Fig 1.
(A) The blue arrows indicate the direction that the sample moves under the objective, acquiring non-overlapping individual fields of view, and pausing only at the end of each ribbon to advance on to the origin of the next ribbon. Each ribbon overlaps by ten percent with its adjacent ribbon. The overlap is used to stitch (green lines) each ribbon following its completion. (B) Completed ribbons are assembled into a (C) composite image that is representative of a single large-area scan. (D) Multiple fields of view elucidate regions or structures of interest within a larger area whereas each (E) individual field of view contains subcellular detail.
Fig 2.
Ribbon scanning confocal microscopy is faster than conventional confocal microscopy and produces equivalent quality and reduced stitch artifacts.
A single 10x10 millimeter dry mount coronal section of rat brain was imaged on (A) the Caliber I.D. ribbon scanning confocal (Olympus 20x, 0.7NA) (B) the Nikon A1R using 4x averaging (Nikon 20x, 0.75NA) and (C) a swept field confocal using high-speed triggered acquisition (Nikon 20x, 0.75NA). Panels D-F are zoomed regions from panel A-C, respectively. The zoomed regions correspond to the red box in panel A. The times of acquisition for various microscopes and modes is recorded in panel G. Acquisition on a (H) Nikon A1 using a Galvano line scanner or (I) swept field confocal using standard sequential acquisition with line averaging led to better quality than the corresponding panels B and C but with additional time penalties.
Fig 3.
Confocal is more effective than multiphoton at multicolor deep tissue imaging in chemically cleared brains.
A mouse was infected subcutaneously with VEEV TrD TaV-cherry (red) and at 96 hours post infection, fluorescent beads (green) were introduced into the vasculature by cardiac perfusion. The brain was harvested, sectioned approximately four millimeters thick, and cleared by CUBIC prior to imaging on the (A) Caliber I.D. ribbon scanning confocal and a (B) Nikon multiphoton. An Olympus 25x, 1.05NA, 2.0WD objective was used to capture both volumes.
Fig 4.
Ribbon scanning confocal microscopy captures subcellular detail throughout large volumes in chemically cleared mouse brains.
A mouse was infected by aerosol with EEEV TaV-GFP. At 96 hours post infection, the brain was harvested, sectioned approximately four millimeters thick, and cleared by CUBIC. The Caliber I.D. ribbon scanning confocal was used to reconstruct a (A) 21.0 x 15.5 x 1.735 millimeter volume. (B) A single optical plane from the volume demonstrates the (C-D) subcellular detail captured throughout the volume.
Fig 5.
Multicolor large-volume imaging by ribbon scanning confocal microscopy.
A mouse was infected subcutaneously with VEEV TrD TaV-cherry (red) and at 96 hours post infection, fluorescent beads (green) were introduced into the vasculature by perfusion. The brain was harvested, sectioned approximately 4mm thick, and cleared by CUBIC. The section was imaged approximately two millimeters deep (the limit of the Olympus 25x, 1.05NA objective) on both sides and reconstructed as (A) one volume. (B) A single optical plane demonstrates that both the microvasculature and virus infected cells were observed throughout the volume. (C-D) Zoomed regions from a single optical place demonstrate that the vasculature in some areas with virus replication were inaccessible to fluorescent beads administered by perfusion.