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      Facile assembly of an affordable miniature multicolor fluorescence microscope made of 3D-printed parts enables detection of single cells

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          Abstract

          Fluorescence microscopy is one of the workhorses of biomedical research and laboratory diagnosis; however, their cost, size, maintenance, and fragility has prevented their adoption in developing countries or low-resource settings. Although significant advances have decreased their size, cost and accessibility, their designs and assembly remain rather complex. Here, inspired on the simple mechanism from a nut and a bolt, we report the construction of a portable fluorescence microscope that operates in bright-field mode and in three fluorescence channels: UV, green, and red. It is assembled in under 10 min from only six 3D printed parts, basic electronic components, a microcomputer (Raspberry Pi) and a camera, all of which can be readily purchased in most locations or online for US $122. The microcomputer was programmed in Python language to capture time-lapse images and videos. Resolution and illumination conditions of the microscope were characterized, and its performance was compared with a high-end fluorescence microscope in bright-field and fluorescence mode. We demonstrate that our miniature microscope can resolve and track single cells in both modes. The instructions on how to assemble the microscope are shown in a video, and the software to control it and the design files of the 3D-printed parts are freely available online. Our portable microscope is ideal in applications where space is at a premium, such as lab-on-a-chips or space missions, and can find applications in basic and clinical research, diagnostics, telemedicine and in educational settings.

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          Most cited references24

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          Mobile Phone Based Clinical Microscopy for Global Health Applications

          Light microscopy provides a simple, cost-effective, and vital method for the diagnosis and screening of hematologic and infectious diseases. In many regions of the world, however, the required equipment is either unavailable or insufficiently portable, and operators may not possess adequate training to make full use of the images obtained. Counterintuitively, these same regions are often well served by mobile phone networks, suggesting the possibility of leveraging portable, camera-enabled mobile phones for diagnostic imaging and telemedicine. Toward this end we have built a mobile phone-mounted light microscope and demonstrated its potential for clinical use by imaging P. falciparum-infected and sickle red blood cells in brightfield and M. tuberculosis-infected sputum samples in fluorescence with LED excitation. In all cases resolution exceeded that necessary to detect blood cell and microorganism morphology, and with the tuberculosis samples we took further advantage of the digitized images to demonstrate automated bacillus counting via image analysis software. We expect such a telemedicine system for global healthcare via mobile phone – offering inexpensive brightfield and fluorescence microscopy integrated with automated image analysis – to provide an important tool for disease diagnosis and screening, particularly in the developing world and rural areas where laboratory facilities are scarce but mobile phone infrastructure is extensive.
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            Contrast in complex images.

            E Peli (1990)
            The physical contrast of simple images such as sinusoidal gratings or a single patch of light on a uniform background is well defined and agrees with the perceived contrast, but this is not so for complex images. Most definitions assign a single contrast value to the whole image, but perceived contrast may vary greatly across the image. Human contrast sensitivity is a function of spatial frequency; therefore the spatial frequency content of an image should be considered in the definition of contrast. In this paper a definition of local band-limited contrast in images is proposed that assigns a contrast value to every point in the image as a function of the spatial frequency band. For each frequency band, the contrast is defined as the ratio of the bandpass-filtered image at the frequency to the low-pass image filtered to an octave below the same frequency (local luminance mean). This definition raises important implications regarding the perception of contrast in complex images and is helpful in understanding the effects of image-processing algorithms on the perceived contrast. A pyramidal image-contrast structure based on this definition is useful in simulating nonlinear, threshold characteristics of spatial vision in both normal observers and the visually impaired.
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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: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 October 2019
                2019
                : 14
                : 10
                : e0215114
                Affiliations
                [001]Unidad Monterrey, Centro de Investigación y de Estudios Avanzados del IPN, Parque PIIT, Apodaca, Nuevo León, Mexico
                Istituto Italiano di Tecnologia Center for Micro BioRobotics, ITALY
                Author notes

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

                Author information
                http://orcid.org/0000-0001-6442-4353
                http://orcid.org/0000-0002-3868-7405
                Article
                PONE-D-19-08507
                10.1371/journal.pone.0215114
                6786622
                31600202
                3eee85d8-c490-4561-a42b-18bc5d75b60e
                © 2019 Tristan-Landin 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
                : 25 March 2019
                : 20 September 2019
                Page count
                Figures: 8, Tables: 0, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003141, Consejo Nacional de Ciencia y Tecnología;
                Award ID: CB-256097
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003141, Consejo Nacional de Ciencia y Tecnología;
                Award ID: AEM-262771
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003141, Consejo Nacional de Ciencia y Tecnología;
                Award ID: CB-286368
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003141, Consejo Nacional de Ciencia y Tecnología;
                Award ID: LN-299051
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100008688, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional;
                Award ID: SEP FIDSC2018/104
                Award Recipient :
                This work was supported by Mexico’s National Council of Science and Technology (CONACyT) Grants No. CB-256097, CB-286368, AEM-262771, LN-299051 and INF-301274; Cinvestav Grant No. SEP FIDSC2018/104.
                Categories
                Research Article
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Bright Field Microscopy
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Engineering and Technology
                Fluidics
                Microfluidics
                Engineering and Technology
                Equipment
                Optical Equipment
                Optical Lenses
                Engineering and technology
                Electronics engineering
                3D printing
                Research and Analysis Methods
                Microscopy
                Electron Microscopy
                Transmission Electron Microscopy
                Bright Field Imaging
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Video Microscopy
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information files.

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