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      An Assemblable, Multi-Angle Fluorescence and Ellipsometric Microscope

      research-article
      1 , 2 , 1 , 2 , 3 , *
      PLoS ONE
      Public Library of Science

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          Abstract

          We introduce a multi-functional microscope for research laboratories that have significant cost and space limitations. The microscope pivots around the sample, operating in upright, inverted, side-on and oblique geometries. At these geometries it is able to perform bright-field, fluorescence and qualitative ellipsometric imaging. It is the first single instrument in the literature to be able to perform all of these functionalities. The system can be assembled by two undergraduate students from a provided manual in less than a day, from off-the-shelf and 3D printed components, which together cost approximately $16k at 2016 market prices. We include a highly specified assembly manual, a summary of design methodologies, and all associated 3D-printing files in hopes that the utility of the design outlives the current component market. This open design approach prepares readers to customize the instrument to specific needs and applications. We also discuss how to select household LEDs as low-cost light sources for fluorescence microscopy. We demonstrate the utility of the microscope in varied geometries and functionalities, with particular emphasis on studying hydrated, solid-supported lipid films and wet biological samples.

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          Single-cell phenotyping within transparent intact tissue through whole-body clearing.

          Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies. Copyright © 2014 Elsevier Inc. All rights reserved.
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            High precision scanning ellipsometer.

            We describe the design, construction, alignment, and calibration of a photometric ellipsometer of the rotating-analyzer type. Data are obtained by digital sampling of the transmitted flux with an analog-to-digital converter, followed by Fourier transforming of the accumulated data with a dedicated minicomputer. With an operating mechanical rotation frequency of 74 Hz, a data acquisition cycle requires less than 7 msec. The intrinsic precision attainable is high because precision is limited only by shot noise or intrinsic source instabilities, even when relatively weak continuum lamps are used as light sources. Precision may be improved by accumulating the data for consecutive cycles at a fixed wavelength. The system allows complex reflectance ratios to be determined as continuous functions of wavelength from the near infrared to the near ultraviolet spectral range. Data reduction programs can be modified to calculate complex refractive index or dielectric function spectra, or film thicknesses and refractive indices, as well as the usual ellipsometric parameters tanpsi, cosDelta.
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              • Record: found
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              Foldscope: Origami-Based Paper Microscope

              Here we describe an ultra-low-cost origami-based approach for large-scale manufacturing of microscopes, specifically demonstrating brightfield, darkfield, and fluorescence microscopes. Merging principles of optical design with origami enables high-volume fabrication of microscopes from 2D media. Flexure mechanisms created via folding enable a flat compact design. Structural loops in folded paper provide kinematic constraints as a means for passive self-alignment. This light, rugged instrument can survive harsh field conditions while providing a diversity of imaging capabilities, thus serving wide-ranging applications for cost-effective, portable microscopes in science and education.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2016
                1 December 2016
                : 11
                : 12
                : e0166735
                Affiliations
                [1 ]Keck Science Department, Scripps College, Claremont, CA, United States of America
                [2 ]Keck Science Department, Claremont McKenna College, Claremont, CA, United States of America
                [3 ]Keck Science Department, Pitzer College, Claremont, CA, United States of America
                Pennsylvania State Hershey College of Medicine, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: VN JR BS.

                • Formal analysis: VN JR BS.

                • Funding acquisition: BS.

                • Investigation: VN JR.

                • Methodology: VN JR BS.

                • Project administration: BS.

                • Resources: VN JR BS.

                • Software: BS.

                • Supervision: BS.

                • Validation: VN JR.

                • Visualization: BS.

                • Writing – original draft: VN BS.

                • Writing – review & editing: VN BS.

                Author information
                http://orcid.org/0000-0002-8265-5988
                Article
                PONE-D-16-31907
                10.1371/journal.pone.0166735
                5132209
                27907008
                55cabaae-92ee-4332-b6ef-7cac4f7b007c
                © 2016 Nguyen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 August 2016
                : 2 November 2016
                Page count
                Figures: 5, Tables: 1, Pages: 15
                Funding
                Funded by: Keck Science Department Startup
                Award Recipient :
                Funded by: S. D. Bechtel, Jr. Foundation and Stephen Bechtel Fund (US)
                Award ID: 5666
                Award Recipient :
                This project was primarily funded through startup funds via the Department of Keck Science ( https://www.kecksci.claremont.edu). Additionally, one of the co-authors (Victoria Nguyen, VN) was funded for a summer through the S.D. Bechtel, Jr. Foundation ( http://sdbjrfoundation.org, Grant ID #5666). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
                Engineering and technology
                Electronics engineering
                3D printing
                Biology and Life Sciences
                Biochemistry
                Lipids
                Engineering and Technology
                Equipment
                Optical Equipment
                Optical Lenses
                Engineering and Technology
                Equipment
                Optical Equipment
                Cameras
                Research and Analysis Methods
                Microscopy
                Inverted Microscopy
                Physical Sciences
                Physics
                Electromagnetic Radiation
                Luminescence
                Fluorescence
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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