Uplansapo 100x 1.4 na

Imaging was performed through Olympus UPlanSApo 100x 1.4 NA oil-immersion objectives mounted on Olympus IX71 inverted microscopes with back optics arranged for oblique incident angle illumination. The microscope contained a custom pentaband dichroic and pentanotch filter (Chroma Technology Corp, Bellows Falls VT) and laser lines at 488/561/647/750 nm (detailed in the Supplemental Experimental Procedures) for excitation of Atto 488, Cy3B, Alexa Fluor 647 and DyLight 750, respectively. A 405-nm laser was used for reactivation of dyes. Images were acquired on an Andor iXon3 897 or 897Ultra EMCCD camera through a QV2 quadview image splitter (Photometrics; Tucson AZ). Each camera pixel corresponded to ∼158 nm in sample space and the total imaging field size was ∼40 μm × 40 μm. Axial focus during imaging was maintained in an automated manner as described previously (Dempsey et al., 2011 (link)).

All imaging experiments were performed on the experimental system shown schematically in supplementary Fig. S1. A 4f optical processing system was built alongside the side-port of an Olympus IX71 microscope frame, with a 100x/1.4 NA oil-immersion objective lens (UPlanSApo 100x/1.4 NA, Olympus). The 4f system consisted of two 150 mm achromat lenses (Edmund Optics), a polarizing beamsplitter (B. Halle) to reject light polarized perpendicular to the axis along which the SLM is capable of modulating phase, a 512x512 pixel SLM (XY Phase Series, Boulder Nonlinear Systems), and assorted mirrors for beam-steering. An EMCCD camera (iXon897, Andor) was used to record data. Imaging experiments were performed using simultaneous illumination by a 641 nm diode laser (CUBE, Coherent), and the 514 nm line of an Ar-ion laser (Innova 90, Coherent). Excitation light was reflected off of a multi-bandpass dichroic (FF425/532/656-Di01-25x36, Semrock), and fluorescence was transmitted through the same dichroic, and passed through an additional multi-bandpass emission filter (Em01-R442/514/647-25, Yokogawa), a notch filter (ZET635NF, Chroma), and 514 long pass filter (Semrock).