Research Paper Complete Parts Of Microscope

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.

Introduction

Microscopes are ubiquitous tools in science, providing an essential, visual connection between the familiar macro-world and the remarkable underlying micro-world. Since the invention of the microscope, the field has evolved to provide numerous imaging modalities with resolution approaching 250 nm and smaller [1]. However, some applications demand non-conventional solutions due to contextual challenges and tradeoffs between cost and performance. For example, in situ examination of specimens in the field provides important opportunities for ecological studies, biological research, and medical screening. Further, ultra-low cost DIY microscopes provide means for hands-on science education in schools and universities. Finally, this platform could empower a worldwide community of amateur microscopists to capture and share images of a broad range of specimens.

Cost-effective and scalable manufacturing is an integral part of “frugal science and engineering” [2]. For example, manufacturing via folding has emerged as a powerful and general-purpose design strategy with applications from nanoscale self-assembly [3] to large-aperture space telescopes [4]. More recently, possibilities of folding completely functional robots have been explored [5]–[7], with actuators, sensors and flexures integrated in a seamless fashion. Modern micro-lens fabrication technology is another prime example of scalable manufacturing. Although the use of high-curvature miniature lenses traces back to Antony van Leeuwenhoek's seminal discovery of microbial life forms [8], manufacturing micro-lenses in bulk was not possible until recently. Modern techniques such as micro-scale plastic molding and centerless ball-grinding have grown to serve numerous applications, including telecommunication fiber couplers, cell phone cameras, and medical endoscopes.

By combining principles of optical design with origami [9]–[11], here we present a novel platform for the fabrication of flat microscopes cheaply in bulk (figure 1). The Foldscope is an origami-based optical microscope that can be assembled from a flat sheet of paper in under 10 minutes (see video S1, figure S8). Although it costs less than a dollar in parts (see Bill of Materials in table 1), it can provide over 2,000× magnification with submicron resolution, weighs less than two nickels (8.8 g), is small enough to fit in a pocket (70×20×2 mm3), requires no external power, and can survive being dropped from a 3-story building or stepped on by a person (see figure 1G and video S2). Its minimalistic, scalable design is inherently application-specific instead of general-purpose, providing less functionality at dramatically reduced cost. Using this platform, we present our innovations for various imaging modalities (brightfield, darkfield, fluorescence, lens-array) and scalable manufacturing strategies (capillary encapsulation lens mounting, carrier tape lens mounting, self-alignment of micro-optics by folding, paper microscope slide).

Figure 1. Foldscope design, components and usage.

(A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.

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The Foldscope is operated by inserting a sample mounted on a microscope slide (figure 1B), turning on the LED (figure 1C), and viewing the sample while panning and focusing with one's thumbs. The sample is viewed by holding the Foldscope with both hands and placing one's eye close enough to the micro-lens so one's eyebrow is touching the paper (figure 1F). Panning is achieved by placing one's thumbs on opposite ends of the top stage (colored yellow in figure 1A–C) and moving them in unison, thus translating both optics and illumination stages while keeping the stages aligned (figure 1B). Focusing is achieved using the same positioning of one's thumbs, except the thumbs are pulled apart (or pushed together). This causes tension (or compression) along the optics stage, resulting in −Z (or +Z) deflection of the micro-lens due to flexure of the supporting structure of the sample-mounting stage (figure 1C). Unlike traditional microscopes, the Foldscope anchors the sample at a fixed location while the optics and illumination stages are moved in sync.

Results

Design Platform

Construction from flat media.

The Foldscope is comprised of three stages cut from paper ― illumination, sample-mounting, and optics ― and assembled via folding (figure 1A–C, video S1). Other primary components include a spherical ball lens (or other micro-lenses), lens-holder apertures, an LED with diffuser or condenser lens, a battery, and an electrical switch (see figure 1D). The three stages are weaved together to form an assembled Foldscope (figure 1B,C,E) with the following features: fully-constrained X–Y panning over a 20×20 mm2 region (figure 1B), flexure-based focusing via Z-travel of the optics stage relative to the sample-mounting stage (figure 1C), and a vernier scale for measuring travel distances across the sample slide with 0.5 mm resolution. The total optical path length from the light source to the last lens surface is about 2.7 mm (figure S1), only 1% that of a conventional microscope. Flat polymeric sheets and filters can also be inserted into the optical path, including diffusion filters for improving illumination uniformity, Fresnel lenses as condensers for concentrating illumination intensity, color filters for fluorescence imaging, and linear polarizers for polarization imaging.

