Tcs sp8 gsted 3 microscope

Cultured cells were imaged using an Axiovert 200 microscope (C-Apochromat 63x/1.22 W objective, Carl Zeiss MicroImaging, Jena, Germany) using Axiovision 4.8 (Carl Zeiss MicroImaging) for acquisition and subsequent image processing. Drosophila nephrocytes were imaged with a Leica TCS SP8 confocal microscope using a 20× air objective with a numerical aperture of 0.75 (PL APO 20x/0.75 DRY, Leica Microsystems). Images were analyzed using Fiji²⁰ (link), and the fluorescence intensity of the nephrocytes was quantified as described previously.¹⁹ (link) For high-resolution stimulated emission depletion (STED) images, a Leica TCS SP8 gSTED 3× microscope (Leica Microsystems) equipped with a white light laser for excitation and hybrid detectors (HyDs) for time-gated detection was used. After acquisition with a 100× oil immersion objective with a numerical aperture of 1.4 (PL APO 100×/1.4 Oil STED, Leica Microsystems), the images were further processed using the Huygens Essential software (Scientific Volume Imaging) for deconvolution. Quantification of the nephrocyte diaphragm (ND) length was done using a previously published Fiji macro.²¹ (link)
Further Methods are provided in Supplementary Methods.

Confocal imaging and three-dimensional (3D) STED nanoscopy were performed on a Leica TCS SP8 gSTED 3× microscope (Leica Microsystems) equipped with white light lasers, HC PL APO 100×/1.40 Oil STED WHITE objective, and 63×/1.40 Oil HC Pl Apo CS2 objective for confocal imaging. A voxel size of (35 to 40) × (35 to 40) × 130 nm (xyz) for ×63 confocal and (10 to 20) × (10 to 20) × 100 nm (xyz) nm for STED images was used. The fluorophore Alexa Fluor 532 was excited at 527 nm, and STED was performed at 660 nm for super-resolution imaging. Alexa Fluor 555 was excited at 555 nm, and STED was performed at 660 nm. Alexa Fluor 594 was excited at 590 nm and ATTO647N at 646 nm, while STED was performed at 775 nm for both fluorophores. Images were deconvolved and, where indicated, rendered into computer-generated 3D surface maps, using Huygens software (Scientific Volume Imaging).