An Assemblable, Multi-Angle Fluorescence and Ellipsometric Microscope

<div><p>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.</p></div

Ellipsometric Sensitivity.

<p>Optimizing ellipsometry sensitivity with angle of incidence and substrate oxide thickness, using an ideal thin-slab dielectric theoretical model. Parameters are for a 532nm wavelength laser and substrates consisting of Silicon with various oxide thicknesses. Sensitivity is measured in degrees of P or A rotation per nanometer of added material, noted by the left colorbar, and also by the gray isolines at 0.5 degrees per nm. The map of which regions are more sensitive in P or A is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166735#pone.0166735.s004" target="_blank">S3 File</a>. <b>a:</b> Sensitivity for 1nm of additional Silicon oxide in air <b>b:</b> Sensitivity for 1nm of lipid material in water.</p

Calculating light-source, filter and fluorphore spectral overlap.

<p><b>a:</b> Emission of three consumer LED bulbs (TCP LED10P20D24/ 41/ 50KNFL) of labelled color temperatures. <b>b:</b> Transmission spectrum of fluorescence excitation filterset (Chroma 59022). <b>c:</b> Excitation spectra of NBD and Texas-Red dyes commonly used in our laboratory. <b>d:</b> The product of multiplying the spectra in a/b/c together. <b>e:</b> 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.</p

Swingscope.

<p><b>a:</b> Superimposed images of the microscope in three geometries, with the imaging ellipsometry components removed for visibility. <b>b:</b> Schematic of optical components in the inverted (gray) and oblique configurations. Components inserted for imaging ellipsometry are outlined in magenta. <b>c:</b> Schematic overlaid with moving and 3D-printed components.</p

Images from the microscope in different geometric configurations.

<p><b>a:</b> Inverted fluorescence image of lipids (97% DOPC, 3% NBD) spreading [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166735#pone.0166735.ref037" target="_blank">37</a>] as a single bilayer out from a lipid-rich stamp on glass, in PBS buffer. <b>b:</b> Upright fluorescence image of lid1+ mRNA in S. pombe fission yeast cells harboring the “green RNA” system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166735#pone.0166735.ref038" target="_blank">38</a>] at 10x and 40x (inset) with a Canon DSLR camera, courtesy of the Tang Lab. <b>c:</b> Side-on imaging of a water droplet on a strip of teflon tape.<b>d: and e:</b> Upright and inverted fluorescence images of the same adult zebrafish brain, revealing distinct morphology. Sample was dissected whole and subjected to PACT (passive clarity technique) with immunohistochemistry for primary antibody mouse gfap (zirc) and secondary antibody goat anti-mouse fitc-488 (abcam) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166735#pone.0166735.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166735#pone.0166735.ref040" target="_blank">40</a>]. All zebrafish protocols were IACUC approved, courtesy of the Spence Lab. Insets are lower magnification (4X) images of the area.</p

Feature critera for microscope camera selection.

<p>Feature critera for microscope camera selection.</p

Ellipsometric Imaging.

<p><b>a:</b> Time-series of contrast ellipsometric images of lipids (82% DOPC, 15%DOPS, 3% NBD-PE) spreading from a source on Silicon with a 100nm of oxide in PBS solution, without a fluorescence filter-cube in the beampath. Images have been corrected for oblique incidence geometric distortion, and the contrast globally enhanced for visibility. <b>b:</b> Fluorescence microscopy image of the same location without moving the sample, by inserting the fluorescence filter-cube and disabling the ellipsometry laser. <b>c:</b> Null-angle as determined by fitting the raw intensity vs. polarizer angle to a parabola. <b>d:</b> Null polarizer angle as a function of position across the profile noted in e. <b>e:</b> Null map of the edge of the a lipid bilayer with the same composition, on bare Silicon in PBS buffer.</p