Shribak
Michael
Shribak
Michael
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ArticleMapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy(SPIE, 2017-01-06) Shribak, Michael ; Larkin, Kieran G. ; Biggs, DavidWe describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100×/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ∼0.5 nm and lateral resolution if ∼300 nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems.
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ArticlePolychromatic polarization microscope : bringing colors to a colorless world(Nature Publishing Group, 2015-11-27) Shribak, MichaelInterference of two combined white light beams produces Newton colors if one of the beams is retarded relative to the other by from 400 nm to 2000 nm. In this case the corresponding interfering spectral components are added as two scalars at the beam combination. If the retardance is below 400 nm the two-beam interference produces grey shades only. The interference colors are widely used for analyzing birefringent samples in mineralogy. However, many of biological structures have retardance <100 nm. Therefore, cells and tissues under a regular polarization microscope are seen as grey image, which contrast disappears at certain orientations. Here we are proposing for the first time using vector interference of polarized light in which the full spectrum colors are created at retardance of several nanometers, with the hue determined by orientation of the birefringent structure. The previously colorless birefringent images of organelles, cells, and tissues become vividly colored. This approach can open up new possibilities for the study of biological specimens with weak birefringent structures, diagnosing various diseases, imaging low birefringent crystals, and creating new methods for controlling colors of the light beam.
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ArticleMachine-learning-based evaluation of intratumoral heterogeneity and tumor-stroma interface for clinical guidance(Elsevier, 2021-09-21) Laurinavicius, Arvydas ; Rasmusson, Allan ; Plancoulaine, Benoit ; Shribak, Michael ; Levenson, RichardAssessment of intratumoral heterogeneity and tumor-host interaction within the tumor microenvironment is becoming increasingly important for innovative cancer therapy decisions because of the unique information it can generate about the state of the disease. However, its assessment and quantification are limited by ambiguous definitions of the tumor-host interface and by human cognitive capacity in current pathology practice. Advances in machine learning and artificial intelligence have opened the field of digital pathology to novel tissue image analytics and feature extraction for generation of high-capacity computational disease management models. A particular benefit is expected from machine-learning applications that can perform extraction and quantification of subvisual features of both intratumoral heterogeneity and tumor microenvironment aspects. These methods generate information about cancer cell subpopulation heterogeneity, potential tumor-host interactions, and tissue microarchitecture, derived from morphologically resolved content using both explicit and implicit features. Several studies have achieved promising diagnostic, prognostic, and predictive artificial intelligence models that often outperform current clinical and pathology criteria. However, further effort is needed for clinical adoption of such methods through development of standardizable high-capacity workflows and proper validation studies.
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ArticleDensity imaging of heterochromatin in live cells using orientation-independent-DIC microscopy(American Society for Cell Biology, 2017-08-23) Imai, Ryosuke ; Nozaki, Tadasu ; Tani, Tomomi ; Kaizu, Kazunari ; Hibino, Kayo ; Ide, Satoru ; Tamura, Sachiko ; Takahashi, Koichi ; Shribak, Michael ; Maeshima, KazuhiroIn eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.
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PreprintOrientation-independent differential interference contrast microscopy( 2006) Shribak, Michael ; Inoue, ShinyaThe image in a regular DIC microscope reflects the orientation of the prism shear direction and the optical path gradients in a phase specimen. If the shear direction lies parallel to the specimen boundary no contrast is generated. Also a bias retardance is generally introduced, which creates a gray background and reduces image contrast. Here we describe the theoretical foundation for a new DIC technique, which records phase gradients independently of their orientation and with the digitally generated gradient magnitude image as well as the optical path distribution image free from the gray background. Separate images can show the magnitude distribution of the optical path gradients and of the azimuths, or the two images can be combined into one picture e.g., with the brightness representing magnitudes and color showing azimuths respectively. For experimental verification of the proposed technique we investigated various specimens such as glass rods embedded in Permount, Siemens star nano-fabricated in 90-nm thick silicon oxide layer, Bovine pulmonary artery endothelial cell, etc, using regular DIC optics on a microscope equipped with a precision rotating stage. Several images were recorded with the specimen oriented in different directions, but with the prism bias unchanged, followed by digital alignment and processing of the images. The results demonstrate that the proposed DIC technique can successfully image and measure phase gradients of transparent specimens, independent of the directions of the gradient. The orientation-independent DIC data obtained can also be used to compute the quantitative distribution of specimen phase or to generate enhanced, regular DIC images with any desired shear direction. We are currently developing a new device using special DIC prisms, which allows the bias and shear directions to be switched rapidly without the need to mechanically rotate the specimen or the prism (US Patent Application 2005-0152030). With the new system an orientation independent DIC image should be obtained in a fraction of a second. A detailed description of the new system will be given in a future publication.