Alignment by Folding.

Folding provides a passive alignment mechanism that is used here to align the micro-lens with the light source. A sharp crease in a thin sheet of inextensible material, such as paper, of thickness h introduces elastic energy of bending of the order ∼h3[12]. Thus, a fold introduces buckling at the inner edge, giving variation in the exact location of the hinge and resulting in random alignment error of the order ∼h. To minimize this error, we introduce folding features that form a closed structural loop between the optics stage and the illumination stage. This improves alignment repeatability through elastic averaging within kinematic constraints (figure 1A; [13]). We characterized alignment accuracy and repeatability by constructing twenty independent Foldscopes out of 350 µm thick black cardstock and manually folding and unfolding them twenty times each (see Materials and Methods section), while measuring absolute X–Y alignment (figure S2). 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. The small assembly accuracy errors (less than 20% of the paper thickness) in both directions are consequences of the design which can be compensated by feature shifts in future designs.

Micro-Optics and Illumination.

The Foldscope design accommodates different optical configurations, including spherical ball lenses, spherical micro-lens doublets (such as a Wollaston doublet), and more complex assemblies of aspheric micro-lenses. While more optical elements generally provide reduced aberration and improved field of view, spherical ball lenses have distinct advantages for high-volume manufacturing, including reduced part count and simplified assembly due to rotational symmetry [14]–[16]. Since magnification varies inversely with ball-lens diameter, commonly available ball lenses provide an ample range of magnifications (under 100X to over 2,000X, as seen in table 2). The back focal length of these lenses varies drastically, thus motivating alternative lens-mounting schemes (above the optics stage, as in figure 1C, or below) and requiring samples with no coverslip for lenses with less than approximately 140 µm back focal length. Equally important for image quality, the illumination source (LED plus diffuser and/or condenser lens) should provide even illumination over the field, ample intensity, narrow intensity profile, and high CRI (color rendering index). The LED used in the Foldscope consumes only 6 mW of electrical power and can operate over 50 hours on a CR2032 button cell battery (figure S3A). Precise control over the illumination profile is required for high-quality microscopy [17], so integration of a condenser lens is crucial for optimal imaging (figure S3C). For low-magnification imaging applications not requiring optimal imaging, the illumination source can be removed and the Foldscope can be operated while facing an external light source.

Design Innovations

High-Resolution Brightfield Microscopy.

For some applications, extending the resolution limit of the Foldscope to submicron length scales is a practical necessity. For this reason, the resolution of the single-ball-lens Foldscope was further optimized and empirically characterized. The analytical optimization was carried out for a single field point at the optical axis to assess the best achievable resolution (see Modeling and Characterization and table 2). A 1,450X Brightfield Foldscope with the configuration depicted in figure 2E was used to capture the image in figure 2A, empirically confirming submicron resolution. As shown in figure S4A, spherical ball lenses have significant wavefront error at the edge of the field defined by the aperture (aperture shown in figure S1). As a result, not all regions can be simultaneously in focus within this field. The center portion of the field, with wavefront error less than 1/5 wavenumber and low curvature and distortion, is denoted the “optimal field of view” (figure S4A–C). Thus, the best achievable resolution is attained at the expense of a reduced field of view. When a digital sensor is used in place of the naked eye, the lens fixture effectively reduces the field of view to roughly the optimal field of view.

Figure 2. Foldscope imaging modalities.

(A) Brightfield Foldscope image of a monolayer of 1 µm polystyrene microspheres (Polysciences 07310-15) using a 1,450X lens. (B) Fluorescent Foldscope image of 2 µm polyfluorescent microspheres (Polysciences 19508-2) using a 1,140X lens with Roscolux gel filters #19 and #80. (C) 2X2 lens-array Brightfield Foldscope image of Giemsa-stained thin blood smear using 1,450X lenses. (D) 140X Darkfield Foldscope images of 6 µm polystyrene microspheres (Polysciences 15714-5), using a 140X lens for the darkfield condenser. Darkfield condenser aperture shown in inset has 1.5 mm inner diameter and 4.0 mm outer diameter. (E–H) Schematic cross-sections of Brightfield, Fluorescence, Lens-Array, and Darkfield Foldscope configurations, showing the respective arrangements of ball lenses, filters, and LEDs. See table 2 for ball lenses used for specific magnifications.

