Mira3 fesem

All samples were fixed for 24 h with 4% paraformaldehyde and 1% glutaraldehyde in PBS, post-fixed for 45 min with 1% osmium tetraoxide in dH₂O, washed and subsequently dehydrated stepwise in ethanol of 25%, 50%, 70%, 95%, 100%, 100% before drying in a critical point dryer (CPD 030, Bal-Tec). Samples were coated with gold-palladium in a Desk V sputter system (Denton Vacuum) and imaged on a field emission scanning electron microscope (Mira3 FE-SEM, Tescan or FE-SEM LEO 1550, Carl Zeiss Inc.). For actin depolymerization studies, cells were treated for 60 min with 10 μM LatA (Cayman Chemical) before fixation, where indicated.

Cells were fixed on TEM grids using freshly prepared Trump’s fixative (4 mL 10x PBS, 10 mL 16% paraformaldehyde, 4 mL 10% glutaraldehyde, 22 mL ddH₂O). First, grids were rinsed with Trump’s fixative at 37°C, which was removed immediately. Fresh Trump’s fixative was added at 37°C and grids were stored at 4°C overnight before removal. Grids were then immersed in 1% osmium tetroxide in 0.05 M cacodylate buffer on ice for 40 minutes. Cacodylate buffer was added in excess for 10 minutes and removed. Fresh buffer was added for another 10 minutes. This process was repeated twice. Grids were washed with 25% and 50% EtOH on ice for 10 minutes, followed by 70% EtOH overnight. Grids were washed with 95%, 100%, 100% EtOH on ice for 10 minutes each before undergoing critical point drying (CPD). Samples were coated with Au-Pd (at 10 mA for 15 seconds) and imaged in a Mira3 FESEM (Tescan, Czech Republic). High-resolution images were acquired at 5 keV and a 3 mm working distance with the in-beam secondary electron detector. Brightness and contrast were adjusted using Adobe Photoshop to optimize visibility of cellular features. Image processing was done consistently across all conditions and regions. The vesicle size distribution shown in Fig. 3F was determined directly from the SEM image by estimating each vesicle as an ellipse and calculating the average of the minor and major axes.

Quantitative microstructural analysis of the different hydrogels was performed on micrographs captured with scanning electron microscopy (SEM). Blank hydrogels prepared as described above were dehydrated using an ethanol gradient. Samples were then placed into hexamethyldisilazane for 10 minutes to preserve collagen microstructure and air dried in a chemical hood. Dry samples were attached to SEM specimen mounts and coated with gold/palladium alloy in a sputter coater (SCD 500, Baltec). All SEM images were taken at 40,500 fold magnification (Mira3FESEM, Tescan). For SEM image analysis, fiber length and diameter were measured utilizing ImageJ (NIH). For each condition, 3 samples were analyzed, images from 5 randomly selected areas were taken and 7 fibers from each image were analyzed. Fiber diameter and length were measured utilizing ImageJ (NIH) following manual tracing of single surface-associated fibers (Supplementary Figure 1).