Coolsnap myo

Variation in membrane tension due to osmotic shock was quantified by using micropipette aspiration. Glass micropipettes of inner diameter of ~5 µm were fabricated by pulling borosilicate glass pipette (BF100-50-10; Sutter Instrument) using a micropipette puller (Sutter Instrument). The micropipette was attached to a custom-made stage with pipette holder assembly (MI-10010; Sutter Instrument). An open chamber was made on coverslip seeded with RPE cells using VALAP sealant. The cells were subjected to osmotic shock, using the aforementioned method. The micropipette was made into contact with the cells with the aid of brightfield imaging using an inverted microscope (Nikon TiS) equipped with 20× objective and a CCD camera (CoolSNAP MYO). A negative hydrostatic pressure of 2.156 kPa was applied to the cell to aspirate the plasma membrane of cells into the pipette. The ratio of equilibrium protrusion length of membrane (L_p) vs. inner radius of pipette was measured for different osmotic conditions.

Tailored non-paraxial 4D fields are analyzed by a fluorescent SAM. For this purpose, the probe, consisting of the monolayer produced on a glass cover slip (refractive index n = 1.33, thickness 170 μm) is placed in the focal plane of the non-paraxial field. Note that the fluorescent molecules are directly irradiated by the light field, thus, the probe is placed with the monolayer at the bottom on its holder (monolayer-glass-order in beam propagation direction, see Fig. 1a). By this, aberration effects occurring when the beam is transmitted trough the cover slip are avoided, as the MO focuses is aberration free in air. Excited fluorescence is observed in transmission, passing through the cover slip. For collecting scattered fluorescence and imaging the fluorescence in the plane of the monolayer (=focal plane) onto a CCD camera (Photometrics CoolSNAP MYO), we apply a high-NA MO (Nikon C-AB Abbe Condenser, ×100, NA = 0.9) in combination with a lens (focal distance: 500 mm). The exciting light field (λ = 532 nm) is filtered in front of the CCD (longpass filter, Thorlabs FEL0550), so that pure fluorescence (λ_fl ~ 594 nm) is observed . Note that noise within recorded fluorescence images is reduced by taking the mean value of ten images and applying background subtraction.