Abstract 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.

Citation: Cybulski JS, Clements J, Prakash M (2014) Foldscope: Origami-Based Paper Microscope. PLoS ONE 9(6): e98781. https://doi.org/10.1371/journal.pone.0098781 Editor: Lennart Martens, UGent/VIB, Belgium Received: January 25, 2014; Accepted: May 7, 2014; Published: June 18, 2014 Copyright: © 2014 Cybulski 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. Funding: Manu Prakash acknowledges support from Terman Fellowship, The Baxter Foundation, Coulter Foundation, Spectrum Foundation (NIH CTSA UL1 TR000093), C-Idea (National Institutes of Health grant RC4 TW008781-01), Bill and Melinda Gates Foundation, Pew Foundation, and Gordon and Betty Moore foundation for financial support. Jim Cybulski is supported by NIH Fogarty Institute Global Health Equity Scholars (GHES) Fellowship. James Clements was supported by NSF Graduate fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: This work is covered under patent application PCT/US2013/025612 (Title: Optical Device). Further details, including the 10,000 microscope project and updates on the latest status of this work, are available on www.foldscope.com. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Discussion and Conclusions By removing cost barriers, Foldscope provides new opportunities for a vast user base in both science education and field work for science and medicine. Many children around the world have never used a microscope, even in developed countries like the United States. A universal program providing “a microscope for every child” could foster deep interest in science at an early age. While people have known for decades that hands-on examination and inquiry is crucial in STEM (Science, Technology, Engineering, and Mathematics) education [19]–[20], the challenge posed by J. M. Bower to engage “all teachers and all children” [21] requires large-scale adoption of practices and broad availability of tools that were previously cost-prohibitive [22]. Moreover, the opportunity to make microscopes both approachable and accessible can inspire children to examine the rich bio-diversity on our planet as amateur microscopists and to make discoveries of their own, as already seen in the field of amateur astronomy ([23]; see images taken by novice user with self-made Foldscope in figure 5H–J). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Mosaic of Foldscope Images. Bright field images of (A) Giardia lamblia (2,180X), (B) Leishmania donovani (1,450X), (C) Trypanosoma cruzi (1,450X), (D) gram-negative Escherichia coli (1,450X), (E) gram-positive Bacillus cereus (1,450X), (F) Schistosoma haematobium (140X), and (G) Dirofilaria immitis (140X). Unstained (H) leg muscles and (I) tarsi of an unidentified ladybug (genus Coccinella). (J) Unstained leg muscles (fixed in formaldehyde) of an unidentified red ant (genus Solenopsis). An LED diffuser (Roscolux #111) was added for (A) and an LED condenser (2.4 mm borosilicate ball lens) was used for (C). Images (H–J) were taken by novice user using a self-made Foldscope (140X). See table 2 for ball lenses used for specific magnifications. White scale bar: 5 µm; black scale bar: 100 µm. https://doi.org/10.1371/journal.pone.0098781.g005 Disease-specific Foldscope designs are an important vision for future development [24]–[25]. Figure 5 depicts early bench-test data, including high-magnification brightfield images of Giardia lamblia, Leishmania donovani, Trypanosoma cruzi (Chagas parasite), Escherichia coli, and Bacillus cereus (figure 5A–E), and low-magnification brightfield images of Schistosoma haematobium and Dirofilaria immitis (figure 5F–G). Note that these include magnifications ranging from 140X to 2,180X, none of which require immersion oil. In the future, darkfield and fluorescence Foldscopes will also be adapted for diagnostics, and sensitivity and specificity will be measured for various disease-specific Foldscopes in the field as clinical validations against existing diagnostic standards. Constructing instruments from 2D media provides other unique advantages and opportunities. Embedding flat rare-earth magnets in paper provides means for magnetic self-alignment, allowing the Foldscope to be reversibly coupled to a conventional smartphone for image capture, for smartphone-based diagnostics, or for telemedicine [26]–[29]. By printing text and images on the paper, this platform provides an efficient information-delivery method for specific staining protocols, pathogen identification guides, or language-free folding instructions (figure S6). Some applications in highly infectious diseases may benefit from a disposable microscope ― or “use-and-throw” microscopy ― where the entire microscope can be incinerated. Also, in place of a glass slide, the 2D media also allows direct addition of the sample to a paper-based micro-fluidic assay [30] for automated staining and/or pathogen-concentration, thus yielding an independent fully-functional diagnostic system. Future work will build upon the key features of this platform. Roll-to-roll processing of flat components and automated “print-and-fold” assembly make yearly outputs of a billion units attainable. Ongoing work with advanced micro-optics and illumination design ― including spherical GRIN lenses [31]–[32], aspheric multi-lens optics, and condenser lens provisions for Köhler illumination ― is expected to improve both resolution and field of view at low cost. International field-work in both diagnostics and education will provide vital inputs for further improvements. Our long-term vision is to universalize frugal science, using this platform to bring microscopy to the masses.