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Fluorescence.

Conventional fluorescence microscopy typically requires an expensive illumination source for high-intensity broad-spectrum illumination and multiple optical elements with precisely defined spectral profiles. The simplified configuration of the Fluorescence Foldscope uses a high-intensity colored LED of narrow spectral width and polymeric sheets inserted in the optical path for a shortpass excitation filter and a longpass emission filter (figure 2B,F). A blue LED light source and commonly available gel filters (with spectral transmissivities plotted in figure S3B) were used to image 2 µm diameter red poly-fluorescent polystyrene beads as shown in figure 2B. For fluorescent imaging requiring higher contrast, small pieces (3 mm square or smaller) of interference filters can be used in place of the polymeric sheets at reasonable cost due to the small size.

Lens-array and Multi-modality.

Since a micro-lens has a very small footprint, multiple optical paths can be independently configured in a single Foldscope (figure 2C,G). Such a lens array may be comprised of identical lenses or of different lenses with different magnifications and/or back focal lengths. This provides for the design of a lens-array Foldscope with several key features. For non-contiguous samples such as blood smears, a larger field of view can be obtained by overlapping a number of small fields of view. Alternatively, an optimal array pitch will give tangential non-overlapping fields of view (as seen in figure 2C), thus reducing the time required to scan a slide for a feature such as a parasite. Since individual lenses have independent optical paths, the novel capability of building multi-modality lens-array microscopes arises. One such combination is a two-by-one array of brightfield and fluorescence modalities, which could be used to scan a sample for the presence of fluorescence markers and then identify the non-fluorescent surrounding structures.

Darkfield.

The Darkfield Foldscope configuration, shown in figure 2H, requires a diffuser, a darkfield condenser aperture (inset in figure 2D), and a condenser lens. The diffuser helps to evenly distribute light from the small LED over the aperture area, while the condenser focuses a hollow cone of light onto the specimen. Since the specimen must be placed at the focal point of the condenser, the slide thickness has to match the back focal length of the condenser plus the spacing from the condenser to the slide. A 140X Darkfield Foldscope was used to image 6 µm polystyrene microspheres as shown in figure 2D.

Capillary Encapsulation Lens Mounting.

The process of precisely mounting micro-optics to an aperture crucially governs lens performance. Therefore, a capillary encapsulation process was developed to automatically mount a ball lens while forming a circular aperture of precisely tunable diameter (see figure 3A). By partially engulfing ball lenses in an opaque polymer held between two glass substrates coated with flat nonstick PDMS (see top left of figure 3A), a precise aperture is self-assembled around the ball-lens (see Materials and Methods section for details). Pressure applied between the substrates is used to precisely tune aperture size, with greater pressure providing a larger aperture. The epoxy encapsulated lens is then adhesively mounted to a paper aperture and inserted in the Foldscope (see bottom right of figure 3A).

Figure 3. Manufacturing innovations for lens- and specimen- mounting.

(A) Fabrication, mounting, and characterization of capillary-encapsulation process for lens-mounted apertures. X and Y error bars for all measurements are 2.5 µm. (B) Reel of polystyrene carrier tape with custom pockets and punched holes for mounting over 2,000 ball lenses with optimal apertures. The first ten pockets include mounted ball lenses. Inset shows sectioned view from CAD model of carrier tape mounted lenses. Note the aperture is the punched hole shown on the bottom side of the ball lens. This tape is 16 mm wide and is designed for 2.4 mm ball lenses (aperture diameter is 0.7 mm). (C) Top: Paper microscope slide shown next to standard glass slide with coverslip, both with wet mount algae specimens. Bottom: Schematic of paper microscope slide, showing specimen containment cavity formed between upper tape and lower tape in middle of slide.

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Carrier Tape Lens Mounting.

Black polystyrene carrier tape is commonly used for low-cost reel-to-reel packaging of electronic components. This pre-existing infrastructure was leveraged to create low-cost mounting structures for ball lenses with optimized apertures. As depicted in figure 3B, the custom thermoformed pocket holds the lens in place with a press fit, and a punched hole in the bottom of the pocket precisely defines the aperture. A single lens is cut from the carrier tape and adhesively attached to a paper aperture which is then inserted into the optics stage of the Foldscope, analogous to that shown for the epoxy encapsulated lens in figure 3A.

