TIRF-SIM of low illumination NA, so called GI-SIM, and astigmatic microscopy was performed on a custom-built system controlled by acquisition software written in LabView25 (link),27 (link). A single 488 nm excitation laser was used (MPB Communications Inc., 2RU-VFL-P-500-488-B1R) to excite both the Lifeact-citrine channel and the fluorescent beads to facilitate rapid acquisition. The excitation beam was first collimated and passed through the acousto-optic tuneable filter (AOTF, AA Quanta Tech, AOTFnC-400.650-TN). The beam was then expanded and sent to a phase-only modulator, consisting of a polarising beam splitter, an achromatic half-wave plate (HWP, Bolder Vision Optik, BVO AHWP3) and a ferroelectric spatial light modulator (SLM, Forth Dimension Displays, QXGA-3DM). The SLM displays a grating pattern with parameters matching the excitation wavelength, which was used to generate diffraction patterns. The diffraction orders of +1 and −1 were focused on opposite sides of back focal aperture of objective lens (Olympus UAPON 100XOTIRF 1.49NA), and then total internal reflected at the cell–gel interface. The evanescent waves generated by +1 and −1 order light interfered to form the illumination pattern. To maximise the pattern contrast, the diffracted light was maintained with s-polarisation using a polarization rotator. The fluorescent images of Lifeact-citrine and fluorescent beads generated by the applied illumination pattern were collected by the same objective, separated by a dichroic beam splitter (Chroma, ZT405/488/560/647tpc), and focused by respective tube lens onto corresponding sCMOS camera (Hamamatsu, Orca Flash 4.0 v3). A cylindrical lens (Optosigma, CLB-3030-1000PM) was introduced in front of the camera to generate astigmatic imaging of the marker beads. For each frame, the acquisition time was 10–100 ms, leading to a super-resolution image (three angles and three phase, nine frames total) every 90–900 ms calculated using a reconstruction method described previously27 (link),43 (link). For live-cell imaging, a micro-incubator (H301, Okolabs, Naples, Italy) at 37 °C and 5% CO2 was used.
During the aTFM acquisition, the TIRF interface at the top of the silicone gel is displaced in the axial direction due to cell generated normal stresses. This will lead to a change in the angle of incidence relative to the gel–sample interface. For the acquired data, the dynamic range of axial displacement was of the order of 500 nm. If this is assumed to act over the contact area of the cell (15 µm), this equates to an angular distortion of the gel of ~1.5°. The excitation light has an incident NA ranging from 1.38 to 1.41; therefore, the incident angle necessary to achieve TIRF illumination was sin−1(1.41/1.515) = 68.5°. If the surface deviates from being flat, the incident angle is 68.5° ± 1.5°, giving a corresponding range in NA from 1.395 to 1.42. TIRF illumination for the described setup is typically achieved for NA > 1.38, indicating that TIRF illumination is maintained during deviations from a flat surface inherent to the aTFM acquisition.
During the aTFM acquisition, the TIRF interface at the top of the silicone gel is displaced in the axial direction due to cell generated normal stresses. This will lead to a change in the angle of incidence relative to the gel–sample interface. For the acquired data, the dynamic range of axial displacement was of the order of 500 nm. If this is assumed to act over the contact area of the cell (15 µm), this equates to an angular distortion of the gel of ~1.5°. The excitation light has an incident NA ranging from 1.38 to 1.41; therefore, the incident angle necessary to achieve TIRF illumination was sin−1(1.41/1.515) = 68.5°. If the surface deviates from being flat, the incident angle is 68.5° ± 1.5°, giving a corresponding range in NA from 1.395 to 1.42. TIRF illumination for the described setup is typically achieved for NA > 1.38, indicating that TIRF illumination is maintained during deviations from a flat surface inherent to the aTFM acquisition.
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