Journal: JACS Au

Article Title: Single-Virus Microscopy of Biochemical Events in Viral Entry

doi: 10.1021/jacsau.4c00992

Figure Lengend Snippet: Microscopy and single-virus detection of fusion events. (a) Flow cell attached to microscope holder with 2 mm tubing inserted in the channel hole for buffer exchange. Inset below shows a schematic of a single channel of the flow cell with labels. (b) Image of a microscope with flow cell setup during image acquisition. (c) Typical image before and 30 s after fusion is triggered by buffer exchange. Note that more Texas Red-labeled viral particles are visible after 30 s, as expected from dilutional dequenching upon fusion. Each individual red dot signifies a TR-labeled influenza viral particle, as illustrated by the cartoon on the right. (d) Fluorescence intensity is plotted versus time for a single viral particle with a dequenching spike apparent upon lipid mixing. The interval from buffer exchange (time = 0) to the dequenching spike (black vertical dashed line) is recorded as the single-event waiting time. (e) Waiting times for all individual fusion events are compiled into a cumulative distribution function. The shape of this distribution contains information about the underlying kinetic process and can be analyzed using gamma functions or randomness-parameter calculations.

Article Snippet: Kapton polyimide tape (Ted Pella) Glass microscope slide (25 × 75 mm, 1.0 mm thickness) Polydimethylsiloxane SYLGARD 184 Silicone Elastomer Kit Glass coverslips (24 × 40 mm, #1.5 thickness) 7X cleaning solution (MP Biomedicals) PLL–PEG (SuSoS) PLL–PEG-Biotin (SuSoS) Reaction buffer (10 mM Sodium Phosphate Monobasic, 90 mM Sodium Citrate and 150 mM Sodium Chloride, pH 7.4 or 5.0 as indicated) Neutravidin (Fisher Scientific) Palmitoyl oleoylphosphatidylcholine (Avanti) Dioleoylphosphatidylethanolamine (Avanti) Cholesterol (Sigma) GD1a (Sigma) DHPE-Biotin (Avanti) DiYO-1 (AAT Bioquest) Virus of interest (X-31 H3N2 influenza is used as an example here) Texas Red-DHPE (Fisher Scientific) HEPES buffer (20 mM HEPES, 150 mM NaCl, buffered to pH 7.2) PD SpinTrap G-25 (Cytiva) Ceramic coverslip rack Scalpel 2 mm biopsy hole punch 2 mm tubing 1 mL Luer-lock syringe

Techniques: Microscopy, Virus, Buffer Exchange, Labeling, Fluorescence