Paper microscope slide.

A low-cost microscope slide (similar to that in [18]) was constructed out of 18mil polystyrene synthetic paper and transparent tape as shown in figure 3C. If tape is placed on only one side (either upper or lower), specimens are conveniently mounted on the exposed sticky surface of the tape. Using both upper and lower pieces of transparent tape creates a cavity for mounting wet specimens such as live algae suspended in water. Since the paper microscope slide is less than half the thickness of a standard glass slide, a spacer is required to elevate the sample closer to the lens. This is achieved by inserting the specimen slide together with two blank paper slides beneath it. Once the specimen has been viewed, the transparent tape can be removed from the synthetic paper and replaced so that the slide can be reused for many specimens. Note that the specimen depicted in figure 2D was mounted on a paper microscope slide.

Modeling and Characterization

Theory and analysis.

For a brightfield Foldscope, basic measures of optical performance can be described in terms of the ball radius (r), index of refraction (n), aperture radius (a), and incident wavelength (λ; see text S1). Assuming the paraxial approximation, these include effective focal length (EFL), back focal length (BFL), and magnification (MAG). For a 300 µm sapphire ball lens: EFL = 172 µm, BFL = 22 µm, and MAG = 1,450X. Thus substantial magnification can be obtained, but the sample must be separated from the lens by only a fraction of the thickness of a human hair. Three additional optical performance metrics include field of view radius (FOV), numerical aperture (NA), and depth of field (DOF). These depend on aperture radius (a), the optimization of which is discussed below. For the previous example, the normalized optimal aperture radius is nOAR = a/r = 0.51, giving: FOV = 88 µm, NA = 0.44, DOF = 2.8 µm (see table 2).

The aperture radius controls the balance between diffraction effects from the edges of the aperture with spherical aberration effects from the lens. Therefore, a complete analytical model was created to predict the normalized optimal aperture radius (nOAR) and optimal resolution (RES), as well as the aberration coefficient (s) for a ball lens (see Supplementary Materials and table 2), yielding:The expressions for nOAR and RES are depicted as 2D design plots as a function of desired MAG in figure 4A,B and figure 4C shows a 3D plot of RES over n and r. For the example discussed earlier, the values for normalized aperture radius and resolution are found by locating the intersection of the lines for r = 150 µm and n = 1.77 in the design plots. This gives nOAR = 0.51 and RES = 0.86 µm, and corresponds to MAG = 1,450X. Note that the regions enclosed by the curves in the 2D design plots represent the available design space for nOAR, RES, and MAG as defined by the range of possible values for n and r. The design curves thus make it a simple exercise to pick optimal design parameters within the space of interest. Also, one can see from figure 4B that the lower limit for the best achievable resolution in ball lenses appears to be near 0.5 µm, based on the range of parameters identified for this figure.

Figure 4. Analytical, numerical, and empirical characterization of Foldscope.

(A,B) Analytical “design curves” for normalized optimal aperture radius (nOAR) and optimal resolution (RES) versus magnification (MAG) over index of refraction (range 1.33–1.91) and ball lens radius (range 40–1200 µm). (C) Comparison of analytical (3D surface) and numerical (plotted as points) results for RES versus index of refraction and ball lens radius. (D) Modulus of the Optical Transfer Function (MTF) over the optimal field of view for a 300 µm sapphire lens with optimal aperture. (E,F) Image of USAF 1951 resolution target taken with 430X ball lens, including an enlarged caption of Group 9, and an intensity profile plot along path denoted by green line in image caption. This demonstrates resolvability for Group 9, Element 4 corresponding to 724 Line Pairs/mm or 1.38 µm resolution. (G,H) Image of USAF 1951 resolution target taken with 140X ball lens, including an enlarged caption of Group 8, and an intensity profile plot along path denoted by green line in image caption. This demonstrates resolvability for Group 8, Element 6 corresponding to 456 Line Pairs/mm or 2.19 µm resolution. The data was taken using GUPPY Pro 503C scientific camera, with 2592×1944 pixels and pixel size 2.2×2.2 µm2.

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Numerical Modeling.

