Figure 5. Super-resolution imaging acquired with anti-epitope tag Fab probes.

(A) Synthesis of fluorescently labeled Fab probes and imaging by their repetitive binding to epitope tags (epitope-tagged actin is shown). Details in STAR methods.

(B) Fab probes showing specific binding to epitope-tagged actin (FLAG-actin, S-actin, or V5-actin) expressed in XTC cells. FLAG-1, FLAG-2, S-1, V5-1,V5-2, and V5-3 Fab probes visualized actin fibers by specific binding to epitope tags. Images are maximum projections of 500 frames acquired every 50 ms. Fab probes, 1 nM. Bars, 5 μm.

(C) Representative image of non-specific binding of Fab probes (V5-6 Fab probe and V5-actin). Fab probe bound diffusely without visualizing actin fibers. Maximum projection of 500 frames acquired every 50 ms. Fab probe, 10 nM. Bar, 5 μm.

(D) Time-lapse images showing repetitive binding of FLAG-2, S-1, and V5-3 Fab probes. Compatible with the fast-dissociating abilities of FLAG-2, S-1, and V5-3 antibodies, bound Fab probes were frequently exchanged. Time lapse recorded every 50 ms but shown every 200 ms (i.e., every 4th frame). Fab probes, 1 nM. Bars, 5 μm and 2 μm.

(E) Super-resolution images acquired with FLAG-2, S-1, and V5-3 Fab probes. These Fab probes dissociate with half-lives of a few seconds (Figure S6). Thin actin fibers were visualized at high-density labeling (between arrowheads) reflecting the frequent binding and dissociation of these Fab probes. Images were reconstructed from 160,000 frames acquired every 50 ms. Fab probes, 1 nM. Bars, 5 μm and 1 μm.

(F) Super-resolution images acquired with FLAG-1, V5-1, and V5-2 Fab probes. These Fab probes dissociate at half-lives of nearly 10 s or longer (Figure S6). Reconstructed from 160,000 frames acquired every 50 ms as performed in (E). Thick actin fibers were visualized, and thin actin fibers were visualized only at low labeling densities (circles). Fab probes, 1 nM. Bars, 5 μm and 1 μm.

See also Figures S5–S7, Table S3, and Video S9.