Polarization microscopy with the LC-PolScope

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

Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

© 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

Mapping birefringence in three dimensions using polarized light field microscopy : the case of the juvenile clamshell

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Royal Microscopical Society for personal use, not for redistribution. The definitive version was published in Journal of Microscopy 271 (2018): 315-324, doi:10.1111/jmi.12721.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.The work was supported by US federal grant GM114274 from the National Institute of General Medical Sciences.2019-06-2

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

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

Image simulation for biological microscopy: microlith

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, including coherently illuminated and incoherent, self-luminous 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.Comment: current: 17 pages, 8 figures, expanded to include biological simulations; previous version: 7 pages, 2 figures; related website: https://code.google.com/p/microlit

Point spread function of the polarized light field microscope

Author Posting. © Optical Society of America, 2022. This article is posted here by permission of Optical Society of America for personal use. Users may use, reuse, and build upon the article, or use the article for text or data mining, so long as such uses are for non-commercial purposes and appropriate attribution is maintained. The definitive version was published in Journal of the Optical Society of America A 39(6), (2022): 1095–1103, https://doi.org/10.1364/JOSAA.458034.We examined the point spread function of the polarized light field microscope and established a computational framework to solve the forward problem in polarized light field imaging, for the purpose of furthering its use as a quantitative tool for measuring three-dimensional maps of the birefringence of transparent objects. We recorded experimental polarized light field images of small calcite crystals and of larger birefringent objects and compared our experimental results to numerical simulations based on polarized light ray tracing. We find good agreement between all our experiments and simulations, which leads us to propose polarized light ray tracing as one solution to the forward problem for the complex, nonlinear imaging mode of the polarized light field microscope. Solutions to the ill-posed inverse problem might be found in analytical methods and/or deep learning approaches that are based on training data generated by the forward solution presented here.National Institute of General Medical Sciences (R01GM114274, R35GM131843)

Living cells and dynamic molecules observed with the polarized light microscope : the legacy of Shinya Inoué

Author Posting. © Marine Biological Laboratory, 2016. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 231 (2016): 85-95.In 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.Preparation of this manuscript was supported by grants from the National Institutes of Health (no. GM100160 to TT; no. GM101701 to MS; and no. GM114274 to RO); and by the Marine Biological Laboratory start-up funds from the Inoue´ Family Endowment, to TT

Polarized light imaging of birefringence and diattenuation at highresolution and high sensitivity

Polarized light microscopy provides unique opportunities for analyzing the molecular order in man-made 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 LC- PolScope 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.Comment: 21 pages, 5 figures, accepted for publication in Journal of Optic

Multiplexed spectral imaging of 120 different fluorescent labels

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)