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ArticlePostnatal structural development of mammalian basilar membrane provides anatomical basis for the maturation of tonotopic maps and frequency tuning(Nature Research, 2021-04-07) Tani, Tomomi ; Koike-Tani, Maki ; Tran, Mai Thi ; Shribak, Michael ; Levic, SnezanaThe basilar membrane (BM) of the mammalian cochlea constitutes a spiraling acellular ribbon that is intimately attached to the organ of Corti. Its graded stiffness, increasing from apex to the base of the cochlea provides the mechanical basis for sound frequency analysis. Despite its central role in auditory signal transduction, virtually nothing is known about the BM’s structural development. Using polarized light microscopy, the present study characterized the architectural transformations of freshly dissected BM at time points during postnatal development and maturation. The results indicate that the BM structural elements increase progressively in size, becoming radially aligned and more tightly packed with maturation and reach the adult structural signature by postnatal day 20 (P20). The findings provide insight into structural details and developmental changes of the mammalian BM, suggesting that BM is a dynamic structure that changes throughout the life of an animal.
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ArticleReal-time polarization microscopy of fibrillar collagen in histopathology(Nature Research, 2021-09-24) Keikhosravi, Adib ; Shribak, Michael ; Conklin, Matthew W. ; Liu, Yuming ; Li, Bin ; Loeffler, Agnes ; Levenson, Richard ; Eliceiri, KevinOver the past two decades, fibrillar collagen reorganization parameters such as the amount of collagen deposition, fiber angle and alignment have been widely explored in numerous studies. These parameters are now widely accepted as stromal biomarkers and linked to disease progression and survival time in several cancer types. Despite all these advances, there has not been a significant effort to make it possible for clinicians to explore these biomarkers without adding steps to the clinical workflow or by requiring high-cost imaging systems. In this paper, we evaluate previously described polychromatic polarization microscope (PPM) to visualize collagen fibers with an optically generated color representation of fiber orientation and alignment when inspecting the sample by a regular microscope with minor modifications. This system does not require stained slides, but is compatible with histological stains such as H&E. Consequently, it can be easily accommodated as part of regular pathology review of tissue slides, while providing clinically useful insight into stromal composition.
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ArticleConstruction and composition of the squid pen from Doryteuthis pealeii(University of Chicago Press, 2019-07-08) Messerli, Mark A. ; Raihan, M. Jahir ; Kobylkevich, Brian M. ; Benson, Austin C. ; Bruening, Kristi S. ; Shribak, Michael ; Rosenthal, Joshua J. C. ; Sohn, Joel J.The pen, or gladius, of the squid is an internalized shell. It serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen’s durability and flexibility are derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from Doryteuthis pealeii. The nanofibrils of the polysaccharide β-chitin are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development, enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid that has a significantly different ionic composition from squid plasma. Two groups of proteins are associated with the pen: those on its surface and those embedded within the pen. Only 20 proteins are identified as embedded within the pen. Embedded proteins are classified into six groups, including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix, and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein.
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ArticleIntranuclear birefringent inclusions in paraffin sections by polychromatic polarization microscopy(Nature Research, 2021-03-18) Vitkunaite, Aiste ; Laurinaviciene, Aida ; Plancoulaine, Benoit ; Rasmusson, Allan ; Levenson, Richard ; Shribak, Michael ; Laurinavicius, ArvydasIntranuclear birefringent inclusions (IBI) found in various cell types in paraffin-embedded tissue sections have long been considered to be a tissue processing artifact, although an association with biological processes has been suggested. We applied polychromatic polarization microscopy to image their spatial organization. Our study provides evidence that IBI are caused by liquid paraffin-macromolecular crystals formed during paraffin-embedding procedures within cells and potentially reflect an active transcriptional status.
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ArticleEntropy-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, RudolfWe 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.
