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

Funding: This project was primarily funded through startup funds via the Department of Keck Science ( https://www.kecksci.claremont.edu ). Additionally, one of the co-authors (Victoria Nguyen, VN) was funded for a summer through the S.D. Bechtel, Jr. Foundation ( http://sdbjrfoundation.org , Grant ID #5666). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2016 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In addition to a specific plan, we explain the customizable design methodology, and demonstrate multi-functionality. Combined fluorescence, imaging ellipsometry and multi-angle imaging implementations are not currently in the literature, as far as we are aware. While markets are subject to change, we demonstrate that the parts required for the combined upright and inverted fluorescence microscope and imaging ellipsometer currently costs less than $16k, and can be assembled from a detailed and provided manual (see Supporting Information, S1 Appendix ) by two undergraduate students in less than a day. This is at least an order of magnitude cheaper than purchasing the three separate comparable commercial systems [ 7 ], and is of sufficient quality to produce research publications [ 9 ]. In this paper we discuss how to customize components in anticipation of rapidly evolving markets of available components (e.g., fluorescence filters, light sources, and cameras). We also include demonstrations of the various functionalities, with samples ranging from molecular films to cells and tissues.

Here we present an assemblable design for a multi-functional optical microscope with upright, side-on and inverted fluorescence, as well as ellipsometric imaging capabilities—a design we name the SwingScope. The multi-functionality of the device enables capabilities beyond cost/space savings. For example, samples can be imaged from multiple angles without being perturbed, and complementary techniques such as fluorescence and label-free ellipsometry simultaneously measure thin-films.

The principle benefit of an assemblable approach is that it requires little manufacturing expertise. Its applicability is enhanced by three recent developments. First, rapid prototyping instruments and services (e.g., additive manufacturing or “3D printing”) enable the ready transfer of fabrication expertise [ 3 ]. This transfer takes the form of design files that can be physically produced with 3D printers, which are now broadly available as part of institutional Maker-spaces [ 4 , 5 ] or through services where the file is uploaded and a physical product is mailed to the customer [ 6 ]. Second, non-printable optical components can be sourced from multiple vendors as roughly comparable off-the-shelf parts, at a fraction of the cost of purchasing complete turn-key microscopes [ 7 ]. Finally, there are free open-source alternatives to otherwise expensive/manufacturer-specific microscope control software packages. These open source projects (e.g., μManager [ 8 ]) enable low cost integration across multiple hardware platforms.

The specificity of a microscope’s design can limit its buildability. The more specifically a design is prescribed (e.g., part numbers from particular vendors), the less useful it will be as component markets change. The more generally a design is prescribed (e.g., a pure optical drawing) the more expertise and infrastructure is required in assembling the system. A successful middle ground is to supplement a component-specified assemblable design with associated design-methodologies to enable readers to adapt the system to their needs and markets [ 1 , 2 ].

Methods and Methodology

Design Overview The salient mechanical feature of the SwingScope design is that the microscope can pivot 180° vertically around the sample, between upright and inverted geometries. This is achieved by mounting the microscope optics to a vibrationally damped rod that has a pivot point roughly aligned with the sample (see Fig 1). An inverted geometry is preferable when imaging samples submerged under water [10], while an upright geometry is useful when studying an air/water interface [11], or non-invertible biological samples. Additionally, side-on imaging enables determining the contact angle and capillary flow of microscopic droplets on surfaces [12, 13], and oblique incident imaging is useful in ellipsometric applications such as thin films [14]. The microscope freely swings through the range of angles. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Swingscope. a: Superimposed images of the microscope in three geometries, with the imaging ellipsometry components removed for visibility. b: Schematic of optical components in the inverted (gray) and oblique configurations. Components inserted for imaging ellipsometry are outlined in magenta. c: Schematic overlaid with moving and 3D-printed components. https://doi.org/10.1371/journal.pone.0166735.g001 At any pivot geometry, the SwingScope converts from a brightfield microscope to a fluorescence microscope by manually swapping filter cubes. When imaging ellipsometry capabilities are desired, a laser-illumination arm manually swings into the beampath and a rotating polarizer slides onto the microscope posts. The system as designed is largely manual, with the exception of camera control and acquisition which are controlled by readily extensible μManager open-source software. We include a detailed manual (S1 Appendix) on the assembly of the instrument. However, the microscope includes several components which strongly impact price and performance, and whose specification is likely to evolve quickly with current markets. To that end, their selection rationale is detailed below. In particular, we discuss how the light source could be a consumer LED bulb, the fluorescence filters could be multi-spectral and swappable, and cameras has several lower-cost options. All parts in the microscope are either off-the-shelf components or rapidly prototyped (i.e., 3D printing/laser-cutting), to reduce required manufacturing expertise.

