Em ccd camera c9100 02

Immunohistochemistry images were recorded with a Olympus microscope equipped with the photoP software, immunofluorescence images with a Leica TCS SP5/AOBS/tandem scanning system with emission detection in sequential mode equipped with the Leica LAS-AF software (v. 2.6.0.7266), and cell migration images with a Leica DMI6000B TIRF MC system equipped with software LAS-AF (v. 2.0.2.2038) and Hamamatsu EM-CCD camera C9100-02 in bright field (TL-PH) mode.

MCF10A cells were infected with pLKO.1 LifeAct-GFP using a M.O.I. equal to 2.
Then cells were seeded at low density on glass bottom plates (Porvair, Norfolk,
UK) coated with 1 μg/ml fibronectin (Sigma-Aldrich, St
Louis, MO, USA). After an overnight starving in medium w/o growth factors (DMEM
supplemented 200 U/ml of penicillin and
200 μg/ml streptomycin), cells were placed on an
inverted microscope equipped with a 37 °C humidified
chamber with 5% CO2, and visualized using True MultiColor Laser TIRF Leica AM
TIRF MC (Leica Microsystems, Wetzlar, Germany) equipped with a 63X oil immersion
objective (HCX PL APO 63x/1.47 OIL CORR TIRF) and Hamamatsu EM-CCD camera
C9100-02. Images were acquired through Leica LAS AF6000 modular system software.
The depth of the evanescent field was kept at 90 nm. Time lapse
movies were performed with a 20 seconds interval and EGF or HGF were
added at the 4^th frame. Cell area was calculated using
ImageJ56 (link).

The impact of both street and standard MXP samples on 5637 and SH-SY5Y cell morphology was examined by wide-field fluorescence microscopy using an Olympus IX-81 microscope (Olympus, Tokyo, Japan). The images were acquired by EM-CCD camera C9100-02 (Hamamatsu, Nakaku, Japan) using xCellence software, 60× oil immersion objective with the NA of 1.4 and high-stability 150 W xenon burner. The images were background-corrected and deconvolved by 2D deconvolution with the no-neighbor algorithm.

Slides were stained with Sirius Red and counter stained with haematoxylin. Slides were mounted on an inverted microscope (Zeiss AxioVert 200 M) equipped with a circularly polariser and coupled to a Hamamatsu EM-CCD C9100-02 camera. Images were taken in grey tone under polarised light were collagen stained with Sirius red appears brightly. Analysis was performed as above and expressed as an area of collagen per airway perimeter.

All microscopy was performed in the Facility for Imaging by Light Microscopy (FILM) at Imperial College London. Time-lapse live-cell microscopy was performed using a CellASIC® ONIX microfluidic platform (Merck-Millipore) using a B04A-03 bacterial plate. Cells were inoculated from glycerol stocks in their appropriate HdB carbon source variant and grown at 37 °C in an orbital shaker at 180 rpm to stationary phase, then diluted 1:200 in fresh medium to mid-exponential phase. Cells were diluted again to an OD₆₀₀ of 0.1 and loaded into the growth chamber of the microfluidic plate. Fresh medium was perfused at a continuous pressure of 1 psi (approximately corresponding to a 5 μl h-1 flow rate) in an environmental chamber at 37 °C. Phase contrast images were captured every 15 min using a Zeiss Axiovert 200M inverted widefield microscope using a 63X oil immersion objective, a motorized stage, and an EM-CCD (C9100-02) camera (Hamamatsu). 4 fields of view per condition (4 conditions per plate) were captured at each timepoint. To ensure in-focus images were obtained, a software autofocus function (Zen) and 10 Z slices at 1 μm intervals were used at each position.