Materials and Methods Ball Lenses The ball lenses used in constructing Foldscopes included material types borosilicate, BK7 borosilicate, sapphire, ruby, and S-LAH79. The vendors included Swiss Jewel Co, Edmund Optics, and Winsted Precision Ball. Part numbers for some select lenses include: 300 µm sapphire lens from Swiss Jewel Co. (Model B0.30S), 200 µm sapphire lenses from Swiss Jewel Co. (Model B0.20S), 2.4 mm borosilicate lenses from Winsted Precision Ball (P/N 3200940F1ZZ00A0), 300 µm BK7 borosilicate lenses from Swiss Jewel Co. (Model BK7-0.30S), and 1.0 mm BK7 borosilicate lenses from Swiss Jewel Co. (Model BK7-1.00). Note that half-ball lenses from both Edmund Optics and Swiss Jewel Co. were also tested for use as condenser lenses for the LEDs. 2D Media and Filters The 2D media used in constructing Foldscopes included black 105 lb card stock (ColorMates Smooth & Silky Black Ice Dust Card Stock, purchased from thePapermillstore.com), polypropylene (PressSense Durapro CC 10mil), and others. Foldscope parts were cut from 2D media using a CO 2 laser (Epilog Elite, Mini24). Copper tape was used for providing connectivity (by soldering) between the LED, battery, and switch. The filters used in constructing Foldscopes included Roscolux colored gel filters (including Primary Blue #80 and Fire Red #19, which approximate an Acridine Orange filter set), Roscolux diffuser filters (Tough Rolex #111), and polymeric linear polarizers (Edmund Optics P/N 86181). Each type of filter is assembled to the Foldscope by cutting out a 3–5 mm square piece and adhesively attaching it to the appropriate stage with single-sided or double-stick Scotch tape. Paper microscope slides were constructed from polypropylene sheets (PressSense Durapro CC 18mil) and transparent scotch tape. LEDs, Switches and Power Sources The LEDs used in constructing Foldscopes included the Avago HSMW-CL25 (now replaced by P/N Avago ASMT CW40) white LED for brightfield Foldscopes, the Kingbright APTD1608QBC/D blue LED for fluorescence Foldscopes. The electrical slider switch was purchased from AliExpress.com (“Off/On MINI SMD Switch”, Product ID: 665019103). The power sources included Duracell 3V CR2032 button cells, Sanyo 3V CR2016 button cells (Sanyo CR2016-TT1B #8565 from Batteriesandbutter.com), and a GW Instek DC power supply (Model GPD-3303D). Button cells were used with no resistors for Foldscopes. Aperture Manufacturing This method produces inexpensive apertures through polymer encapsulation of ball lenses while preserving the optical quality of the lens and allowing multiple lenses to be encapsulated at once. The experimental setup shown in the top left of figure 3A was used to encapsulate 300 µm sapphire ball lenses with aperture diameters ranging from 100 µm to 214 µm. The lens was sandwiched between parallel substrates (glass or silicon) coated with planar films of PDMS with thickness greater than 1 mm (formed from Dow Corning Sylgard 184 PDMS). A micrometer stage was used to precisely apply pressure between the substrates to adjust the diameter of the resulting elastic deformation of the PDMS film. This diameter was measured in situ using phase contrast microscopy to set the target value for the aperture. A fast-curing opaque polymer (Smooth-On Smooth-Cast Onyx Fast Polyurethane) was then injected into the cavity and allowed to cure. Reflected light microscopy was used to measure the dimensions of the final aperture formed. Once removed from the non-stick PDMS films, the encapsulated lens was attached to the underside of the optics stage of a Foldscope Characterization of Self-Alignment by Folding Twenty independent microscopes were cut out of black 105 lb cardstock, each marked with a cross-hair in both the optics and illumination stages (see figure S2C). After folding, alignment was measured using a dissection microscope (Olympus upright, 30× magnification) via digitizing the cross-hair images, drawing lines through the center of each cross-hair (X and Y cross-hairs on both stages), and digitally measuring the X and Y displacements to characterize the alignment. Every Foldscope was iteratively folded, imaged to record X–Y alignment, and unfolded twenty times. The data was then used to assess accuracy and repeatability (see figure S2A,B). Sample Preparation Thin-blood smears of Plasmodium falciparum (ring stage), Trypanosoma cruzi, Giardia lamblia, Leishmania donovani, and Dirofilaria immitis were freshly prepared from cultures provided by Center for Discovery and Innovation in Parasitic Diseases (CDIPD) at UCSF. The samples were fixed in methanol and stained in freshly prepared Giemsa solution (Sigma Aldrich, #48900-500ML-F) using standard protocols before imaging. Once fixed, the slides could be