Imagemx2 digital em ccd cameras

To measure adhesion dynamics, single-wavelength temporal image correlation microscopy (tICM) and dual-wavelength temporal image cross correlation microscopy (tICCM) assays were carried out using a TIRF microscope platform. Cells on 35 mm cover glass bottom dishes were placed in a stage top environmental control chamber (Live Cell Instrument, CU-501) at 37 °C under a 5% CO₂ environment with a flow rate of 50 mL/min. TIRF was achieved using a Spectral Diskovery unit (Spectral Applied Research, Richmond Hill, ON) attached to an inverted Leica DMI6000B microscope (Leica Microsystems, Wetzler, Germany) with a Leica Plan ApoChromat 63x/1. 47 NA TIRF oil immersion objective lens. The platform incorporated a 488 nm diode laser, 561 nm diode-pumped solid-state laser (Spectral Applied Research) and was equipped with two ImagEMX2 Digital EM-CCD Cameras (Hamamatsu, Hamamatsu City, Japan). An EGFP filter cube (ET 525/50 nm), mCherry filter cube (ET 620/60 nm), and EGFP/mCherry dual cube allow for single wavelength or dual-wavelength simultaneous pixel-by- pixel image acquisitions. A 100 Watt X-Cite 120 LED (370–700 nm) source was also integrated to allow visualization of fluorescence proteins by eye. The platform was integrated with MetaMorph 7.1 image acquisition software (Molecular Devices Inc.) and custom designed TIRF controls (Quorum Technologies Inc., Guelph, ON).

To measure protein dynamics, the tICM were implemented using a TIRF (total internal reflectance fluorescence) microscope. Caco-2 cells were transient transfected with CD13-mCherry and GFP-Rab11 in 2D culture. After 24 h, cells were seeded on 8 well Ibidi plate supplemented with 5% GelTrex-media at 37 °C in 5% CO₂ in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin for 24 h prior to performing live imaging. TIRF was obtained using a Spectral Diskovery unit (Spectral Applied Research, Richmond Hill, ON) attached to an inverted Leica DMI6000B microscope (Leica Microsystems, Wetzler, Germany) with a Leica Plan ApoChromat 63x/1. 47 NA TIRF oil immersion objective lens. The platform incorporates a 488 nm diode laser, 561 nm diode-pumped solid-state laser, 642 nm didode laser (Spectral Applied Research) with two ImagEMX2 Digital EM-CCD Cameras (Hamamatsu, Hamamatsu City, Japan). A 100-Watt X-Cite 120 LED (370-700 nm) source was applied to allow visualization of fluorescence proteins by eye. The platform was integrated with MetaMorph 7.1 image acquisition software (Molecular Devices Inc.). Each image was set to collect 1000 frames with a 30 ms interval.