Ixon ultra du 897u

Fluorometry and patch-clamp fluorometry (PCF) experiments were performed on an inverted IX71 microscope (Olympus, Tokyo, Japan) equipped with a 60× water immersion objective and an additional 1.6x magnification lens for PCF. Fluorescent indicators were excited with LED light (Spectra X light engine, Lumencor, Beaverton, OR). Excitation and emission filters used were 543/22 nm and 568LP nm for pHrodo-Red and 472/30 nm and 525/35 nm for CalBryte-520 and ANG-2. An EMCCD camera (iXon Ultra DU-897U, Andor Technology, Belfast, Ireland) controlled by the Andor Solis software was used for imaging.
An Axopatch 200B amplifier connected to Digidata 1440 A acquisition board controlled by the software ClampEx (Molecular Devices, Union City, CA) was used for electrophysiological recordings. Patch pipettes were pulled from borosilicate capillaries (Hilgenberg, Malsfeld, Germany) using a DMZ puller (Zeitz Instruments GmbH, Martinsried, Germany). All experiments were conducted at RT.

For single colour GSDIM, wafers were placed on droplets of a 1:1 solution of glycerol (80%) and imaging buffer containing an oxygen scavenging system (200 mM Phosphate buffer containing 10% glucose, 0.5 mg/ml glucoseoxidase, 40 ug/ml catalase, 15 mM beta-Mercapto-ethylamine hydrochloride (MEA HCI), pH 8.0). For double labelling the wafers were placed on droplets of a mixture of 20% Vectashield – 80% TRIS-Glycerol (5% v/v 1 M TRIS pH 8.0 in Glycerol)^{37 (link)}. A glass bottom Petri dish as described in the confocal microscopy section was used, for super-resolution microscopy in addition, silicon strips were added to stabilize the imaging.
Super-resolution imaging was performed on a Leica SR-GSD 3D microscope (Leica Microsystems) using a 160x/1.43 NA 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). Images of 180 × 180 pixels were then taken with an integration time of 7 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 X software (Leica Microsystems), by applying a detection threshold of 30 to 40 photons for each channel and a rendering pixel size of 4 nm.

Live-cell calcium mobilization imaging was performed on a home-built TIRF microscope consisting of the IX73 frame (Olympus), UApo N, 100×1.49 Oil immersion TIRF objective (Olympus) and OptoSplit II image splitter (CAIRN Optics) mounted on the camera port. Samples were illuminated using 200 mW 488 nm laser (Sapphire, Coherent) in a TIRF mode and the intensity was regulated by acousto-optic tunable filter (AOTFnC-400.650-TN, AA Optoelectronics). Fluorescence emission was detected by an EMCCD camera (iXon ULTRA DU-897U, Andor) with EM gain set to 200. Images were taken in 500 ms intervals with the exposure time set to 50 ms.