used for several weeks. Bacterial samples of Bacillus cereus and Escherichia coli were provided by KC Huang Lab at Stanford University. The samples were heat fixed onto glass slides using standard procedures and gram stained using standard protocols (Fisher Scientific Gram Stain Set, Catalog No. 23-255-959). Plasmodium-infected red blood cells were taken from cultures provided by the Center for Discovery and Innovation in Parasitic Diseases (CDIPD). Schistosoma haematobium were provided by the Michael Hsieh Lab at Stanford University. Insects used for imaging were caught on Stanford campus and imaged after fixing in formaldehyde without any stain. No human samples were utilized in the current work. Image-Capture Protocol Brightfield images were taken using a Canon EOS 5D Mark II with the Foldscope placed 3 cm away from the 100 mm focal length lens and using the following settings: F/3.2, 1/30 sec. exposure, ISO-2000. An initial image was first captured using automatic white-balance and then used as a reference white balance image during data collection. Fluorescence images were taken in a similar fashion to the brightfield images with typical camera settings: F/2.8, 15 sec. exposure, ISO-1000. Although not presented, images were also obtained by coupling the Foldscope to cell-phones including an iPhone using a custom magnetic coupler. USAF 1951 resolution target data was taken using GUPPY Pro 503C scientific camera, with 2592×1944 pixels and pixel size 2.2×2.2 µm2, using the setup shown in Figure S7. Numerical Model Zemax software was used to model the Foldscope optics to assess optimal aperture radius and resolution. The basic model of the system consists of a ball lens, an aperture, an object at infinity, and an image plane (see figure S5A). This model requires two parameters to be independently optimized ― lens-image distance and aperture radius. The analysis is carried out in four steps: 1) optimize lens-image distance in model by minimizing focusing metric (figure S5C); 2) determine search space for aperture radius as defined by empirically chosen limits on Strehl Ratio, 0.75–0.98; 3) optimize aperture radius using resolution metric (figure S5D); and 4) use Matlab surface-fitting tool to fit data for optical performance parameters as functions F(n,r, λ) and compare with analytical model.

Supporting Information Figure S1. Foldscope Schematics. (A) Real image formation via projection. (B) Virtual image formation via direct observation with the eye. Note the drawings are not to scale. The indicated lengths are example values that show the versatility of this design as well as its extreme space efficiency. For example, the same system can be used for projecting or imaging simply by changing the object-lens distance by about 20 µm. Also, notice the total path length from the LED to the lens is almost an order of magnitude smaller than the size of the human eye. https://doi.org/10.1371/journal.pone.0098781.s001 (TIF) Figure S2. Characterization of Self-Alignment by Folding. Twenty independent Foldscopes were constructed out of 350 µm thick black cardstock and manually folded and unfolded twenty times each, with alignment measured after each assembly. The data was used to produce plots of (A) assembly repeatability (distribution of all 400 values, adjusted to give zero mean for each Foldscope) and (B) assembly accuracy (distribution of 20 mean values calculated per Foldscope) using (C) cross-hair alignment features on the optics and illumination stages. Note that the span of the data in both plots is less than the thickness of the paper used to construct the Foldscopes. Based on the data shown in the plots, assembly repeatability was assessed as the mean value of twice the standard deviation for each Foldscope (65 µm in X and 25 µm in Y), while assembly accuracy was assessed as the mean value of all trials (59 µm in X and 67 µm in Y). A higher skew in X-axis repeatability results from structurally distinct constraints implemented for the X- and Y-axes, while the assembly accuracy errors in both directions are consequences of the design which can be compensated by feature shifts in future designs. Note that the X and Y error bars for all measurements are 8.4 µm. https://doi.org/10.1371/journal.pone.0098781.s002 (TIF) Figure S3. Component Characterization. (A) LED voltage and intensity versus time for a white LED (Avago HSMW-CL25) powered by a Duracell CR2032 battery with no resistor. (B) Filter transmission spectra of three Roscolux filters ― Tough Rolex diffuser (#111), Fire Red (#19), and Primary Blue (#80) ― measured with Ocean Optics Photo spectrometer USB4000. (C) Intensity profile of a white LED (Avago HSMW-CL25) as visualized in water with dissolved fluorescein. The