Apo tirf 100 1.49 na oil immersion objective

Wide-field imaging was performed using an inverted epifluorescence microscope equipped with an Apo TIRF ×100/1.49-NA oil immersion objective (Nikon). An iChrome MLE-LFA multilaser engine (Toptica Photonics) containing four lasers emitting at 405, 488, 561 and 640 nm was used as the light source and fiber coupled into the microscope using a TIRF illumination module (Nikon). Focus stabilization in time-lapse imaging was achieved using a perfect focus system (PFS3, Nikon). Excitation and emission light were separated using a quad-edge dichroic beamsplitter, and emitted light was further filtered using bandpass filters (AHF Analysetechnik). Images were acquired using an iXon+ 897 Ultra electron-multiplying CCD camera (Oxford Instruments Andor), which was also used as a timing device to synchronize excitation lasers and camera exposures during imaging with alternating laser excitation. The microscope and all connected devices were controlled using the Micromanager software platform⁴⁴ . Typically, images were acquired with a 50-ms exposure at 5–10 W cm^–2 illumination intensity and at a 95- or 146-nm pixel size. Multispectral images were acquired using a motorized filter wheel equipped with 525/50-nm (eGFP), 605/70-nm (mCherry, TMR) and 685/70-nm (SiR) bandpass filters.

The two-color, live-cell TIRF movies were all acquired using a custom-built microscope in the Princeton University Lewis-Sigler Imaging Core Facility, consisting of the following components: 488 nm (Coherent) and 561 nm (CrystaLaser) excitation lasers, an acousto-optical tunable filter (AA Optoelectronic), a Plan Apo 60×/1.49 NA oil immersion objective (Olympus), a 37°C heated stage and coverslip holder, a multiband filter set (Semrock, LF488/561-A-000), an Andor iXon EMCCD camera, and custom control software written in Matlab (Mathworks). Fluorescence emission bands are as follows: pHluorin, 523/40 nm emission; mRFP, 610/52 nm emission.
Three-color, live-cell TIRF movies were acquired on a Nikon Ti-E microscope in the Princeton University Molecular Biology Confocal Microscopy Facility. This microscope is equipped with 405 nm, 488 nm, and 561 nm excitation lasers (Agilent), an Apo TIRF 100×/1.49 NA oil immersion objective (Nikon), an Andor iXon Ultra EMCCD camera, a 37°C heated stage, and Nikon NIS Elements software. Fluorescence emission bands are as follows: mTurquoise2, ∼450/60 nm; pHluorin, ∼525/50 nm emission; mRFP, ∼605/50 nm emission.