Fastcam sa3 camera

We measured the bending rigidity of E.coli PLE GUVs by fluctuation analysis of the thermally induced motion of the membrane. Membrane fluctuations were enhanced by osmotic shock (250–300 mOsm glucose) and recorded at 22 °C. Experiments were performed on an inverted Nikon Eclipse 2000Ti (Nikon, Japan) microscope equipped with a 100 W TI- 12 DH Pillar Illuminator, an LWD 0.52 collimator, and a PlanApo VC 100× (NA1.4) oil immersion objective. Images were captured with a FASTCAM SA3 camera (Photron, San Diego, CA, USA) using an effective pixel-size of 50 nm × 50 nm at 500 frames per second to provide images with an optimal signal-to-noise ratio (SNR). The GUV contours were automatically tracked at the equatorial plane with a custom-made algorithm [18 (link)]. The description of the normal deformation h in a series of discrete Fourier modes, h(t) = Σ_(q)h_q(t) exp.(iqx), allowed the calculation of the fluctuation spectrum P(q), $P\left(q\right)=\frac{k_{\mathrm{B}}T}{\left(\sigma q^2+\kappa q^4\right)}$ where q = l/R is the equatorial projection of the fluctuation wave-vector (with R as the radius and l = 2, 3, 4, … ∞ the azimuthal number), κ is the bending rigidity, and σ is the surface tension. The mechanical parameters κ and σ were obtained by fitting the experimental mode amplitudes to the theoretical fluctuation spectrum P(q) particularized to the equatorial fluctuations [19 (link)].