Mechanical Pivot Our design methodology includes a pivot aligned with the anticipated sample position, which allows changes in angle with only a minimal change in sample focus position. The base of the pivot and the base of the sample-holder cannot attach to the table at the same place, and so must be offset in the direction transverse to the rotation, while the optical elements must still align at the sample. We chose to place the microscope mount further away from user, to keep the sample more accessible. We chose a freely-swinging, sturdy pivot with attached platforms on both ends. This is convenient for easy imaging with 40X objectives or below, however a damped pivot would have enable higher magnification objectives to be used with less concern about table vibrations. Concerns about safety and a desire to have a continuous range of available angles led us to introduce a secondary angle-stabilization mechanism. A post taller than the microscope is mounted on the same table and affixed with a pulley. Steel cable runs from the top of the microscope post through the pulley to a manual winch, which allows us to continuously set the angle of the microscope, and provides a measure of safety should the single pivot fail.

3D Printed Stabilization and Adapter Necessary mechanical components that cannot be sourced commercially must be fabricated. Our design includes two such components: an alignment piece between the focus block and the rest of the assembly, and a mechanical adapter for our light source. These components were designed procedurally using an open-source software package called OpenSCAD [15] and printed on a consumer additive manufacturing (3D printing) system (Afinia 3D, Chanhassen, MN). The OpenSCAD design files and printable stereolithograph (.stl) files are included in S2 File.

Fluorescence and Brightfield Microscopy Lightpaths A broad-band white light source is mounted on a pathway perpendicular to the microscope post, and passes through two lenses that focus it on the back focal plane of the objective, in a configuration (Köhler [16]) that evenly illuminates the sample. In between the lenses is an aperture that is imaged onto the sample so that the illumination is “field apertured”. This feature is particularly useful for geometric exposure techniques such as Fluorescence Recovery After Photobleaching (FRAP [17]). The light then passes through a manually interchangable cube that filters the white light to the absorption spectrum of the fluorescent molecules at the image plane. A diagonal beamsplitting filter in the cube, called a dichroic, reflects the beam towards the objective/sample while also partially filtering it. This filter cube can be readily swapped to accommodate different fluorophores, or be replaced with unfiltered glass to enable a non-fluorescence bright-field imaging mode. The excitation-filtered light passes through the filter-cube to an objective lens attached to a manual focus-block. The alignment between the focus block and the illumination/detection portion of the microscope is critical, and requires another custom 3D-printed component for rigidity. If the sample illumination is uneven, it is often this alignment that needs adjustment, even with the rigidity enhancement. The light returning through the objective is re-filtered by the filter-cube to reject excitation wavelengths and isolate the emission from the sample fluorophores. It passes through a high quality tube lens before striking a camera placed at its focal length. We have experimented with a plano-convex lens, a fused doublet lens and a lateral and axial chromatically corrected fused doublet lens, and found the performance of the corrected doublet lens to merit the greater price (2.0μm vs 3.5μm resolution at 10X, see S3 File). Selection of the objective lens is also critical. Because fluorescence absorption and emission wavelengths are significantly different, objectives that correct for chromatic aberration are preferable for fluorescence applications. Such objectives can be expensive, and must be chosen with applications in mind. In our design we specify a 10X objective and a long working distance 40X objective with a built-in adjustment collar for imaging through different thicknesses of coverglass. Objectives beyond 40X are often significantly more expensive (e.g., up to the cost of our entire microscope) and are more prone to revealing table vibrations. Additionally, we chose infinity-corrected objectives, which do not tightly prescribe the distance between the objective and the rest of the optics, which simplifies microscope customization.

Filters and light sources Our approach is to target two spectrally distinct fluorophores and optimize the filters and light sources accordingly. The common fluorophores in our laboratory are Texas-Red and NBD (N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl), which have largely separate excitation and emission spectra (see Fig 2). We selected optical filters from a manufacturer (Chroma, Bellows Falls, VT) that could excite and detect emission from both of these dyes at the same time, to reduce cost with a tolerable efficiency diminishment. Our instrument design allows for rapid swapping of filter-cubes without realignment should higher efficiency single-fluorophores filter sets be required in the future. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Calculating light-source, filter and fluorphore spectral overlap. a: Emission of three consumer LED bulbs (TCP LED10P20D24/ 41/ 50KNFL) of labelled color temperatures. b: Transmission spectrum of fluorescence excitation filterset (Chroma 59022). c: Excitation spectra of NBD and Texas-Red dyes commonly used in our laboratory. d: The product of multiplying the spectra in a/b/c together. e: Relative efficiency of the different light sources for the two probes, determined by integrating the area under the curves of the spectra in d. Here the 5000K bulb is best suited for our fluorophores. https://doi.org/10.1371/journal.pone.0166735.g002 Recent advances [18] in consumer LED light bulbs has availed a broad range of efficient, long-lasting and low-cost options [19]. We found a form-factor that fit well in our system (PAR20) and mounted it onto a 3D printed cage-adapter with an E26 light cord (Ikea). We measured the spectra of several LED light bulbs using a spectrophotometer (CC100, Thorlabs) and determined the best spectral overlap with our two fluorophores, including the fluorescence filtering of the microscope. A comparison of three color temperature bulbs from the same manufacturer is shown in Fig 2. This approach is prone to some ripple flicker. Comparable LEDs from the same manufacturer have a 191Hz 3.75% ripple [20], but the fluctuations are negligible for exposures longer than 50ms.

