Ribbon scanning confocal for high-speed high-resolution volume imaging of brain

<div><p>Whole-brain imaging is becoming a fundamental means of experimental insight; however, achieving subcellular resolution imagery in a reasonable time window has not been possible. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. Further, using this technology, we reconstruct large volumes of mouse brain infected with encephalitic alphaviruses and demonstrate that regions of the brain with abundant viral replication were inaccessible to vascular perfusion. This reveals that the destruction or collapse of large regions of brain micro vasculature may contribute to the severe disease caused by Venezuelan equine encephalitis virus. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.</p></div

Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes

ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection

Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes

ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection

Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes

ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection

Ribbon scanning confocal microscopy is faster than conventional confocal microscopy and produces equivalent quality and reduced stitch artifacts.

<p>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.</p

Multicolor large-volume imaging by ribbon scanning confocal microscopy.

<p>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.</p

The ribbon scanning approach.

<p>(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.</p