Lsm880 axioobserver z1 confocal microscope

The effect of interactions of hollow or various curcumin-loaded nano complexes on the structural integrity of zwitterionic GUV was visualized with confocal and widefield fluorescence microscopes. Each solution of calcein-loaded GUV (500 µL) was mixed with a solution of the nanoparticles (150 µL, 0.28 mg/mL) or 10% Triton X-100 (150 µL) prepared in a leakage buffer, vortexed for 1 min, and allowed to stand for 15 min before mounting on glass microscope slides. To image calcein-loaded and empty GUV in the solution after leakage, the morphology was visualized separately in the rhodamine B (λ_ex 540, λ_em 625) and calcein (λ_ex 494, λ_em 517) channels, with the Axio Imager 2 fluorescence microscope equipped with an Axiocam 506 monochromatic camera (Carl Zeiss, Oberkochen, Germany) and Zeiss LSM880 AxioObserver Z1 confocal microscope equipped with AiryScan FAST (Carl Zeiss, Oberkochen, Germany). Zen 2.3 pro and 3.4 software (Carl Zeiss, Oberkochen, Germany) were used for image processing and optimization.

FRET experiments were performed on HEK293 cells expressing equimolar amounts of Sig‐1R‐mCh and Kv1.2‐GFP using a Zeiss LSM880 AxioObserverZ1 Confocal Microscope (Zeiss GmbH, Oberkochen, Germany), with excitation wavelengths of 561 nm and 488 nm for mCh and GFP, respectively. Cells were imaged in Phenol Red free MEM (Wisent Bioproducts) containing 10% FBS on a prewarmed 37°C stage with 5% CO₂, using a 63× (NA 1.4) oil immersion objective (Zeiss). A resolution of 512 × 512 pixels was used, with a dwell time of 0.5 μsec/pixel. Following five frames of prebleach baseline, a square region of interest (ROI) was bleached at 80% 561 nm laser power for 2.5 sec. 15 frames were captured postbleach. FRET efficiency was measured via increased donor (GFP) emission following acceptor photobleaching (mCh), using the formula: $E=1-(I_{\mathrm{pre}}/I_{\mathrm{post}})$ where I_pre and I_post are the fluorescent intensities of the donor before and after photobleaching (Bajar et al. 2016). Mean fluorescent intensities within the ROI were determined using Fiji (Schindelin et al. 2012) and were background subtracted prior to analysis. As an internal control, FRET efficiency was calculated within a nonbleached ROI of each cell to account for false‐positive FRET signals.