Imageem emccd camera

Single colonies grown overnight on LB plates with antibiotics were used to inoculate the supplemented M63 medium and the cultures were grown at 30 °C (for better folding of fluorescent proteins) to log phase (OD_600nm 0.3). They were diluted two-fold in the same medium to which 0.2% arabinose or 20 ng/ml aTc was added and the growth was continued 90 min or 45 min, respectively. 5 μl of cell culture was spotted on the center of a glass P35 dish (MatTek corporation, Ashland, MA) and the spot was overlaid with 1% agarose gel pad made with the same supplemented M63 medium. Time-lapse microscopy was performed starting at 90 min after inducer addition, with images taken every 15 min, unless otherwise specified, on a Nikon TiE inverted microscope with Nikon 100x/1.4 Oil Plan Apo Ph3 DM lens, Lumencor sola light engine (Beaverton, OR), and ImageEM EMCCD camera (Hamamatsu, Japan). The fluorescence from GFP was detected using 10% laser output and 300 msec exposure, and from td-Tomato using 30% laser output and 700 msec exposure.

The cells were imaged in their living state by epifluorescence (Scientifica Slice Scope, Cairn Research OptoLED 470 nm illumination, 60× water Olympus immersion objective, NA 1.0, Hamamatsu ImageEM EMCCD camera, Metafluor software). Using ImageJ, the extent of dye loading was measured by drawing a region of interest (ROI) around individual cells and calculating the mean pixel intensity for the ROI. The mean pixel intensity of the background fluorescence was also measured in a representative ROI, and this value was subtracted from the measures obtained from the cells. All of the images displayed in the figures reflect this procedure in that the mean intensity of the pixels in a representative background ROI has been subtracted from every pixel of the image. At least 40 cells were measured in each condition per independent repetition. The mean pixel intensities from all independent repetitions were plotted in one cumulative probability distribution. Statistical comparisons of dye loading under different conditions were performed by comparing the individual median values from each independent repetition via the Mann–Whitney U-test. As we have used non-parametric statistical comparisons, we show the median values together with the upper and lower quartiles in the figures.

Cells were grown in 1× M63 medium supplemented with 1 mM CaCl₂, 1 mM MgSO₄, 0.001% vitamin B1, 0.2% fructose, 0.1% casamino acids at 30°C until an OD_600 nm of 0.3. When required, kanamycin was added to a final concentration of 12.5 μg/ml. Expression of ParA2:GFP or ParA2 K124X:GFP (where X = Q, E or R) was induced by adding 0.0008% arabinose for 1 h at 30°C with shaking. 10 μl of the culture was plated on the centre of a glass P35 dish (MatTek corporation, Ashland, MA), and overlaid with a 1% agarose disc prepared with the same M63 medium described above but supplemented with 0.02% arabinose. Images were taken every 30 secs on a Nikon Ti-Eclipse inverted microscope with Nikon 100×/1.4 Oil Plan Apo Ph3 DM objectives, imageEM EMCCD camera (Hamamatsu, Japan) and Lumencor sola light engine (Beaverton, OR) set to 5% 475 nm laser output and 300 ms exposure.

A time series of 2D multichannel images of strain JN2101 was used to test the detection and the tracking performance of the proposed method for 2D time lapse images. The experimental conditions for sample preparation and stimulation are same as Data 2 except stimulation period (51 to 151 frames after initiation of the experiment). The fluorescence in the CFP and YFP channels was observed simultaneously using DMI6000B inverted microscope with HCX PL APO 63x (NA 1.40) objective lens (Leica), a dual-view FRET imaging system DV2 (Photometrics) and an ImageEM EM-CCD camera (Hamamatsu Photonics) [46 (link)]. The sizes of the image along the x₁ and x₂ axes were 512 and 256, respectively. The sizes of a voxel along the x₁ and x₂ axes were 0.254 and 0.254 μm, respectively. The frame rate was 1.83 per second, and 241 2D frames were recorded (about 131 s).

To image Ca²⁺ changes in tanycytes, hypothalamic slices were incubated with the Ca²⁺ indicator Fura‐2 (12.5 µg/ml in 0.125% DMSO and 0.025% pluronic) for 90–120 min in 1.0 mM glucose aCSF. Loaded slices were transferred to a flow chamber containing circulating 1.0 mM glucose aCSF and imaged with an Olympus BX51 microscope using a 60× water immersion objective (NA 0.95). An Andor Ixon EM‐CCD camera was used to collect the images. A ratiometric image of Fura‐2 loading was achieved by illuminating at 340 and 380 nm with a xenon arc lamp (Cairn Research) and a monochromator (Optoscan, Cairn Research).
For live imaging of slices derived from mice expressing GCaMP3, the slices were mounted on a Scientifica Slicescope and observed via an Olympus 60x water immersion objective (NA 1.0). Illumination was provided via a 470 nm LED (OptoLED, Cairn Research) and a Hamamatsu ImageEM EM‐CCD camera was used to collect the images. Metafluor imaging software was used to control the illumination and camera in all experiments.