Elyra ps 1 super resolution microscope

Images captured on an Axioskop2 (Zeiss) fluorescent microscope used a 63 x DC magnification lens and images were acquired with ZEN software (Zeiss). For images captured on an Olympus IX71 DeltaVision Core System (Applied Precision, GE), a 1.40/100 x lens was used, and images were acquired using SoftWoRx suit 2.0 (Applied Precision, GE). Z stacks were acquired (no more than 10 μm thick) and images de-convolved (conservative ratio; 1024x1024 resolution) using the SoftWoRx software. Super-resolution structural illuminated images were captured on an Elyra PS.1 super resolution microscope (Zeiss), and using images were acquired using ZEN software as Z stacks. Fiji (Schindelin et al., 2012 (link)) was used to subtract the background of images and for counting cells. The brightness and contrast for counting were set relative to unstained controls. False colors were assigned to fluorescent channels and the signal enhanced for clear visualization. 3D images were generated using IMARIS software (V8.2; Oxford Instruments) using super-resolution Z stacked images. Scale bars are as stated on images or in legends.

Confocal fluorescence imaging was performed with a Zeiss Elyra PS-1 Super Resolution Microscope (Oberkochen, Germany), using a white-light laser source and a 60× objective. Briefly, Caco-2 cells (seeding density of 1 × 10⁵ cells/well) were incubated with rhodamine B-MSN at a concentration of 50 µg/mL in DMEM. After 4 h incubation, the supernatant was discarded, and the cells were washed 3x with PBS and fixed with 4% paraformaldehyde. Before imaging, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged at an emission wavelength of 461 nm (excitation wavelength 358 nm) and the cell membrane was stained with Alexa-488 at an emission wavelength of 525 (excitation wavelength of 490 nm) which appeared as blue and green, respectively.

To image transformed bacteria, mating experiments, and the soil survival experiment, we used a Zeiss AxioImager M1 microscope. To gain further insight into enzyme localization in E. coli, we performed structured illumination microscopy (SIM) using a Zeiss Elyra PS.1 Super Resolution microscope. We used cryotome sectioning and confocal microscopy using a Zeiss LSM710 to probe enzyme localization in EPS. For TEM, cell cultures were stained with 2% methanolic uranyl acetate and imaged using a Tecnai 10 TEM.

Acid-washed high performance coverslips (#1.5H, thickness 170±5 μm, Schott) were used, processed for immunofluorescence as above with the exception of using eight times the concentration of fluorescently labelled phalloidin (1:50) and mounting on unfrosted glass microscope slides (Thermo Fischer Scientific) followed by curing for three days at room temperature in the dark prior to imaging. Z-stacks were acquired at five phases and five rotations of the illumination grid on an ELYRA PS.1 superresolution microscope (Carl Zeiss Microscopy). The images were processed and channel aligned using ZEN Black ELYRA edition (Carl Zeiss Microscopy). Line profiles on single slices were generated in ZEN Black and normalized for each channel.

The proximity ligation assay (PLA) was performed using the Duolink Red Starter Kit for Rabbit/Mouse (Sigma #DUO92101) according to the manufacturer’s specifications. Briefly, cells were fixed with 3.7% formalin (Sigma-Aldrich #F8775) for 25 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich #X-100) for 5 min, and blocked using 0.05% Tween20 (Fisher Scientific #BP337) in 2mg/mL in PBS for 1 hour (all steps performed at room temperature). Samples were then incubated with primary antibodies against acetyl-p53 K373 (EMD Millipore #04–1137) and Bax (Santa Cruz #sc-7480), followed by incubation with the PLA probes. The Duolink PLUS and MINUS probe was used against the anti-acetyl-p53 K373 and anti-Bax antibodies, respectively. If the two proteins are interacting, the oligonucleotide PLA probes would be in proximity of each other. Ligation of the co-localized probes was performed using a ligase and amplification of the ligated oligonucleotides was achieved through rolling circle replication with a polymerase. Samples were then incubated with fluorescent probes specific for the oligonucleotides. Slides were imaged on the Zeiss Elyra PS1 Super-Resolution Microscope using structured illumination (SIM) at the Delaware Biotechnology Institute Bioimaging Center.