Detector/Camera selection Fluorescence microscopy cameras tend to be one of the most expensive components of a microscope. There are several key features to consider. Camera sensitivity is crucial for fluorescence detection efficiency, while frame-rate strongly affects user-experience and noise/resolution characteristics determine image quality. However, there are options in adjacent consumer fields with tolerable compromises that are orders of magnitude lower in cost. We experimented with low cost cameras from consumer photography (Canon 20D, 60D) and consumer astronomy (QSI 628s), with success. Cameras are rapidly evolving products and market surveys are prudent at the time of microscope assembly [21]. We currently use the astronomy camera, largely due to its higher grayscale sensitivity and its higher frame-rate that facilitates focussing. To aid in future searches, we list some key camera characteristics and our design methodologies in Table (1). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Feature critera for microscope camera selection. https://doi.org/10.1371/journal.pone.0166735.t001

Imaging Ellipsometry Ellipsometry is a mature, label-free, non-contact optical technique for determining the properties of thin films by how they change the polarization of obliquely incident light. Its operational and modeling aspects are deeply explored elsewhere [14, 22], but briefly parallel polarizations interact with a surface differently than transverse polarizations. This difference manifests as a change in the total polarization of the light, and is sensitive to surface properties and adherent thin films. In practice, we measure the surface-induced change by finding the right combination of incident polarizations such that the reflected polarization is a single linear polarization, which is readily detectable by minimizing intensity at the detector with a polarizer. This approach is called “nulling,” and the polarization angles of nulling can be computationally modeled to determine surface parameters such as the thickness and refractive index of an adhering film. While the spatial resolution is limited by diffraction optics, the thickness-resolution is typically sub-nm. The technique has applications in semiconductor industries [23] and biology, particularly supported lipid bilayers [24, 25]. Imaging ellipsometry is a variant of the technique which spatially resolves thin films on the surface [26]. To control the incident polarization we use a combination of a laser, linear polarizer (P) and birefringent compensator (C) to produce the namesake elliptical polarization that strikes the sample at an oblique angle. In “nulling” conditions the polarizer and compensator are rotated so that after striking the surface the light is linearly polarized. It then passes through a analyzing polarizer (A) that is rotated 90 degrees from the reflected polarization, resulting in a minimum signal passing through (“null”). Mechanically, in our multifunctional microscope the optical components of ellipsometry should be removable from the light path, as these optical elements are not necessary during fluorescence imaging. Our design places the incident optical components on a rigid cage system attached to a ball-mount that can freely enter and exit the light path (see Fig 1). A low power 532nm wavelength collimated laser diode module (CPS532, Thorlabs) is the light source, and angle alignment is performed by way of a digital tilt sensor (iGaging, San Clemente, CA). This design, coupled with the SwingScope’s 180 degree range of motion allows for a large range of incident angles for optimizing imaging ellipsometry applications. A rotatable polarizer near the detector also slides on and off the four-post rails in the optical path immediately after the tube lens. Three of the optical components of imaging ellipsometry need to be rotatable. There are many commercial systems for rotating optical components, both manual and motorized. For contrast, a key design parameter is the resolution that the angle can determined. For quantitative ellipsometry suitable for modeling (which we do not pursue here), motorization of the polarization angles (and focus knobs) would be beneficial. To perform imaging ellipsometry with underwater samples the optical geometry is vastly simplified by orienting the windows of the wet sample-cell to be perpendicular to the incident beam. Our approach is to laser-cut two triangles out of acrylic as ends of a sample-cell, which are then placed on a laser-engraved base with two 22x50mm coverslips leaning on the triangles as the laser windows. The sample is enclosed in the cell with rapid-curing silicone polymers (Microset 101, Leicestershire, U.K.). The laser-cutting documents are included in the S1 File, and like 3D printing there are online services for laser-cutting parts from files if a local instrument is not available [27].