A ray-tracing numerical model was developed for the Foldscope using Zemax software to confirm the results of the analytical model and to evaluate points across the field of view (see Materials and Methods section for details). The results for nOAR and RES show very good agreement, with correlation coefficients of R2 = 0.985 for nOAR and R2 = 0.998 for RES (see figure 4C). The numerical modeling results across the field of view are shown in figure S4A, where the optimal field of view used for calculating the MTF (Modulus of the Optical Transfer Function) is defined. The MTF for this system is plotted in figure 4D for a field point on the optical axis and another at the edge of the optimal field of view. The field point at the optical axis shows near-diffraction-limited response, and the tangential and sagittal curves for the edge of the field drop to half of their low-frequency value at a spatial frequency of about 300 cycles/mm.

Empirical Characterization of Resolution.

The resolution of the 430X and 140X lenses were empirically characterized using a standard USAF 1951 resolution target and a GUPPY Pro 503C scientific camera (with bare sensor) as shown in figure S7. Images taken with these lenses are shown in figure 4E,G, and the relevant intensity profiles are shown in figure 4F,H. The formula for the resolution (in line pairs/mm) of a given group and element of the USAF 1951 target is given by RESLP = 2Group+(Element−1)/6, and the center-to-center distance between the lines in microns is given by RESCC = 1000/RESLP. From table 2, we see the theoretical values for the resolution of the lenses are 1.44 µm and 1.90 µm, respectively. Based on the critically resolved intensity profiles in figure 4F,H, the empirical values for RESCC are found to be 1.38 µm and 2.19 µm.

For the 430X lens, note that the empirical value is 4.2% smaller than the theoretical value. A less conservative theoretical model (see text S1) predicts values 11.5% smaller than those in table 2, indicating the empirical value is reasonable and near the limits predicted by theory. For the 140X lens, note that the empirical value is 15.3% larger than the theoretical value. This difference is attributed to the fact that this particular lens was selected for its low cost at the expense of larger tolerances on diameter and sphericity (grade 48) compared to the 430X lens (grade 10). For example, diameter tolerance per ball sphericity is 0.25 µm for grade 10 and scales linearly with grade number.

Since the USAF 1951 resolution target has a glass covering with thickness comparable to a standard coverslip (140 µm), ball lenses with magnification higher than 430X could not be assessed with this resolution target due to short back focal length. Instead, a monolayer of 1 µm polystyrene beads was imaged using a 1450X ball lens (figure 2A) to demonstrate sub-micron resolution for this lens (theoretical value is 0.86 µm).

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).

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.

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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 CO2 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

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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

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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

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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

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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

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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

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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

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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.

References

  1. 1. Keller E, Goldman R (2006) Light Microscopy. Woodbury, NY: Cold Spring Harbor Laboratory Press. p. 8.
  2. 2. Whitesides GM (2011) The Frugal Way. The Economist - The World in 2012
  3. 3. Rothemund PW (2006) Folding DNA to create nanoscale shapes and patterns. Nature 440: 297–302.
  4. 4. Hyde RA (1999) Eyeglass, a large-aperture space telescope. Appl Opt 38(19): 4198–4212.
  5. 5. Hoover AM, Fearing RS (2008) Fast scale prototyping for folded millirobots. IEEE International Conference on Robotics and Automation 5: 886–892.
  6. 6. Whitney JP, Sreetharan PS, Ma KY, Wood RJ (2011) Pop-up book MEMS. J Micromech Microeng 21(115021): 1–7.
  7. 7. Sreetharan PS, Whitney JP, Strauss MD, Wood RJ (2012) Monolithic fabrication of millimeter-scale machines. J Micromech Microeng 22(055027): 1–6.

Microscopy

(This passage was adapted from Microbiology: A Laboratory Manual,5th edition, Cappuccino, J.S. and Sherman, N., Benjamin/CummingsScience Publishing.)

Purposes

1.To become familiar with the history and diversity of microscopeinstruments.

2.To understand the components, use, and care of the compoundbrightfield microscope.

3.To learn the correct use of the microscope for observation andmeasurement of microorganisms.

 

INTRODUCTION

 

Microbiology, the branch of science that has so vastly extendedand expanded our knowledge of the living world, owes its existence toAntony van Leeuwenhoek. In 1673, with the aid of a crude microscopeconsisting of a biconcave lens enclosed in two metal plates,Leeuwenhoek introduced the world to the existence of microbial formsof life. Over the years, microscopes have evolved from the simple,single-lens instrument of Leeuwenhoek, with a magnification of 300,to the present-day electron microscopes capable of magnificationsgreater than 250,000. Microscopes are designated as either lightmicroscopes or electron microscopes. The former use visible light orultraviolet rays to illuminate specimens. They include brightfield,darkfield, phase-contrast, and fluorescent instruments. Fluorescentmicro-scopes use ultraviolet radiations whose wavelengths are shorterthan those of visible light and are not directly perceptible to thehuman eye. Electron microscopes use elec-tron beams instead of lightrays, and magnets instead of lenses to observe submicro-scopicparticles.