left image is taken with the bare LED while the right image is taken with a condenser lens (2.4 mm borosilicate ball lens) placed adjacent to the LED in the optical path, demonstrating that a ball lens can be used to effectively collimate the light emitted by this LED. https://doi.org/10.1371/journal.pone.0098781.s003 (TIF) Figure S4. Numerical Modeling Characterization of Optimal Field of View. (A) Plot of Wavefront Error over the full field of view defined by the aperture for a 300 µm Sapphire ball lens with a 147 µm aperture. With increasing field coordinate, the Wavefront Error becomes very large and the image will be out of focus. An “optimal field of view” is defined at a field coordinate of 21 µm, where the Wavefront Error is approximately 1/5 wave number. (B,C) Plots of Field Curvature and Distortion over the optimal field of view. (D) Plot of RMS spot size over the optimal field of view depicting four cases: optimized solution treated as reference with zero defocus (red line), defocus of 3 µm (green line), defocus of 3 µm (green line), diffraction limit (dashed black line). The reference solution provides the best achievable resolution at the center of the field of view (approximately equal to the diffraction limit for this choice of aperture), while other plots show that increasing defocus moves the region of best resolution radially out from the center in an annular ring. (E) Plot of RMS spot size over the optimal field of view depicting optimal aperture predicted by analytical model (red lines) and adjusted aperture giving uniform RMS spot size over the field of view (purple lines). https://doi.org/10.1371/journal.pone.0098781.s004 (TIF) Figure S5. Diagrams and Plots for Numerical and Analytical Models. (A) Schematic of time-reversed Zemax model showing collimated light coming from an object at infinity, passing through aperture, and focused by the ball lens onto a focal point in the image plane. (B) Schematic of time-reversed model showing key parameters used in some derivations for the analytical model. (C) Plot of Focusing Metric versus Lens-Image Distance for λ = 0.55 µm, r = 150 µm, n = 1.517. This illustrates how focusing metrics FM1, FM2, and FM3 select different values for the optimal lens-image distance. (D) Plot of Resolution Metric versus Aperture Radius for λ = 0.55 µm, r = 150 µm, n = 1.517. This illustrates how resolution metrics RM1 and RM2 select nearly the same aperture radius but yield different values for resolution. https://doi.org/10.1371/journal.pone.0098781.s005 (TIF) Figure S6. Artistic Layout of Foldscope Paper Components. Artistic version of Foldscope layout with integrated universal folding instructions based on color coding, where like colors are matched during the folding process to leave a single solid color in the final folded configuration. https://doi.org/10.1371/journal.pone.0098781.s006 (TIF) Figure S7. Foldscope Image Capture Setup for Resolution Metric. Picture of the experimental setup used to caputure images of the USAF 1951 resolution target viewed with the Foldscope. The data was taken using GUPPY Pro 503C scientific camera, with 2592×1944 pixels and pixel size 2.2×2.2 µm2. https://doi.org/10.1371/journal.pone.0098781.s007 (TIF) Figure S8. Quick Reference Guide for Foldscope Assembly. One-page handout to facilitate users in guided assembly of a Foldscope. https://doi.org/10.1371/journal.pone.0098781.s008 (PDF) Video S1. Foldscope Assembly. A short video of 140X Brightfield Foldscope assembly process. https://doi.org/10.1371/journal.pone.0098781.s009 (M4V) Video S2. Foldscope Drop Test and ruggedness. A short video of a three story drop test and ruggedness of Foldscope demonstrated by stomping under feet. https://doi.org/10.1371/journal.pone.0098781.s010 (AVI) Text S1. Supplementary information. Analytical expressions and derivations related to optical performance metrics. https://doi.org/10.1371/journal.pone.0098781.s011 (DOCX)

Acknowledgments We thank all members of the Prakash lab for valuable suggestions. We acknowledge Ioana Urama for assistance with supplementary figure S6, Anika Radiya for assistance with magnetic couplers and Marisa Borja for taking images in figure 5H–J, and Center for Discovery and Innovation in Parasitic Diseases (CDIPD) at UCSF and the Michael Hsieh Lab at Stanford University for supplying samples. This work is covered under patent application PCT/US2013/025612 (Title: Optical Device). Further details, including the 10,000 microscope project and updates on the latest status of this work, are available on www.foldscope.com.

Author Contributions Conceived and designed the experiments: JSC MP. Performed the experiments: JSC JC MP. Analyzed the data: JSC JC MP. Wrote the paper: JSC MP.