Emccd camera

A subset of mice from each group was euthanized 24 h post laser irradiation for histological analysis. Tumors were extracted and sectioned for staining with FITC-labeled caspase-3 antibody (BD Biosciences, San Diego, CA, USA) to assess apoptosis. Fluorescence emission (524 ± 24 nm) in response to 485 ± 35 nm excitation, filtered from a Nikon Mercury/Xenon arc lamp, was captured by the EM-CCD camera with exposure time set at 0.1 s. Mean and SDs of the image intensities (n = 3 images) were quantified using ImageJ.

Hippocampal neuronal cultures were prepared from embryonic day 18 Sprague Dawley rats following a method previously described [49] (link), [50] (link). On the day of seeding (0 DIV) cells were co-electroporated (4D Nucleofector) with GluA1-mEos and Homer-1-Cerulean plasmids and maintained for 18–21 DIV. Then neurons were mounted on an open chamber containing Tyrode's solution (containing in mM: 10 D-glucose, 120 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, pH = 7.4) and were observed on a Nikon inverted microscope equipped with EMCCD camera using total internal reflection geometry (TIRF). GluA1-mEos subunits of AMPARs were imaged every 5 minutes till 30 minutes after cLTP induction following a brief control (before cLTP induction) acquisition. In some experiments cells were preincubated with GM6001 for 30 minutes then cLTP was induced. GluA1 subunit of AMPARs tagged with mEos were imaged with 20 millisecond exposure time at the rate of 20 frames per second and in each recording session 3000 frames were collected. Images were acquired using high numerical aperture (100×/1.49, oil) objective to avoid inducing detrimental effect to living cells. Differential interference contrast microscopy (DIC) images were taken prior to and after excitation to serve as a reference point for cell viability.

Fluorescence images were acquired using a Nikon spinning disk confocal system with a 60x oil-immersion objective, equipped with an Andor Ixon EMCCD camera, under the control of the Nikon elements software. Images were processed using the image analysis software ImageJ (http://rsb.info.nih.gov/ij/). Representative confocal micrographs displayed in the figures are maximal intensity projections of the 3D data sets, unless otherwise noted.
Live cell imaging of inclusions expressing the fluorescent protein Clover (Lam et al., 2012 (link)) under the control of the hctA promoter (Grieshaber et al., 2012 (link); Chiarelli et al., 2020 (link)) was achieved using an automated Nikon epifluorescent microscope equipped with an Okolab (http://www.oko-lab.com/live-cell-imaging) temperature controlled stage and an Andor Zyla sCMOS camera (http://www.andor.com). Images were taken every fifteen minutes for 48 hours. Multiple fields of view of multiple wells of a glass bottom 24 well plate were imaged. The fluorescence intensity of each inclusion over time was tracked using the ImageJ plugin Trakmate (Tinevez et al., 2016 (link)) and the results were averaged and plotted using python and matplotlib.

For fluorescence time-lapse microscopy, cells were imaged using a confocal spinning-disk inverted microscope (Nikon TI-E Eclipse) equipped with an Evolve EMCCD camera. The optical sectioning was performed by a Yokogawa motorized confocal head CSUX1-A1. Images were acquired using an illumination system from Roper Scientific (iLasPulsed) with a CFI Plan APO VC oil immersion objective (60X, N.A 1.4) or CFI Plan Fluor oil immersion objective (40X, N.A. 1.3). Z-series were generated using a motorized Z-piezo stage (ASI) by acquiring images with a step size of 0.4 μm. Microscope was controlled with MetaMorph software (Molecular Devices). Temperature, CO₂, and humidity control was performed using a chamlide TC system (TC-A, Quorum technologies). Solid-state 405, 491 and 561 nm lasers (iLas, Roper Scientific) and ET 460/50M (Chroma), ET 525/50M (Chroma), FF01-605/54 (Semrock) emission filters were used for excitation and emission of Hoechst 33342, EGFP and mCherry fluorescence, respectively.

Tissue preparations containing urothelium, lamina propria and smooth muscle bundles were loaded with the Ca²⁺-indicator, fluo-4AM (1μM). Preparations were imaged with an EMCCD camera (Nikon, London, UK) and images acquired at 20fps with WinFluor software (University of Strathclyde, Glasgow, UK). Changes in fluorescence intensity (ΔF/F₀) were calculated off-line by normalising the background-corrected fluorescence (F) to baseline (F₀) which was obtained by averaging 150 frames during quiescent periods in active cells. Data analysis and statistical tests were performed with Microsoft Excel, Prism (Graphpad Prism, v5.02) and Clampfit (pClamp, v10.3). The amplitude, frequency and area under the curve (AUC) of spontaneous Ca²⁺ events were measured during 120s of continuous recording. Peak fluorescence intensity occurring during the recording window was noted; events with fluorescence intensities greater than 10% of the peak were accepted for analysis. Amplitude was measured as fluorescence intensity (ΔF/F₀); event duration was measured when ΔF/F₀ exceeded 10% of the peak amplitude and recovered to this level and frequency was calculated as number of events/min. AUC is reported as ΔF/F₀.min. Unpaired t-tests were used with p<0.05 considered significant.