Confocal imaging.

Fixed-cell images were acquired with a Mariana system (Intelligent Imaging Innovations, Denver, CO) based on a Zeiss Axio-Observer inverted microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa Electric, Tokyo, Japan), a piezo-driven z-translation, and linear encoded x and y translations and controlled with SlideBook V6.0 (Intelligent Imaging Inc., Denver, CO). Excitation wavelengths were 488, 561, and 640 nm (lasers were obtained from Cobolt, Solna, Sweden); the emission filters were 525/50-, 607/36-, and 680-nm long-pass filters (Semrock, Rochester, NY). z-series covering the full volume of the cells were acquired with a z-step of 0.5 μm. All wavelengths were acquired with 100-ms exposure times at 100% laser power.

For live-cell imaging, BSC-1 cells were grown on 8-chambered number 1.5 cover glass slides (Cellvis) in a volume of 400 μl supplemented DMEM and allowed to grow overnight to 50% confluence. Prior to imaging, all 8 chambers were washed three times with 400 μl Fluorobrite DMEM (Invitrogen) containing 5% FBS and 25 mM HEPES (pH 7) (Invitrogen), and a final volume of 200 μl of this imaging medium was added to the wells. For Baf A1 experiments, this 200 μl of medium also contained 1% DMSO and 100 nM Baf A1, which was incubated for 1 h prior to the addition of virus. The slides were then mounted on a Nikon Ti motorized inverted microscope with a Perfect Focus system enclosed in an OkoLab cage microscope incubator at 37°C with 5% CO₂.

After the addition of virus to a chamber of interest (MOI = 10 for Rhod5F-labeled rcTLPs; MOI = 50 for Atto 565- and pHrodo Red-labeled rcTLPs), live-cell images were acquired every 10 s with 200-ms (642 nm), 300-ms (488 nm and 562 nm for Atto 565 and Rhod5F), and 500-ms (562 nm for pHrodo Red) exposure times for 30 min. All images were collected using a 60×, 1.4-numerical-aperture (NA) objective with a Yokagawa CSU-X1 spinning-disk confocal microscope with Spectral Applied Research Borealis modification. Images were acquired with a Hamamatsu Flash4.0 V3 sCMOS camera (2-by-2 binning) controlled by MetaMorph software using the Spectral Applied Research LMM-5 laser merge module with solid-state lasers controlled by acousto-optic tunable filter (AOTF) at the following wavelengths: 488 nm (100 mW), 561 nm (100 mW), and 642 nm (101 mW).

pHrodo Red-labeled rcTLPs on glass were imaged in 200 μl Fluorobrite DMEM containing 5% FBS and 25 mM HEPES (pH 7) as single z-series over 15 z-planes with a 0.5-μm step size with the exposure times outlined above. Two hundred microliters of 1 M sodium acetate at pH 5.2 was then added, and particles were once again imaged.