 

Essential Features of Various Microscopes

 

Brightfield Microscope

This instrument contains two lens systems for magnifyingspecimens: the ocular lens in the eyepiece and the objective lenslocated in the nose-piece. The specimen is illuminated by a beam oftungsten light focused on it by a sub-stage lens called a condenser,and the result is that the specimen appears dark against a brightbackground. A major limitation of this system is the absence ofcontrast between the specimen and the surrounding medium, which makesit difficult to observe living cells. Therefore, most brightfieldobservations are performed on nonviable, stained preparations.

 

Darkfield Microscope

This is similar to the ordinary light microscope; however, thecondenser system is modified so that the specimen is not illuminateddirectly. The con-denser directs the light obliquely so that thelight is deflected or scattered from the spec-imen, which thenappears bright against a dark background. Living specimens may beobserved more readily with darkfield than with brightfieldmicroscopy.

 

Phase-Contrast Microscope

Observation of microorganisms in an unstained state is possiblewith this microscope. Its optics include special objectives and acondenser that make visible cellular components that differ onlyslightly in their refractive indexes. As light is transmitted througha specimen with a refractive index different from that of thesurrounding medium, a portion of the light is refracted (bent) due toslight varia-tions in density and thickness of the cellularcomponents. The special optics convert the difference betweentransmitted light and refracted rays, resulting in a significantvari-ation in the intensity of light and thereby producing adiscernible image of the struc-ture under study. The image appearsdark against a light background.

 

Fluorescent Microscope

This microscope is used most frequently to visualize speci-mensthat are chemically tagged with a fluorescent dye. The source ofillumination is an ultraviolet (UV) light obtained from ahigh-pressure mercury lamp or hydrogen quartz lamp. The ocular lensis fitted with a filter that permits the longer ultravioletwavelengths to pass, while the shorter wavelengths are blocked oreliminated. Ultraviolet radiations are absorbed by the fluorescentlabel and the energy is re-emitted in the form of a differentwavelength in the visible light range. The fluorescent dyes absorb atwavelengths between 230 and 350 nanometers (nm) and emit orange,yellow, or greenish light. This microscope is used primarily for thedetection of antigen-antibody reactions. Antibodies are conjugatedwith a fluorescent dye that becomes excited in the presence ofultraviolet light, and the fluorescent portion of the dye becomesvisible against a black background.

 

Electron Microscope

This instrument provides a revolutionary method of microscopy,with magnifications up to one million. This permits visualization ofsubmicroscopic cel-lular particles as well as viral agents. In theelectron microscope, the specimen is illu-minated by a beam ofelectrons rather than light, and the focusing is carried out byelec-tromagnets instead of a set of optics. These components aresealed in a tube in which a complete vacuum is established.Transmission electron microscopes require speci-mens that are thinlyprepared, fixed, and dehydrated for the electron beam to pass freelythrough them. As the electrons pass through the specimen, images areformed by direct-ing the electrons onto photographic film, thusmaking internal cellular structures visi-ble. Scanning electronmicroscopes are used for visualizing surface characteristics ratherthan intracellular structures A narrow beam of electrons scans backand forth, producing a three-dimensional image as the electrons arereflected off the specimen's surface.

 

While scientists have a variety of optical instruments with whichto perform routine laboratory procedures and sophisticated research,the compound brightfield micro-scope is the "workhorse" and iscommonly found in all biological laboratories. Although you should befamiliar with the basic principles of microscopy, you probably havenot been exposed to this diverse array of complex and expensiveequipment. Therefore, only the compound brightfield microscope willbe discussed in depth and used to examine specimens.

 

USE OF THE MICROSCOPE

 

PURPOSES

To become familiar with the:

 

1.Theoretical principles of brightfield microscopy.

2.Component parts of the compound micro-scope.

3.Use and care of the compound microscope.

4.Practical use of the compound microscope for visualization ofcellular morphology from stained slide preparations.

