Deltavision softworx program

HeLa cells were seeded onto eight chamber glass slides at 5 × 10⁴ cells per chamber and allowed to attach overnight. The media was replaced with either free DOX or DOX-SPIOs at 1 μg/ml DOX. At 2 and 8 hours of incubation, cells were washed with PBS, fixed with paraformaldehyde, and stained with DAPI. Cells were imaged with a DeltaVision deconvolution fluorescent microscope equipped with DAPI, TRITC, and Cy5 filters (Applied Precision, Issaquah, WA). Confocal image processing was done using the DeltaVision Softworx program (Applied Precision, Issaquah, WA). The fluorescent intensity of the images was normalized using ImageJ (NIH, Bethesda, MD). The iron internalization was measured by trypsinizing the cells, performing a cell count, and measuring the iron content of the cell population with a ferrozine assay and subtracting the endogenous iron measured from an untreated sample.

E. amylovora cells were inoculated onto imaging pads in welled microscopy slides. Pads were made up of 1% agarose, 25% LB, 2 μg/mL FM4–64, and 0.1 μg/mL DAPI. Between 0–1% arabinose was used to induce expression from the pHERD-30T plasmid, depending on the construct. The slides were incubated at 30°C for 3 hours, then moved to room temperature for infection with 10 μL undiluted RAY lysate. Slides were imaged using the DeltaVision Elite deconvolution microscope (Applied Precision) and deconvolved using the aggressive algorithm in the DeltaVision softWoRx program (Applied Precision). Image analysis was performed on images prior to deconvolution.