Multi-Channel Total Internal Reflection Fluorescence

The qDF set up and the theory of qDF have been described previously in detail15 (link). Here, we expended qDF to three channels (TqDF). The set up consisted of an IX71 inverted TIRF research microscope (Olympus America) with a 100 × NA 1.45 plan-apochromatic oil immersion TIRF microscopy objective and 10 mW blue (λ=488 nm), 10 mW yellow-green (λ=561 nm), and 5 mW red (λ=641 nm) diode-pumped solid-state lasers (CVI Melles Griot) as TIRF excitation light sources. Images were captured at a rate of 0.2–1 frames per second using a QV2 (Photometrics) QuadView video coupler and a 16-bit digital charge coupled device camera (Hamamatsu C10600-10B ORCA-R2). The laser shutters and camera were controlled with the SlideBook5.5 software (Intelligent Imaging Innovations). The absorption and emission peaks of the fluorochromes used in this study were, respectively, 493 and 518 nm for DL488, 562 and 576 nm for DL550, 649 and 666 nm for CellMask DeepRed. There was no bleed-through between channels. A TIRF incidence angle of θ=70° was used for all three lasers in all TqDF experiments.