 

PRINCIPLE

Microbiology is a science that studies living organisms that aretoo small to be seen with the naked eye. Needless to say, such astudy must involve the use of a good compound microscope. Althoughthere are many types and variations, they all fundamentally consistof a two-lens system, a variable but controllable light source, andmechanical adjustable parts for determining focal length between thelenses and specimen.

 

Components of the Microscope

 

Stage

A fixed platform with an opening in the center allows for thepassage of light from an illu-minating source below to the lenssystem above the stage. This platform provides a surface for theplacement of a slide with its specimen over the central opening. Inaddition to the fixed stage, most microscopes have a mechanical stagethat can be moved vertically or horizontally by means of adjustmentcontrols. Less sophisticated micro-scopes have clips on the fixedstage, and the slide must be positioned manually over the centralopening.

 

Illumination

The light source is positioned in the base of the instrument.Some microscopes are equipped with a built-in light source topro-vide direct illumination. Others are provided with a mirror; oneside flat and the other concave.

An external light source, such as a lamp, is placed in front ofthe mirror to direct the light upward into the lens system. The flatside of the mirror is used for artificial light, and the concave sidefor sunlight.

 

Abbe Condenser

This component is found directly under the stage and contains twosets of lenses that collect and concentrate light passing upward fromthe light source into the lens sys-tems. The condenser is equippedwith an iris diaphragm, a shutter controlled by a lever that is usedto regulate the amount of light entering the lens system.

Body Tube

Above the stage and attached to the arm of the microscope is thebody tube. This structure houses the lens system that magnifies thespecimen. The upper end of the tube contains the ocular or eyepiecelens. The lower portion consists of a movable nosepiece containingthe objective lenses. Rotation of the nosepiece posi-tions objectivesabove the stage opening. The body tube may be raised or lowered withthe aid of coarse-adjustment and fine-adjustment knobs that arelocated above or below the stage, depending on the type and make ofthe instrument.

 

Theoretical Principles of Microscopy

 

To use the microscope efficiently and with minimal frustration,you should understand the basic principles of microscopy:magnification, resolution, numerical aperture, illumination, andfocusing.

 

Magnification

Enlargement or magnification of a specimen is the function of atwo-lens system; the ocular lens is found in the eyepiece, and theobjective lens is situated in a revolving nose-piece. These lensesare separated by the body tube. The objective lens is nearer thespecimen and magnifies it, producing the real image that is projectedup into the focal plane and then magnified by the ocular lens toproduce the final image.

 

The most commonly used microscopes are equipped with a revolvingnosepiece containing four objective lenses possessing differentdegrees of magnification. When these are combined with themagnification of the ocular lens, the total or overall linearmagnification of the specimen is obtained.

 

Resolving Power or Resolution

Although magnification is important, you must be aware thatunlimited enlargement is not possible by merely increasing themagnifying power of the lenses or by using additional lenses, becauselenses are limited by a property called resolving power. Bydefinition, resolving power is the ability of a lens to show twoadjacent objects as discrete entities. When a lens cannotdiscriminate, that is, when the two objects appear as one, it haslost resolu-tion. Increased magnification will not rectify the loss,and will, in fact, blur the object. The resolv-ing power of a lens isdependent on the wave-length of light used and the numericalaperture, which is a characteristic of each lens and imprinted oneach objective. The numerical aper-ture is defined as a function ofthe diameter of the objective lens in relation to its focal length.It is doubled by use of the substage condenser; which illuminates theobject with rays of light that pass through the specimen obliquely aswell as directly. Thus, resolving power is expressed mathematically,as follows:

 

Resolving power = Wavelength of Light .

2 (Numerical Aperture)

 

Based on this formula, the shorter the wave-length, the greaterthe resolving power of the lens. Thus, short wavelengths of theelectromag-netic spectrum are better suited than longer wavelengthsin terms of the numerical aperture.

However; as with magnification, resolving power also has limits.You might rationalize that merely decreasing the wavelength willautomati-cally increase the resolving power of a lens. Such is notthe case, because the visible portion of the electromagnetic spectrumis very narrow and borders on the very short wavelengths found in theultraviolet portion of the spectrum.

