Megaview

Cryo-TEM measurements were performed on a FEI Tecnai G2 20 cryo-Transmission Electron Microscope (Jena Center for Soft Matter, Jena, Germany). The acceleration voltage was set to 200 kV. Samples were prepared on Quantifoil grids (3.5/1) after cleaning by argon plasma treatment for 120 s; 8.5 µL of the solutions were blotted by using a Vitrobot Mark IV. Samples were plunge-frozen in liquid ethane and stored under nitrogen before being transferred to the microscope utilizing a Gatan transfer stage. TEM images were acquired with a 200 kV FEI Tecnai G2 20 equipped with a 4k x 4k Eagle HS CCD and a 1k x 1k Olympus MegaView camera.

Neurons were maintained at 37°C in the sample holder of the microscope stage capable of moving in X and Y directions with nanometer precision and imaged through 100 X oil immersed, 1.4 NA objective lens mounted on an inverted microscope (IX80, Olympus). Stacks of phase contrast images of neurons from DRG ganglia were obtained by Charge couple device (CCD) camera (Olympus Megaview) and by moving the objective lens vertically. Each stack contains images obtained in the focal plane of the objective, focused on the coverslip where neurons were cultured i.e. at height 0 and at 1, 2, 3, 4, 5 and 6 micron above the coverslip. Stacks of images were acquired with 0.1–1 Hz frequency to quantify the 3D motion of lamellipodia. Then, for a further analysis, the time lapse image sequence for each height was extracted by using Xcellence software (Olympus) to create videos of different height. Two algorithms were developed to quantify the dynamics of lamellipodia. Algorithm I was designed to quantify in a semi-automatic way the time course of protrusion/retraction cycles by using an improved version of the Kymograph [52 (link),53 (link)]. Algorithm II was designed to quantify the vertical motion of lamellipodia during these cycles.

Cryo-TEM images were acquired with a 200 kV FEI Tecnai G2 20 transmission electron microscope equipped with a 4k × 4k Eagle HS CCD and an Olympus MegaView camera (1379 × 1024 pixels) for overview images. Sample preparation was performed by plunge-freezing the samples with a Vitrobot Mark IV system. 8.5 µL of the aqueous solutions were blotted (blot force − 2; blotting time 1 s) on Quantifoil grids (R2/2, Quantifoil, Jena, Germany) and were vitrified in liquid ethane. The grids were rendered hydrophilic by Ar-plasma cleaning for 30 s (Diener Electronics, Germany). Samples were stored in liquid nitrogen until transfer to the cryo holder (Gatan 626). Transfer to the microscope was performed with a Gatan cryo stage and the temperature was maintained below − 172 °C at all times after vitrification.

For TEM measurements, copper grids were rendered hydrophilic by Ar plasma cleaning for 2 min (Diener Electronics, Ebhausen, Germany). Ten microliters (10 µL) of either AgNPs/PB-b-PDMAEMA sample or control sample (AgNPs prepared in the absence of the PB-b-PDMAEMA diblock copolymer) were applied onto the grid and an excess of the sample was blotted with a filter paper. TEM images were acquired with a 200 kV FEI Tecnai G2 20 transmission electron microscope equipped with a 4 k × 4 k Eagle HS CCD and a 1 k × 1 k Olympus MegaView camera for overview images.