660 nm center wavelength collimated led

QPI of HeLa and M202 cells was performed on an Axio Observer A1 inverted microscope (Zeiss) with a SID4Bio quadriwave lateral shearing interferometry (QWLSI) camera (Phasics). A temperature and CO₂ regulated stage-top cell incubation chamber (ibidi) was fit to a motorized xy stage (Thorlabs) to maintain environmental homeostasis and enable QPI at multiple locations. A Zeiss LD Plan Neofluar 20x NA 0.4 objective was heated and maintained at 37°C with a custom built copper objective heater driven by a heat controller (Thorlabs). Trans-illumination was by a 660 nm center wavelength collimated LED (Thorlabs). Image collection occurred every 10 min for 24 h at 15 randomly selected imaging locations per well containing cells plated with sufficient spacing to enable automated image processing and biomass segmentation. Imaging locations were selected within the central part of each well, where the highest quality quantitative phase information can be obtained without aberrations caused by the cell culture apparatus or optical hardware. The selected imaging fields cover a range of locations within each imaged well, to obtain a sample that is representative of the cell population (Fig. S3).

Quantitative phase microscopy (QPM) was used to determine decorrelation rate as follows. Cardiac fibroblasts were isolated from Col5a1 floxed mice, immortalized and then treated with a Cre expressing or GFP lentivirus to generate Col5a1CKO or control fibroblasts. Cells were imaged every 5 min for 20 h at 20x with a SID-4 Bio (Phasics) camera to acquire QPM data via quadriwave lateral shearing interferometry(Bon et al., 2009 (link)). This was on an Axio-vision Observer Z1 (Zeiss) equipped 0.4 numerical aperture objective lens with illumination was provided by a 660 nm center wavelength collimated LED (Thorlabs). The Col5a1CKO fibroblasts and the control fibroblasts were imaged with enough spacing between cells to allow for automated particle tracking (Crocker and Grier, 1996 ; Zangle et al., 2013a (link)) and cell or cell cluster segmentation (Otsu, 1979 ; Zangle et al., 2013b (link)). Automated detection of cell division events were done by pattern matching (Zangle et al., 2014 (link)) of quantitative phase images. Using experimentally determined cell average specific refractive index, quantitative phase shifts is related to the dry biomass of cells (Barer, 1952 (link); Davies and Wilkins, 1952 (link)). All image processing was performed using custom MATLAB (MathWorks) scripts.