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An acquisition and analysis pipeline for scanning angle interference microscopy

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Figure 1: Implementation of SAIM pipeline to measure sample topography.

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Acknowledgements

We would like to thank G. Bhabha and S. Niekamp for help with electron microscopy, axoneme and microtubule experiments and J. Sung and S. Vo (director of the Leia3d Advance Lithography Center in Palo Alto, California) for ellipsometry measurements. We thank T. Goddard and E. Meng for help generating topography models of SAIM data using Chimera software. We are grateful to V. Weaver and M. Rubashkin for initial help implementing SAIM and to M. Paszek and W. Shin for useful discussions about the SAIM software and calibration device. We thank G. Bhabha and A. Jain for comments on the manuscript. The authors acknowledge funding from the National Institutes of Health (R01EB007187, R.D.V.) and the Howard Hughes Medical Institute.

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Correspondence to Ronald D Vale.

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R.D.V. and N.S. are shareholders of Open Imaging, Inc., a company that provides support for the Micro-Manager software used in this manuscript.

Integrated supplementary information

Supplementary Figure 1 Height error introduced by calibration error.

(a) Correctly calibrated data. SAIM Data from a DiI labeled supported lipid bilayer fit correctly. Intensity measurements were taken at 1−degree intervals from −42° to 42°. (b) Incorrectly calibrated data. The same data as in a, fit with a false calibration wherein the measurements were aligned to a start angle of −44.5° and an angle step size of 1.05°.

Supplementary Figure 2 Calibration using supported lipid bilayers.

(a) Periodicity of SAIM function at differing oxide heights. Theoretical intensity is plotted vs. angle for 488 nm (green lines) or 561 nm (red lines) laser illumination at 25 (light lines) nm or 50 nm (dark lines) from the surface of the oxide. Four oxide heights are represented: 500 nm, 1000 nm, 1500 nm and 1900 nm. (b) Measurement of oxide layer thickness. Three separate wafers were measured by ellipsometry at 8 positions spaced at increments of 1 cm apart. Error bars represent standard deviation of these eight measurements. “All” depicts the mean and standard deviation of 24 measurements across three wafers (1930.5 ± 2.0 nm). “SAIM” depicts the mean and standard deviation for the deduced oxide height at three wavelengths (488 nm, 561 nm, 647 nm), on two different microscopes and on five separate days (1928.5 ± 5.6 nm) for wafer #3. (c) Using SAIM to deduce oxide thickness. A triple labeled bilayer (DiO, DiI, DiD) was measured by SAIM at three wavelengths (488 nm, 561 nm, 647 nm). These data were then fit using oxide thicknesses ranging from 1915 nm to 1935 nm, which is plotted here versus the resultant sample height. Vertical dashed line represents the oxide thickness measured by ellipsometry (1930.5 ± 2.0 nm). Horizontal dashed line represents the expected height of the supported lipid bilayer (6.4 nm) [Supplementary reference 10]. Deduced oxide height is the intersection of the horizontal line for true bilayer height and the data. (d) Daily wavelength-dependent variation in oxide height measurements. Data for “SAIM” in b are shown as individual data points to highlight day-to-day variability.

Supplementary Figure 3 Imaging fluorescent nanobeads.

(a) SAIM prediction for 200 nm fluorescent nano-sized beads. Model of the SAIM intensity function a 200nm sphere, stained throughout generated by dividing the sphere in 9 parts, calculating the SAIM curve for each part, and averaging the predicted intensity for the 9 parts at each angle (weighted by the fraction of the sphere's volume contained in each part) (blue line), compared to the SAIM curve of a point source at the radius of the bead (orange line). (b) Histogram of fluorescent nanobead radii measured by SAIM. Left, histogram of yellow-green nanobeads. Measured heights of nominal “20 nm” radius (company provided specifications) nanobeads shown in light green (n = 1330, mean = 33 ± 13 nm), and nominal “50 nm” radius nanobeads shown in dark green (n = 823, mean = 63 ± 11 nm). Right, histogram of red nanobeads. Measured heights of nominal 20 nm radius nanobeads shown in light red (n = 451, mean = 28 ± 8 nm), and nominal 50 nm radius nanobeads shown in dark red (n = 1391, mean = 64 ± 7 nm). (c) Comparison of nanobead radius measured by SAIM or electron microscopy. Color-coding corresponds to panel b, radii measured by SAIM are solid colored, radii measured by electron microscopy are indicated by hatch marks. Error bars denote standard deviation.

Supplementary information

Supplementary Figures 1–3, Supplementary Notes 1–5, Supplementary Protocol and Supplementary Methods

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Supplementary Software

Source code and binaries for calibration and analysis software. (ZIP 69094 kb)

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Carbone, C., Vale, R. & Stuurman, N. An acquisition and analysis pipeline for scanning angle interference microscopy. Nat Methods 13, 897–898 (2016). https://doi.org/10.1038/nmeth.4030

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