Scmos camera

In-house imaging was performed on a Nikon Eclipse Ti-E wide-field epi-fluorescence microscope, equipped with a sCMOS camera (Hamamatsu) and X-cite LED for fluorescence imaging. We imaged through ×60 and ×100 1.4 NA oil-immersion PH3 objectives. The microscope was controlled by NIS-Elements v.3.30.02. Cell samples were spotted on a 3% (w/v) agarose pad placed on a microscope slide. The microscope chamber was heated to 30 °C or 37 °C when needed for timelapse experiments.
Several images in our dataset were taken by two other laboratories using three distinct microscope/camera configurations. The Brun laboratory provided images of C. crescentus acquired on a Nikon Ti-E microscope equipped with a Photometrics Prime 95B sCMOS camera. Images were captured through a ×60 Plan Apo λ ×100 1.45 NA oil Ph3 DM objective. The Wiggins laboratory provided E. coli and A. baylyi timelapses from both a Nikon Ti-E microscope using NIS-Elements v.4.10.01 as well as a custom-built tabletop microscope using Micro-Manager v.1.4, both of which are described in previous studies^{48 (link),49 (link)}.

Optical projection tomography (OPT) system was used to analyze the 3D microstructure of the SF hydrogels and to evaluate the distribution of the cell-laden SF hydrogels. SF hydrogel samples were prepared by filling fluorinated ethylene propylene (FEP) tubes with 100μL of the un-laden or cell-laden SF/HRP/H₂O₂ mixture. The specimens were immersed in an index-matching liquid (distilled water) and rotated through a series of angular positions. The center of rotation and the alignment of the samples were adjusted using a manual x-y-stage (Standa, Lithuania) in conjunction with the sample-positioning module (Standa, Lithuania) by using the 4 available axes (3 translational, 1 rotational). The acquisition consists of rotating a sample 360° in 0.9° steps and capturing an image at each rotation angle, ranging from 0° to 359.1°. A total of 400 images were acquired per sample. The OPT system was used in brightfield mode. The images were captured with an sCMOS camera (Hamamatsu, Japan) and the exposure time was adjusted from 4 ms to 20 ms depending on the transparency of the sample. Projections collected in each orientation were used to create the 3D reconstructions of each sample and the visualization of the 3D-volume was obtained using Avizo software (FEI, USA).

Confocal immunofluorescence images were taken using an inverted Nikon Eclipse Ti2 microscope attached to a spinning disk unit (CSU-W1, Yokogawa, Melville, NY, USA) and Hamamatsu sCMOS camera (Hamamatsu City, Japan). A Nikon oil immersion objective (Plan Fluor 40X, NA 1.30) was used for all imaging experiments. The excitation wavelengths used were 405, 488, and 561 nm for blue, green, and red fluorophores, respectively. Further image processing and analysis was conducted using Fiji software (version 2.1.0/1.53c, open source). The fluorescence intensity of the respective signal or Lysotracker counts was obtained from at least three independent replicates (3–5 different fields/replicate). The mean fluorescence intensity (MFI) and average puncti count were calculated accordingly.

Size and concentration distributions of exosomes were determined using nanoparticle tracking analysis (NS-300 NanoSight Instrument, Malvern Instruments Ltd., Malvern, UK). NTA determines the Brownian motion of nanoparticles in real-time to assess size and concentration utilizing a laser-illuminated microscopic technique equipped with a 405 nm laser and a high sensitivity digital camera system (sCMOS camera, Hamamatsu Photonics, Hamamatsu, Japan).
EXO samples were diluted in vesicle-free PBS. Samples were administered and recorded under controlled flow, using the NanoSight syringe pump. Data acquisition and processing were performed using NTA software version 2.3 build 0025. Background extraction was applied, and automatic settings were employed to determine the minimum expected particle size, minimum track length, and blur settings. Since samples were diluted in ultrapure DPBS 0.0095 M (PO₄) w/o Ca and Mg (Lonza, Basel, Switzerland), viscosity settings for water were applied and automatically corrected for the temperature used. Data were obtained at camera-level 12 (shutter:600, gain: 350). For each sample, three videos of 30 s duration at 25 frames per second were recorded and assigned a single measurement in triplicates. Three sets of samples were run, from which exosome distribution, size and mean concentration were calculated.