Dp73 digital microscope camera

For routine-, differential interference contrast light microscopy (DIC-LM) and fluorescence microscopy (FLM), cells were viewed with an Olympus BX60 (Olympus America Inc., Melville, NY, USA) equipped with an Olympus DP73 digital microscope camera, using DP Controller 3.2.1.276 software. Confocal laser scanning microscopy (CLSM) was performed with an Olympus FluoView™ 300 or 1200 Confocal Microscope using Fluoview 5.0 with O3D software.

Cell shape were preliminarily observed using a Stemi 2000C anatomical lens (Carl Zeiss AG, Oberkochen, Germany). Brightfield images were collected on a BX51 microscope equipped with 4× 0.13 NA, 10× 0.3 NA, 20× 0.5 NA, 40× 0.75 NA, and 100× oil 1.3 NA objectives lenses (Olympus, Osaka, Japan). Brightfield Images were captured using an DP73 digital microscope camera (Olympus, Osaka, Japan) and analyzed using cellSens imaging software version 1.16. Fluorescence images were collected on an Axio Imager A2 microscope equipped with 5× 0.16 NA, 10× 0.3 NA, 20× 0.5 NA, 40× 0.95 NA, 63× oil 1.25 NA and 100× oil 1.3 NA objectives lenses (Carl Zeiss AG, Oberkochen, Germany). Fluorescence images were captured using a pco.edge 4.2LT sCMOS camera (PCO AG, Kelheim, Germany) and analyzed using Zen 2.3 (blue edition).
Cell shape in control and RNAi cells were compared as follows: for each cell, the widest part of the cell outline was assumed to represent the OA, and the part farthest away from the OA was assumed to represent the holdfast [11 (link)]. The size of the cell was defined as the distance between the OA and the holdfast and calculated using the scale bars of the imaging software. Live, fully extended cells were used as controls for cell shape.

This method was performed only on PVDF nanofibers because of the inconvenient results from the disk diffusion method. Disks were removed from the Petri dish and placed on a glass slide. This was followed by dyeing them with fluorescent dye (SYTO^®9 and propidium iodide) for 10 s and then covering them with a square coverslip. The fluorescence microscope Olympus BX53 (Olympus, Tokyo, Japan) equipped with Microscope Digital Camera DP73 (Olympus, Tokyo, Japan) and the cell Sens Standard 1.18 (Olympus, Tokyo, Japan) software was used to determine this method. The live and dead bacteria cells were absorbed according to the dye’s interaction with the cell membrane.

The samples (25 mm²) were washed with sterile saline solution after the cultivation (see Section 2.4.1) and placed on a glass slide. They were dyed by fluorescence dye (SYTO^®9 and propidium iodide) for 10 s and then covered with a square coverslip. Fluorescence microscopy was performed using a fluorescence microscope Olympus BX53 (Olympus, Tokyo, Japan) equipped with Microscope Digital Camera DP73 (Olympus, Tokyo, Japan) and the cell Sens Standard 1.18 (Olympus, Tokyo, Japan) software. The analysis was performed on a minimum of 20 positions in three replicates. LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Thermo Fisher, Waltham, MA, USA), based on the protocol [31 ], was executed using slight modifications. SYTO^®9 dyed plasma membranes of all bacteria, while propidium iodide can color DNA of only dead cells. The excitation/emission maxima for these dyes are 480/500 nm for SYTO^®9 stain and 490/635 nm for propidium iodide. Thus, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes (dead) stain fluorescent red.

MET gene status was evaluated using a commercially available FISH assay [26 (link)], with Vysis MET Spectrum Red FISH Probe (Abbott Molecular, Chicago, IL, USA) and control Vysis CEP7 Centromere Spectrum Green Probe (Abbott Molecular) on 4 μm-thick TMA sections. The signals of each sample were counted in at least 50 well-defined nuclei using a fluorescence microscope (BX43, Olympus, Tokyo, Japan) equipped with a Microscope Digital Camera (DP73, Olympus, Tokyo, Japan). An average MET gene copy number ≥ 5 and a MET/CEP7 ratio ≥ 2 (true MET amplification) were regarded as MET FISH positive [22 (link)].