basic LC-PolScope technology can be adapted to most research grade microscopes and is available commercially from Cambridge Research and Instrumentation (CRI, http://www.cri-inc.com) in Woburn, Massachusetts, under the trade name LC-PolScope., Financial support from the National Institute of General Medical Sciences and from the National Institute of Biomedical Imaging and Bioengineering through grants GM49210 and EB002045, respectively.

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 23 (2012): 3133-3142, doi:10.1091/mbc.E12-04-0314., We report on experiments directly in living cells that reveal the regulation of kinetochore function by tension. X and Y sex chromosomes in crane fly (Nephrotoma suturalis) spermatocytes exhibit an atypical segregation mechanism in which each univalent maintains K-fibers to both poles. During anaphase, each maintains a leading fiber (which shortens) to one pole and a trailing fiber (which elongates) to the other. We used this intriguing behavior to study the motile states that X-Y kinetochores are able to support during anaphase. We used a laser microbeam to either sever a univalent along the plane of sister chromatid cohesion or knock out one of a univalent's two kinetochores to release one or both from the resistive influence of its sister's K-fiber. Released kinetochores with attached chromosome arms moved poleward at rates at least two times faster than normal. Furthermore, fluorescent speckle microscopy revealed that detached kinetochores converted their functional state from reverse pac-man to pac-man motility as a consequence of their release from mechanical tension. We conclude that kinetochores can exhibit pac-man motility, even though their normal behavior is dominated by traction fiber mechanics. Unleashing of kinetochore motility through loss of resistive force is further evidence for the emerging model that kinetochores are subject to tension-sensitive regulation., This work was supported by a grant to R.O. from the National Institute of Biomedical Imaging and Bioengineering (R01EB002045).

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 22 (2011): 4801-4808, doi:10.1091/mbc.E11-06-0494., 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., This study was supported by a grant to R.O. from the National Institute of Biomedical Imaging and Bioengineering (R01EB002583-17).

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 193 (2011): 1065-1081, doi:10.1083/jcb.201012143., The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals., This work was supported by the National Science Foundation under grant No. MCB-0719126 to A.S. Gladfelter, the National Institute of Biomedical Imaging and Bioengineering under grant No. EB002583 to R. Oldenbourg, a Drexel CURE grant from the State of Pennsylvania Tobacco Settlement Fund, and National Institute of Neurological Disorders and Stroke grant NS48090- 06A to E.T. Spiliotis.

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.