Ec plan neofluar

Frozen grids were fixed into FIB C-clip rings to increase mechanical stability. Samples were transferred to a CorrSight shuttle (FEI) and mounted on the CorrSight cryo-stage (FEI) maintained at liquid nitrogen temperature for cryo-light microscopy. Samples were imaged using wide-field or spinning-disk confocal, epifluorescence microscopy using FEI MAPS 2.1 software. Images were acquired with a 1,344 × 1,024 pixel camera (pixel size 6.4 μm, Hamamatsu Digital Camera C10600 ORCA-R2). Grids were imaged in wide-field mode at low magnification with a 5× air objective (Plan-ApoChromat, NA 0.16; Carl Zeiss) and in spinning-disk confocal mode at high magnification with a 40× air objective (EC Plan-Neofluar, NA 0.9; Carl Zeiss) for identification of cells and/or aggregates. Image acquisition and further SEM correlation was performed by three-point correlation using FEI MAPS 2.1 software.

Cells upon treatment were transferred to ice, washed twice with ice-cold PBS, and fixed with ice-cold 3.6% paraformaldehyde for 15 min. After three washes with PBS, cells were immunostained, as described previously [44 (link)–46 (link)].
Slides were scanned using a ZEISS LSM 710 confocal microscope with EC Plan-Neofluar 40 × 1.3 NA oil immersion objective. ZEN 2009 software (Zeiss) was used for image acquisition. At least ten 12-bit images with resolution 1024 × 1024 pixels were acquired per experimental condition. Images were then analyzed by MotionTracking software (http://motiontracking.mpi-cbg.de) with respect to integral intensity and number of LTβR- and EEA1-positive vesicles [47 (link)–49 (link)]. Images were then assembled in Photoshop (Adobe) with only linear adjustments of contrast and brightness.

The periphery of hippocampus was coronally sectioned into slices of 1–3 mm-thickness for 3D imaging. After treating the slices for different lengths of times according to their thickness, they were embedded in 2% agarose, trimmed, put into AICI reagent again, and placed on the shaker for 1–2 h at 25 °C. The excitation source was a λ = 488 nm laser for GFP detection. 3D-images of brain slices were taken by light sheet fluorescence microscopy (LSFM, Lightsheet Z.1, Carl Zeiss, Germany). The illumination lens was a 5x, 0.1 NA at air and the objective of emission part was a 5x, 0.16 NA, EC Plan-Neofluar by Zeiss. The stitched 3D-images were 3 × 3 tiles with field of view of 2.47 × 2.47 mm (1920 × 1920 pixel), z-step size was a 5 μm. All acquisitions were controlled by ZEN (Carl Zeiss) software. The experiment for fine neuronal structure preserving was implemented by confocal microscopy (A1Rsi, Nikon, Tokyo, Japan) under the control of NIS (Nikon) software ver 5.0. The objective was a 60x, Plan Apochromat from Nikon. A field of view of images was a 212 × 212 μm (1024 × 1024 pixel) with 0.2 μm z-step varying focus depth.

Micro particle image velocimetry (μPIV) was performed using deionized water as working fluid, seeded with 3.55 μm fluorescent polystyrene particles (530/607 nm, microParticles GmbH). The experimental set-up consisted of an epi-fluorescence microscope system including a 3D stage system (LaVision FlowMaster Mitas) in combination with a 532 nm Nd:YAG laser (New Wave, Solo II-15) and a 2048 × 2048 pixel CCD camera (LaVision Imager ProX 4 M). For image acquisition a 5X, 0.16 NA microscope objective was employed (Zeiss EC “Plan-Neofluar”). For data post-processing the commercial software package Davis 7 (LaVision) was used. The volume flow of fluids was controlled with a peristaltic pump (P1500, Harvard Apparatus) and measured with a flow sensor (PVDF Chemical Flowmeter 0.025–2.5 l/min, B.I.O-TECH e.K., Germany). For each flow condition and measurement position 25 two-frame experimental images were acquired at a trigger rate of 4 Hz. The cross-correlation technique was used for analysis. The final interrogation window of 128 × 128 pixels overlapped by 50%, the corresponding vector spacing was 62.5 μm. The depth of correlation was determined to be about 124 μm, following ref. 61 . After processing, the 25 experimental images were time-averaged. Further details on the experimental set-up have been previously reported in ref. 62 (link).