Ultra emccd camera

Neurons were incubated for 10 min in culture media containing 5.6 µM Fluo-4 AM (Life Technologies, Grand Island NY). Coverslips were then removed and washed for 2 min prior to recording with Tyrode's solution containing (in mM) 150 NaCl, 4 KCl, 1.25 MgCl₂, 2 CaCl₂ and 10 TES buffer, pH adjusted to 7.4. Where solutions are noted to have 0 mM Ca⁺ the solution is prepared from deionized water with no added Ca²⁺. All solution changes except +100 mM hypertonic sucrose include 2 min of wash time in the new solution to ensure full application of the new conditions. Neurons were imaged using a 40× objective on a Nikon TE2000-U microscope. Images were collected at 10 frames per second using an Andor xION Ultra EMCCD camera for a duration of ∼ 2 min. Event frequencies per ROI were estimated using the population average obtained from 72 ROIs monitored per experiment. Our analysis, therefore, refers only to average per ROI frequency per experiment. Illumination was provided by a Sutter DG-4 arc lamp using a 470 ± 40 nm bandpass excitation filter. Post experiment synapse visualization used a 548 ± 10 nm filter to excite Syb2-mO and Tyrode's solution containing 50 mM NH₄Cl to maximize fluorescence. The emission filter in place allowed 515 ± 15 nm and 590 ± 20 nm bands to pass. Fluo-4 traces were generated by measuring circular ROI, 3 µm radius centered over Syb2-mO puncta.

Live cell TIRF imaging was performed on a Nikon N-STORM microscope using a Nikon 100x Plan Apo 1.49 NA objective and an Andor iXon Ultra EMCCD camera. Cells in metaphase were identified using the H2B mCherry channel, following which images were acquired every 10–30 s in TIRF in the green channel. Cells were imaged until they had completed telophase and initiated cell spreading. A camera gain of 110 was used. The hardware was controlled using Nikon Elements AR software.

Total internal reflection fluorescence microscopy (TIRFM) images were acquired in Nikon Ti2Eclipse inverted microscope with a 100x/1.50 NA oil immersion lens and a iXON Ultra EMCCD camera at 37°C. B-cells expressing LifeAct-mCherry were plated on Ag-coated glass chambers (Nunc™ Lab-Tek™ II). Images were acquired for 30 min at 15 s per frame for spreading.