Ix83 tirf microscope

TIME-mNGrActin cells were either left untreated or fixed using PFA + glutaraldehyde (as described above). Both live and fixed cells were then imaged at 37°C using an Olympus IX83 TIRF microscope equipped with a Z-Drift Compensator and a UAPO 100x/1.49 NA oil-immersion TIRF objective (Olympus, Tokyo, Japan). The microscope was equipped with an iXon 888 1k × 1k EMCCD Camera (Andor; Oxford Instruments). With an additional 1.6x magnification in place, the pixel size in the recorded image was 81 nm × 81 nm. Using the Olympus CellSens software, excitation light of 491 nm from an Olympus CellTIRF-4Line laser system was directed to the sample by a TRF8001-OL3 Quad-band dichroic mirror. The penetration depth was set to 70 nm via the cellSens software (Olympus). Laser power was 2.31 mW at the coverslip. FSM videos were acquired at a frame rate of 0.2 Hz for 109 frames (9 min). This was equivalent to the FSM arm of the SMI-FSM data acquisition scheme described above, although with longer acquisition time. Fluorescence was collected, filtered with an emission filter of ET520/40m (Chroma Technology) and projected onto a section of the camera chip by an OptoSplit III 3-channel image splitter (Cairn Research, Faversham, UK). Camera EM gain was set to 100 for all acquisitions.

TIME-mNGrActin cells plated on fibronectin coated glass bottomed dishes were fixed with 4% PFA (as described above). Following fixation, sample was incubated with Alexa Fluor^™ 647 phalloidin (ThermoFisher Scientific, A22287) at 1:20 dilution in blocking buffer (1% BSA, 5% NGS in wash buffer). After washing, cells were incubated in imaging buffer and imaged at 37°C using an Olympus IX83 TIRF microscope (microscope described in previous section). For excitation, light of 640 nm (0.2 mW at coverslip) and 491 nm (2.3 or 5.1 mW at coverslip) from an Olympus CellTIRF-4Line laser system was directed to the sample. The penetration depth was set to 100 nm via the cellSens software (Olympus). First, the 640 channel was imaged for an exposure time of 99 ms. Then, the 491 channel was used for acquiring FSM videos at 10 Hz for 5 frames. Fluorescence of each wavelength was collected, filtered with emission filters of ET520/40m and ET705/72m (Chroma Technology), and projected onto different sections of the camera chip by an OptoSplit III 3-channel image splitter (Cairn Research). Camera EM gain was set to 100 for all acquisitions.