Journal: Nature methods

Article Title: Kilohertz two-photon brain imaging in awake mice

doi: 10.1038/s41592-019-0597-2

Figure Lengend Snippet: (a) Conventional epi-fluorescence image in motor cortex of an anesthetized mouse after intravenous administration of rhodamine-B. Dashed box encloses the artery in b . (b) Image time series (2.5 ms per frame; 200 kHz laser pulse rate; 2.2 mW per beamlet) taken by high-speed two-photon imaging reveals flow of injected, fluorescent HEK-293 cells. Arrowheads mark a cell’s progress. (c) Flow speed map for the vessel in b . (d) Trajectories of individual HEK-293 cells. Each trajectory is encoded in color and superposed on an epi-fluorescence image of the vasculature. (e) Flow speeds in neocortical arteries had a parabolic cross-sectional profile. For 15 different cross-sections chosen within 3 different arteries (>50 μm in diameter; N = 3 mice), we computed flow speeds, V ( r ), as a function of the radial deviation, r , from the vessel’s longitudinal axis. We fit (red curve) the data (blue points) to V/V max = 1 – ( r/R ) n , where V max is each vessel’s peak flow speed and R is its radius. The fitting parameter, n = 2.0 ± 0.3 (95% C.I.), revealed the flow speed’s quadratic profile. Error bars: s.e.m. ( N = 15 cross-sections). (f) Flow speed map determined by 1-kHz-two-photon imaging (200 kHz laser repetition rate; 2.9 mW per beamlet; 450 × 110 μm 2 field of view). (g) Trajectories of individual HEK-293 cells, determined from the same dataset used for f . (h) Periodic fluctuations in blood flow, as computed within the encircled area in f . The heart rate of ~150 beats·min −1 determined by high-speed imaging matches conventional measurements in ketamine-xylazine-anesthetized mice . (i) Sketch of the mouse superior sagittal sinus (SSS). Inset : We targeted areas near bregma for imaging (dotted rectangle). (j) Example time traces of bridging vein diameter determined by 200-Hz-imaging (450 × 300 μm 2 field of view; 200 kHz laser pulse rate; 2.2 mW per beamlet) after intravenous injection of rhodamine-B. During wakefulness, the vein diameter (blue trace) exhibited fast constriction (red dots) and dilation (cyan dots), which anesthesia abolished (gray trace). (k) Negative- and positive-going changes in vein diameter, relative to each vessel’s mean diameter, during constriction (red curve) and dilation (blue curve) in 4 awake mice. Constriction and dilation rates were, respectively, −6.5 ± 0.9% and +4.0 ± 0.7% per 100 ms (mean ± s.e.m; 36 events of each type). Shading: s.e.m. Scale bars: 50 μm in a – d, f , g .

Article Snippet: We incubated the samples at 37°C for 1–2 h and washed the quantum-dot-labeled HEK-293T cells twice with phosphate buffered saline (PBS); after each wash we collected the cells by centrifugation at 200 G-force for 5 min. We re-suspended the final cell pellet in PBS at a concentration of ~2.5 · 10 7 cells/mL.

Techniques: Fluorescence, Imaging, Injection

Journal: eLife

Article Title: Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus

doi: 10.7554/eLife.80156

Figure Lengend Snippet:

Article Snippet: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml +/+ or Pml -/- (from Dr. Lallemand-Breitenbach, and whose cell identity was authenticated by STR profiling) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37 °C under 5% CO2 and humid atmosphere.

Techniques: Transduction, Recombinant, Plasmid Preparation, Clone Assay, Mutagenesis, Sequencing, Quantitative RT-PCR, Concentration Assay, In Situ, Software, ChIP-sequencing