Quantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation

Author Posting. © Optical Society of America, 2013. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Optical Society of America A: Optics, Image Science, and Vision 30 (2013): 769-782, doi:10.1364/JOSAA.30.000769.We 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.This publication was made possible by Grant No. R01-GM101701 from the National Institute of General Medical Sciences, National Institutes of Health

Polychromatic polarization microscope : bringing colors to a colorless world

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 5 (2015): 17340, doi:10.1038/srep17340.Interference 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.This publication was made possible by Grant Number R01-GM101701 from the National Institute of General Medical Sciences, National Institutes of Health

Orientation-independent differential interference contrast microscopy

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Applied Optics 45 (2006): 460-469, doi:10.1364/AO.45.000460.The 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

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

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

Author Posting. © Society of Photo Optical Instrumentation Engineers, 2017. This article is posted here by permission of Society of Photo Optical Instrumentation Engineers for personal use, not for redistribution. The definitive version was published in Journal of Biomedical Optics 22 (2017): 016006, doi:10.1117/1.JBO.22.1.016006.We 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.This publication was made possible by Grant No. R01-GM101701 from the National Institute of General Medical Sciences, National Institutes of Health

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

Quantitative orientation-independent differential interference contrast (DIC) microscopy coupled with orientation-independent Polarization microscopy

Author Posting. © Microscopy Society of America, 2007. This article is posted here by permission of Cambridge University Press for personal use, not for redistribution. The definitive version was published in Microscopy and Microanalysis 13 Suppl. 2 (2007): 10-11, doi:10.1017/S1431927607075186.Differential 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

Entropy-driven formation of a chiral liquid-crystalline phase of helical filaments

Author Posting. © The Authors, 2006. This article is posted here by permission of American Physical Society for personal use, not for redistribution. The definitive version was published in Physical Review Letters 96 (2006): 018305, doi:10.1103/PhysRevLett.96.018305.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.M. S. and R. O. are supported by NIH Grant No. EB002583

Machine-learning-based evaluation of intratumoral heterogeneity and tumor-stroma interface for clinical guidance

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Laurinavicius, A., Rasmusson, A., Plancoulaine, B., Shribak, M., & Levenson, R. Machine-learning-based evaluation of intratumoral heterogeneity and tumor-stroma interface for clinical guidance. American Journal of Pathology, 191(10), (2021): 1724–1731, https://doi.org/10.1016/j.ajpath.2021.04.008.Assessment 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.Supported by the European Social Fund grant 09.3.3-LMT-K-712

Real-time polarization microscopy of fibrillar collagen in histopathology

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Keikhosravi, A., Shribak, M., Conklin, M. W., Liu, Y., Li, B., Loeffler, A., Levenson, R. M., & Eliceiri, K. W. Real-time polarization microscopy of fibrillar collagen in histopathology. Scientific Reports, 11(1), (2021): 19063, https://doi.org/10.1038/s41598-021-98600-w.Over 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.This work was supported by NIH R01 CA238191 (KWE), NIH P41GM135019 (KWE), NIH R01 GM101701 (MS), funding from the Morgridge Institute for Research (KWE), the Semiconductor Research Corporation (SRC) (KWE), and the William T. Golden Endowment (MS)