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ArticleSingle nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.(Rockefeller University Press, 2019-03-01) Nagashima, Ryosuke ; Hibino, Kayo ; Ashwin, S. S. ; Babokhov, Michael ; Fujishiro, Shin ; Imai, Ryosuke ; Nozaki, Tadasu ; Tamura, Sachiko ; Tani, Tomomi ; Kimura, Hiroshi ; Shribak, Michael ; Kanemaki, Masato T. ; Sasai, Masaki ; Maeshima, KazuhiroAlthough chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.
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PreprintPolarized light imaging of birefringence and diattenuation at high resolution and high sensitivity( 2013-07) Mehta, Shalin B. ; Shribak, Michael ; Oldenbourg, RudolfPolarized 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.
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ArticleQuantitative orientation-independent differential interference contrast (DIC) microscopy coupled with orientation-independent Polarization microscopy(Cambridge University Press, 2007-08-05) Shribak, Michael ; LaFountain, James R. ; Biggs, David ; Inoue, ShinyaDifferential interference contrast (DIC) microscopy is widely used to observe structure and motion in unstained, transparent living cells and isolated organelles, producing a monochromatic shadowcast image of optical phase gradient. Polarized light microscopy (Pol) reveals structural anisotropy due to form birefringence, intrinsic birefringence, stress birefringence, etc. DIC and Pol complement each other as, for example, in a live dividing cell, the DIC image will clearly show the chromosomes while the Pol image will depict the distribution of the birefringent microtubules in the spindle. Both methods, however, have the same shortcomings: they require the proper orientation of a specimen in relation to the optical system in order to achieve best results.
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ArticleQuantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation(Optical Society of America, 2013-03-27) Shribak, MichaelWe describe a quantitative orientation-independent differential interference contrast (DIC) microscope, which allows bias retardation to be modulated and shear directions to be switched rapidly without any mechanical movement. The shear direction is switched by a regular liquid-crystal cell sandwiched between two standard DIC prisms. Another liquid-crystal cell modulates the bias. Techniques for measuring parameters of DIC prisms and calibrating the bias are shown. Two sets of raw DIC images with the orthogonal shear directions are captured within 1 s. Then the quantitative image of optical path gradient distribution within a thin optical section is computed. The gradient data are used to obtain a quantitative distribution of the optical path, which represents the refractive index gradient or height distribution. Computing enhanced regular DIC images with any desired shear direction is also possible.
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ArticleLiving cells and dynamic molecules observed with the polarized light microscope : the legacy of Shinya Inoué(Marine Biological Laboratory, 2016-08) Tani, Tomomi ; Shribak, Michael ; Oldenbourg, RudolfIn 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at the Marine Biological Laboratory (MBL), which quickly became Inoué's scientific home. Primed by his Japanese mentor, Katsuma Dan, Inoué followed Dan's mantra to work with healthy, living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion that there are dynamic, fine structures inside living cells, in which molecular assemblies such as mitotic spindle fibers exist in delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 1980s, Inoué and others at the MBL were instrumental in conceiving video microscopy, a groundbreaking technique which married light microscopy and electronic imaging, ushering in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. Here, we recount some of Inoué's accomplishments and describe how his legacy has shaped current activities in polarized light imaging at the MBL.
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ArticleThe common morphospecies Cypridopsis vidua (O.F. MÜLLER, 1776) (Crustacea, Ostracoda) is not an obligate parthenogen(Royal Belgian Zoology Society, 2023-01-19) Martens, Koen ; Shribak, Michael ; Arkhipova, Irina ; Schön, IsaThe common non-marine ostracod Cypridopsis vidua (O.F. Müller, 1776) is used as a proxy in various biological disciplines, such as (palaeo-)ecology, evolutionary biology, ecotoxicology and parasitology. This morphospecies was considered to be an obligate parthenogen. We report on the discovery of the first population of C. vidua with males from Woods Hole (MA, USA) and determine that it is a population with mixed reproduction. We describe the morphology of the males and of the sexual and asexual females. We illustrate a copula of a male and a sexual female as well insemination in a sexual female, showing that males are functional. Therefore, Cypridopsis vidua is a morphospecies with mixed reproduction, not a full apomictic parthenogen. We use, for the first time, polychromatic polarization microscope technology to illustrate soft parts of ostracods. In addition, we compare the sexual species C. bisexualis, C. okeechobei, C. howei and C. schwartzi and conclude that these species, especially the latter three, are morphologically very close to C. vidua.