Cms 196

Grids were transferred to the cryostage (CMS-196, Linkam Scientific, UK) and suitable regions on the grid were found by searching whilst illuminating with 488 nm light. When a region was found, its position on the grid was noted for later correlation. For samples that were not pre-deactivated at room temperature, first a deactivation step was performed, by illuminating the sample with 488 nm light for up to 15 min. Next, SR imaging was initiated by first illuminating the sample with an activation pulse (405 nm, 28.5 W/cm²), see also Supplementary Fig. S3. Each activation pulse was followed by 2.8 sec of imaging, corresponding to 40 acquisition frames each of 50 ms exposure time with a 20 ms read-out time, under constant exposure to 488 nm light (28.5 W/cm²). For the work with cells, laser intensity was reduced to 20.8 W/cm² to account for the thicker carbon layer necessary for cell adherence to grids, and to speed up imaging, 10 acquisition frames per imaging cycle were acquired. For each position, the activation pulse started at 200 ms length, and was increased incrementally to 2 sec, to counter the switching fatigue of the fluorophores. During imaging, regular refocussing was necessary, since axial drift cannot be corrected post acquisition. After cryoFLM acquisition, grids were stored under liquid nitrogen until cryoEM imaging.

We adapted a standard upright widefield fluorescent microscope (Leica DM RXA) for SMLM acquisition. A 405 nm laser (LuxX + 405–120, Omicron) and a 488 nm laser (LuxX + 488–100, Omicron) were used for activation and excitation, respectively, which were coupled in using a polarising-maintaining optical fibre, all from Omicron-Laserage Laserprodukte GmbH, Germany. Standard Leica fluorescent cubes were used (FITC filter or N2.1 filter), with the excitation filters removed from the filter blocks. Fluorescence was collected using either a long-working distance lens (HCX PL Fluotar L 100x/0.75, Leica) in combination with a cryostage (CMS-196, Linkam Scientific, UK), or an oil-immersion lens for experiments at 294 K (PL Fluotar 100x/1.3, Leica). Fluorescent images were recorded by a sCMOS camera (pco.edge 4.2, DVision, Oostakker, Belgium).
Fluorescence emission spectra were recorded by a home-made set-up that was mounted on an extra camera port via a beam-splitter (100-50-0%, Leica). The emitted light was coupled in a multimode optical fibre (M15L01, Thorlabs Inc, USA) by an aspheric glass lens (C340 TMD-A, Thorlabs Inc, USA) and connected to a fibre-optic spectrometer (AvaSpec-2048L, Avantes, the Netherlands). Software written in-house was utilised to read-out the camera, and to synchronise the lasers and camera acquisition.