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. Author manuscript; available in PMC: 2017 Apr 30.
Published in final edited form as: Nat Methods. 2016 Oct 31;13(11):897–898. doi: 10.1038/nmeth.4030

Figure 1.

Figure 1

Implementation of SAIM pipeline to measure sample topography. (a) Schematic representation of sample setup for SAIM. NA, numerical aperture; PDMS, polydimethylsiloxane. (b) Schematic of SAIM theory. (c) Schematic of SAIM tools. (d) SAIM height of a supported lipid bilayer labeled with DiI. Left, average fluorescence intensity across all angles sampled. Right, SAIM reconstruction showing a height s.d. of 1.44 nm across a 10,000-μm2 field. Relation between color and height is shown in bar at the right of SAIM heightmap. (e) Representative single-pixel fit for supported lipid bilayer measurements. Raw data shown in light red; fit shown in dark red. A.U., arbitrary units. λ, wavelength; h, pixel height obtained from fit; R2, coefficient of determination. (f) SAIM height of a microtubule crossing an axoneme. Left three images, average fluorescence intensity for a Cy5-labeled axoneme (red) and Alexa-647-labeled microtubule (cyan). Right, SAIM reconstruction of microtubule height showing a representative deflection at the microtubule’s intersection with the axoneme and intersection with other microtubules. (g) 3D model of data shown in f. Microtubules are shown as cylinders with a radius of 12.5 nm along spline fits through SAIM data (cyan). The axoneme is modeled as a straight cylinder with a radius of 100 nm (red). Axes represent x-, y-, and z-scales. (h) Schematic of membrane interface showing relative sizes of proteins used to form partitioned membrane regions based on published Protein Data Bank structures 2h5q and 3fap. (i) SAIM height of a membrane interface between a giant unilamellar vesicle (GUV) and a supported lipid bilayer (SLB). Left, average fluorescence intensity for a GUV-tethered triple-length mCherry (red), GUV-tethered Alexa-647-labeled FKBP binding SLB-tethered FRB (cyan), Atto390-labeled GUV membrane (blue) and the merged mCherry and FKBP fluorescence images. Right, SAIM reconstruction of GUV membrane derived from Atto390-labeled lipid fluorescence showing representative deflection at mCherry clusters. (j) 3D model of data shown in i. Z-scale is exaggerated to clearly depict membrane deformations.