Du 897u

Wafers were mounted section side down in a petri dish (glass bottom, thickness 170 μm +/−5 μm, Ibidi) onto a drop of a 1:1 mixture of glycerin (80%) and an imaging buffer containing an oxygen scavenging system (200 mM Phosphate buffer containing 10% glucose, 0.5 mg/ml glucoseoxidase, 40 μg/ml catalase, 15 mM beta-Mercaptoethylamine (MEA), pH 8.0). Excess buffer was removed until the wafer was brought as close as possible to the cover glass.
Super-resolution imaging was performed on a Leica SR-GSD 3D microscope (Leica Microsystems) using a 160x (NA 1.43) oil immersion objective (HC PL APO, Leica Microsystems). The system was equipped with 488 nm/300 mW, 532 nm/500 mW and 647 nm/500 mW continuous wave lasers and an EMCCD camera (iXon Ultra, DU-897U, Andor).
The sample was first illuminated with the 642 nm laser at maximum laser power to ensure an efficient transfer of the fluorescent molecules into the off-state. Images of 180 × 180 pixels were then taken with an integration time of 15 ms in epifluorescence mode. A total of 30,000 frames were collected for each reconstruction.
Image reconstruction and visualization via Gaussian fitting was performed with the LAS AF software (Leica Microsystems).

The tensile stage sits on an optical microscope. We illuminate (355 nm laser) the sample from the bottom and likewise detect phosphorescence from the bottom. For single-molecule imaging the crosslinks are diluted (phosphorescent: non-phosphorescent ~1:1000). For calibration of macroscopic force-extension relations, every crosslink was phosphorescent.
Circularly polarized light is used for best excitation efficiency, except for experiments in which we use linearly-polarized light to monitor the polarization dependence. The light emitted is recorded using an EMCCD camera (DU-897U, Andor) with a large pixel density, 512 × 512, and a ×100 oil objective (Olympus). The field of view is 80 by 80 µm, pixel size 160 nm, and depth of focus 1.5 µm. Video images are analyzed using 2D Gaussian feathering to remove background, using IDL codes written in-house, to give the absolute intensity and positions of the optical probes. To measure the phosphorescence spectrum, the tensile stage is fiber-coupled to an Ocean Optics spectrometer. Photoluminescence (PL) is quantified by integrating emission between wavelengths 500 and 700 nm.