Fig. 2: Principle of 3D super-resolution biplane imaging using dTRABI.
From: Approach to map nanotopography of cell surface receptors
a Overview of the experimental setup applied to perform 3D dTRABI imaging of T cells. HILO illumination of the sample (blue beam; only shown in enlarged box) triggers fluorescence emission (orange), which is split by a 50/50 non-polarizing beamsplitter (50/50 BS) to acquire biplane images on two separate EM-CCD cameras. The respective imaging lenses are shifted along the optical axis to induce a relative defocus of the image detection on synchronized cameras. Spots, apparent in both detection planes, are fitted by a Gaussian with identically set FWHM. b Using a piezo stage, the focal plane was linearly moved through the sample plane while imaging a single-molecule surface under dSTORM conditions. Hereby, both cameras were synchronized. Fitting the raw PSFs by independent Gaussians with invariable FWHM yielded axially dependent single-molecule intensity curves (upper panel). The relative change of position of the imaging lens in the reflection path is mirrored by the relative shift of the respective intensity curve (indicated by circles). Data points were spline interpolated to guide the eye (solid lines). An axially precise calibration function γ is derived directly from the raw intensities (ICamera1, ICamera2) of corresponding localizations from both cameras as γ(z) = (ICamera2 − ICamera1)(ICamera2 + ICamera1)−1. The running median of the raw data (gray squares) is fitted with a high-order polynomial (black line) to generate the basis of the axial lookup table (lower panel). c A two-dimensional high-resolution data set is generated from both image stacks (transmission and reflection path) to create a three-dimensional dTRABI data set according to the calibration. Finally, the transmission localization set is used to render a high-resolution, axially color-coded image of the focused target structure. Scale bars, 5 μm.
