Csu x1 5000rpm spinning disk confocal head

Confocal imaging was performed on a Nikon inverted confocal microscope with 20x air objective and 60x oil immersion objective, outfitted with a Yokogawa CSU-X1 5000RPM spinning disk confocal head, and Ti2-ND-P perfect focus system 4, a compact 4-line laser source: 405 nm (100 mW) 488 nm (100 mW), 561 nm (100mW) and 640-nm (75 mW) lasers. The following combinations of laser excitation and emission filters were used for various fluorophores: EGFP/Alexa Fluor 488 (488 nm excitation; 525/36 emission), mCherry (568 nm excitation; 605/52 emission), Alexa Fluor 647 (647 nm excitation; 705/72 emission), and differential interference contrast (DIC). The acquisition time for all images was 1 second with 50% laser power intensities. ORCA-Flash 4.0 LT+sCMOS camera. 20x objective or 60x objective was used for all HEK293T cell experiments. 10x objective was used for mouse brain slice images. All images were collected using Nikon NIS-Elements hardware control and processed using NIS-Elements General Analysis 3 software.