The relationship between wavelength and numerical aperture isvalid only for increased resolving power when light rays areparallel. Therefore, the resolving power is dependent on anotherfactor, the refractive index. This is the bending power of lightpassing through air from the glass slide to the objective lens. Therefractive index of air is lower than that of glass, and as lightrays pass from the glass slide into the air, they are bent orrefracted so that they do not pass into the objective lens. Thiswould cause a loss of light, which would reduce the numericalaperture and diminish the resolving power of the objective lens. Lossof refracted light can be compensated for by interposing mineral oil,which has the same refractive index as glass, between the slide andthe objective lens. In this way, decreased light refraction occursand more light rays enter directly into the objective lens, producinga vivid image with high resolution.

Illumination

Effective illumination is required for efficient magnification andresolving power. Since the intensity of daylight is an uncontrolledvariable, artificial light from a tungsten lamp is the most commonlyused light source in microscopy. The light is passed through thecon-denser located beneath the stage. The condenser contains twolenses that are necessary to produce a maximum numerical aperture.The height of the condenser can be adjusted with the con-denser knob.Always keep the condenser close to the stage, especially when usingthe oil-immersion objective.

 

Between the light source and the condenser is the iris diaphragm,which can be opened and closed by means of a lever; therebyregulating the amount of light entering the condenser. Excessiveillumination may actually obscure the specimen because of lack ofcontrast. The amount of light entering the microscope differs witheach objec-tive lens used. A rule of thumb is that as themag-nification of the lens increases, the distance between theobjective lens and slide, called working distance, decreases, whereasthe numerical aperture of the objective lens increases.

Use and Care of the Microscope

 

You will be responsible for the proper care and use ofmicroscopes. Since microscopes are expensive, you must observe thefollowing regu-lations and procedures.

 

The instruments are housed in special cabinets and must be movedby users to their laboratory benches. The correct and only acceptableway to do this is to grip the microscope arm firmly with the righthand and the base with the left hand, and lift the instrument fromthe cabinet shelf. Carry it close to the body and gently place it onthe laboratory bench. This will prevent collision with furniture orco-workers and will protect the instrument against damage.

 

Once the microscope is placed on the laboratory bench, observe thefollowing rules:

1.Remove all unnecessary materials such as books, papers, purses, and hats from the laboratory bench.

2.Uncoil the microscope's electric wire and plug it into an electrical outlet.

3.Clean all lens svstems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency ot the microscope. The ocular; scan-ning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper tow-eling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with methanol is used to wipe it clean. If the lens is very dirty it may be cleaned with xylol however the xylol cleansing procedure should be performed only by the instructor, and only if necessary. Consistent use of xylol may loosen the lens.

 

The following routine procedures must be followed to ensurecorrect and efficient use of the microscope while focusing.

 

1. Place the microscope slide with the specimen within the stage clips on the fixed stage. Move the slide to center the specimen over the opening in the stage directly over the light source.

2. Rotate the scanning lens or the low power lens into position. While watching from the side to insure that the lens doesn't touch the specimen, turn the coarse focus knob to move the stage as close as it can get to the lens without touching the lens. (Always watch from the side whenever you move a specimen towards any objective lens to make sure the lens doesn't crash through the specimen and get damaged!)

3. Now, while looking through the ocular lens, turn the coarse focus knob carefully, and slowly move the stage away from the lens until the specimen comes into vague focus. Then, use the fine focus knob to bring the specimen into sharp focus.

4. If this is the first specimen of the day, you should Kohler your microscope at this point (while it is in focus). Otherwise, if your microscope has already been Kohlered you won't need to do it again

5. Routinely adjust the light source by means of the light source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

6. Our microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without further major adjustment. In practice, however; usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.

7. Once you have brought the specimen into sharp focus with a low-powered lens, preparation may be made for visualizing the spec-imen under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Care should be taken not to allow the high-power objective to touch the drop of oil.The slide is observed from the side as the objective is rotated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is readjusted to bring the image into sharp focus.

8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is accomplished by scanning the slide with-out the application of additional immersion oil. This will require continuous, very fine adjustments by the slow, back-and-forth rotation of the fine adjustment knob only.

 

On completion of the laboratory exercise, return the microscope toits cabinet in its original condition. The following steps arerecommended:

 

1.Clean all lenses with dry, clean lens paper. If you need to, you can use a drop or two of methanol to help clean the lens. Use xylol to remove oil from the stage only.

2. Place the low-power objective in position and bring the stage and objectives close together.

3.Center the mechanical stage.

4.Coil the electric wire around the body tube and the stage.

5.Carry the microscope to its position in its cabinet in the manner previously described.

 

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