Leica tcs sp8 smd

Continuous photobleaching was performed using a confocal microscope (Leica TCS SP8 SMD, Leica Microsystems, Wetzlar, Germany). For illumination, the PicoQuant Picosecond Pulsed Diode Laser Head of 40 MHz 470 nm was used (laser intensity was set to ~1 µW). The light is focused onto a small confocal volume through a 63x water immersion objective lens with NA = 1.2 (Leica HC PL APO 63x/1.20 W CORR). The signal is detected by sensitive detectors (Leica HyD SMD) within the detection window (500–600 nm). A specific point was chosen in the nuclear interior and a ‘point measurement’ was performed for measuring the intensity at a high frequency (~1 KHz) for approximately 60 s per cell. CP data analysis was performed with Matlab and a custom-made algorithm (Bronshtein et al., 2015 (link)).
Fluorescence correlation spectroscopy (FCS) curves were extracted from the region where the fluorescence intensity is constant (approximately 30 s after beginning of the measurement) in order to avoid inaccuracy due to bleaching. The FCS analysis was done by SymPho Time software (PicoQuant, Berlin, Germany). The best fit was achieved with the FCS Triplet 3D fitting model: $G\left(t\right)=\left[1+\sum _{j=0}^{n_{Trip}-1}T\left[j\right]\left[\exp \left(-\frac{t}{{\tau }_{Trip}\left[j\right]}\right)-1\right]\right]\sum _{i=0}^{n_{Diff}-1}\frac{\rho \left[i\right]}{\left[1+{\left[\frac{t}{{\tau }_{Diff}\left[i\right]}\right]}^{a\left[i\right]}\right]{\left[1+\frac{{\left[\frac{t}{{\tau }_{Diff}\left[i\right]}\right]}^{a\left[i\right]}}{k^2}\right]}^{0.5}}+G_{Inf}$ $w_0={\left[\frac{V_{Eff}}{k}\right]}^{\frac{1}{3}}{\pi }^{-0.5}$ $D\left[k\right]=\frac{w_0^2}{4{\tau }_{Diff}\left[k\right]}$

Macroscopic contact angles of purified water (Milli-Q, Type 1), ethylene glycol (99.5%, Sigma-Aldrich) and hexadecane (99%, Sigma-Aldrich) were measured using a goniometer with drop shape analysis (OCA40, Dataphysics GmbH). Roll-off angles (RA) were determined by measuring the tilt angle leading to roll-off for 10 μL drops. The tilt angle was increased with a rate of 0.3° s⁻¹. Advancing and receding contact angles were measured by slowly (1 μL s⁻¹) increasing the drop volume from 5 to 25 μL and then decreasing the volume back to < 1 μL. All contact angles were determined using the tangent fitting method in the SCA 20 software (Dataphysics GmbH) and the results of five individual measurements at different positions on the sample were averaged.
An inverted laser scanning confocal microscope (Leica TCS SP8 SMD, Leica Microsystems) with a HC PL APO CS2 40×/1.10 water objective was used to image a water droplet resting on the superamphiphobic surface.