Ixon3 887 emccd

Disintegration and bubble expansion images of NAMs are performed on a TIRF microscope built with a Ti-E Eclipse stand (Nikon Instruments). The objective used is an Apo TIRF 100X (N.A. 1.49). CUBE diode 405 nm laser (Coherent), Sapphire OPSL 488 nm, 514 nm, and 561 nm lasers (Coherent), and OBIS 647 nm (Coherent) are combined into a fiber optic cable and into a TIRF illuminator (Nikon) attached to the microscope stand. Shuttering of the laser illumination is controlled by an acousto-optic tunable filter (AA Optoelectronics) before the fiber coupler. Disintegration images are acquired with an iXon3 + 887 EMCCD (Andor Technology) camera and bubble expansion images are taken by a high-speed camera (Fastec Imaging). Synchronization between these components was achieved using μManager⁶¹ with a microcontroller (Arduino). We image the disintegration and bubble expansion of the NAMs by exposing sample slides to each activation (405, 488, 514, 561, 647 nm) with incident laser power at 0.5, 0.75, 1, 1.5, and 2 mW in a temperature controlled chamber (HaisonTech) at 25 °C. For bubble expansion images, the time and information of the bubble size are exported to Morphometric and Matlab program files for father data analysis and plotting purpose.

Imaging was performed on a total internal reflection fluorescence (TIRF) microscope built with a Ti-E Eclipse stand (Nikon Instruments, Inc., Melville, NY, USA). A Plan Apo Lambda 100X DM (NA 1.45) (Nikon) objective was used to acquire phase-contrast images concurrently with TIRF images. CUBE diode 405-nm and Sapphire OPSL 561-nm lasers (Coherent, Santa Clara, CA, USA) were combined into an optical fibre and into a TIRF illuminator (Nikon) attached to the microscope stand. Shuttering of laser illumination was controlled with an acousto-optic tuneable filter (AA Optoelectronics, Orsay, France) before the fibre coupler. Images were acquired with an iXon3+ 887 EMCCD (Andor Technology, South Windsor, CT, USA) camera, and synchronization between components was achieved using μManager 1.4 (ref. 43 ) with a microcontroller (Arduino, Almuñécar, Spain).