Ixon du 897 emccd camera

For time-lapse microscopy, cells were plated onto 22 × 22 mm (No. 1.5) glass coverslips (Corning) and cell culture medium was changed to DMEM with 10% FBS (without phenol red and supplemented with HEPES buffer) 6-12 h before mounting. Coverslips were mounted onto 1-well Chamlide CMS imaging chambers (Microsystem AB; Sweden) immediately before imaging. All live-cell imaging experiments were performed at 37°C using temperature-controlled Nikon TE2000 microscopes equipped with a modified Yokogawa CSU-X1 spinning-disc head (Yokogawa Electric), an electron multiplying iXon+ DU-897 EM-CCD camera (Andor) and a filter-wheel. Three laser lines were used for excitation at 488, 561 and 647nm. All live cell imaging experiments were performed using an oil-immersion 100x 1.4 NA Plan-Apo DIC (Nikon), with the exception of the experiments conducted with co-expression of Fab311-Cy3 and eGFP-Nup153, that were performed using an oil-immersion 60x 1.4 NA Plan-Apo DIC (Nikon). All image acquisition was controlled by NIS Elements AR software. Images were collected every 30 s: 9 × 2 μm z stacks spanning a total volume of 16 μm.

For super-resolution image, we used super-resolution structured illumination microscopy (SIM; Nikon N-SIM). The raw images were reconstructed to three-dimensional SIM images using NIS-Elements software (Nikon). Images were acquired using 100× oil objective lens [numerical aperture (NA), 1.49] and equipped with an iXon DU-897 EMCCD camera (Andor Technology). Multicolor fluorescence was acquired using a diode laser (488 nm, 561 nm). For colocalization percentage analysis, acquired whole images were analyzed using Fiji (https://imagej.net/Fiji). For FRAP analysis, we used a Nikon A1R⁺ inverted confocal microscope with 60× oil objective lens (NA, 1.49). A 3 μm × 3 μm photobleaching spot was chosen at a dendrite. Cells were maintained in a humidified, 5% CO₂ environmental chamber at 37°C (Live Cell Instrument), and images were acquired every 20 s for 10 min using NIS-Elements. Maximum intensity projection images were used, and each fluorescence intensities before bleaching were set to 100%. For dendritic spine density analysis, spines were defined on the basis of morphology from GFP volume filling. To be considered a spine, the compartment must be a clearly defined protrusion from the dendrite, extending at least 1 μm away from the dendritic shaft. Acquired images were analyzed using ImageJ [National Institutes of Health (NIH), https://imagej.nih.gov/ij/].

To identify the modification of cerebral capillaries by senile plaque, we used the super-resolution Structured Illumination Microscope (Nikon N-SIM, Nikon). 3D-SIM images of each fixed brain slices were taken by moving the stage in the z-direction with a step size of 0.15 μm. The sequential z-sections were reconstructed to create a 3D-SIM image (z axis; brain slices thickness about 5.0±0.4 μm) and produce the 3D-deconvolution with alpha blending function using NIS-E software (Nikon). Images were taken with an Eclipse Ti-E inverted research microscope, using CFI Apo TIRF × 100 oil objective lens (NA=1.49, Nikon) and 512 × 512-pixel resolution iXon DU-897 EMCCD camera (Andor Technology, Belfast, UK). Multicolor fluorescence analysis was performed using a diode laser (488, 561 nm; exposure time, 40 ms; EM gain, 150; conversion gain, × 1), and images were processed with the NIS-E software (Nikon, Tokyo, Japan) and exported to the Adobe Photoshop program.

Single-molecule imaging was performed 24–48 h after RNA injection using total internal reflection fluorescence (TIRF) microscopy. Manually devitellinized oocytes were placed on high-refractive-index coverglass (n = 1.78) and imaged through a 100× 1.65-NA oil immersion objective (Olympus). 13 × 13–µm frames showing low density (50–250 individual puncta per frame) to minimize the risk of false colocalization were imaged. EGFP-tagged VSDs and full-length subunits were excited with a PhoxX 488 (60 mW) laser, and mCherry-tagged PDs and full-length subunits were excited with a 593-nm diode-pumped solid-state (DPSS) laser. z488/594-rpc polychroic (Chroma) was used as the excitation filter; 525/50 and 629/53 emission filters were used for EGFP and mCherry, respectively. mCherry and EGFP were excited sequentially. Videos of 800 frames (∼200 for mCherry and 600 for EGFP) were captured at the rate of 20 Hz with the iXon DU-887 EMCCD camera (Andor Technology).
The bleaching videos of the PD tagged with EGFP were acquired in a similar way, using Nikon 100× 1.49 NA oil immersion TIRF objective. Devitellinized oocytes were placed on 35-mm µ-dishes with glass bottom (n = 1.52; IBIDI). They were excited with a 488-nm laser. The light path contained a 525/50-nm emission filter. The videos were captured with iXon DU-897 EMCCD camera (Andor Technology) at 10 Hz.

Chromaffin cells were imaged at room temperature using an inverted microscope (Ti-E, Nikon). TIRF microscopy was done using a MLC400 monolithic laser combiner (Agilent Technologies), ZT405/488/561/640rpc dichroic filter (Chroma), and Plan Apo Lambda 100× 1.45 N.A. oil objective (Nikon). Rapid FFN imaging was done using ET455/50 m emission filter (Chroma) and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Dual imaging of FFN and BDNF-pHluorin was done using ZET488/561/635m emission filter (Chroma) and iXon DU-897 EMCCD camera (ANDOR). Dual imaging of FFN with Alexa Fluor 488 (ThermoFisher) or CF488A (Biotium) was done using Dual-View filter cube (Optical Insights) mounted with T510lpxrxt beam-splitter (Chroma), ET460/36 m and ET545/40 m emission filters (Chroma), and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Confocal imaging of FFN and Alexa Fluor 488 was done using an integrated laser engine (ANDOR), Borealis CSU-W1 spinning disk (ANDOR), ZT405/488/561/640rpcv2 dichroic filter (Chroma), Plan Apo VC 100× 1.4 N.A. oil objective (Nikon), ZET405/488/561/635 emission filter (Chroma), and iXon DU-888 EMCCD camera (ANDOR). Cells were imaged in modified Tyrode's solution, and exocytosis was stimulated with 45 mM K⁺ by adding an equal volume of stimulation buffer (in mM, 54.5 NaCl, 10 HEPES-NaOH, pH 7.4, 10 glucose, 90 KCl, 5 CaCl₂, 1 MgCl₂).