Fig 1.
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.
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.
Table 1.
Feature critera for microscope camera selection.
Fig 3.
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 S3 File. a: Sensitivity for 1nm of additional Silicon oxide in air b: Sensitivity for 1nm of lipid material in water.
Fig 4.
Images from the microscope in different geometric configurations.
a: Inverted fluorescence image of lipids (97% DOPC, 3% NBD) spreading [37] as a single bilayer out from a lipid-rich stamp on glass, in PBS buffer. b: Upright fluorescence image of lid1+ mRNA in S. pombe fission yeast cells harboring the “green RNA” system [38] at 10x and 40x (inset) with a Canon DSLR camera, courtesy of the Tang Lab. c: Side-on imaging of a water droplet on a strip of teflon tape.d: and e: 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) [39, 40]. All zebrafish protocols were IACUC approved, courtesy of the Spence Lab. Insets are lower magnification (4X) images of the area.
Fig 5.
a: 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: Fluorescence microscopy image of the same location without moving the sample, by inserting the fluorescence filter-cube and disabling the ellipsometry laser. c: Null-angle as determined by fitting the raw intensity vs. polarizer angle to a parabola. d: Null polarizer angle as a function of position across the profile noted in e. e: Null map of the edge of the a lipid bilayer with the same composition, on bare Silicon in PBS buffer.