Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems.

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

Dorsiflexion is more feasible than plantar flexion in ultrasound evaluation of the calcaneofibular ligament: a combination study of ultrasound and cadaver.

PURPOSE: Ultrasound (US) is a valuable tool for the evaluation of chronic lateral instability of the ankle; however, the feasibility of US for calcaneofibular ligament (CFL) assessment remains unknown. This study aimed to depict and compare CFL on US in various ankle positions to determine the optimal method for evaluating CFL with US and to interpret US findings using cadaveric specimens. METHODS: The US study included 43 ankles of 25 healthy individuals. The CFL was scanned with US in 20° plantar flexion, neutral position, 20° dorsiflexion and maximum dorsiflexion. The distances between fibula and CFL were compared. The cadaveric study included macroscopic qualitative observation of the dynamic change of CFL in 7 ankles and quantitative observation of the directions of CFL and footprints in 17 ankles. RESULTS: In the US study, the mean distance (mm) between fibula and CFL was 7.3 ± 1.3 in 20° plantar flexion, 6.7 ± 1.6 in neutral position, 4.3 ± 2.5 in 20° dorsiflexion and 3.1 ± 2.1 in maximum dorsiflexion. The more dorsiflexed the ankle was, the shorter the distance between fibula and CFL was (Jonckheere's trend test p < 0.001). In the cadaveric study, the CFL fibres were aligned parallel between the mid-substance and the fibular attachment in maximum dorsiflexion, whilst CFL was reflected and rotated in plantar flexion. CONCLUSIONS: The whole length of the CFL, including its fibular attachment, is more likely to be visualized with US in dorsiflexion than in plantar flexion due to the direction of the CFL at the fibular attachment, which is parallel with the mid-substance in maximum dorsiflexion. LEVEL OF EVIDENCE: IV.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

A circuit motif in the zebrafish hindbrain for a two alternative behavioral choice to turn left or right.

Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies. Key inhibitory neurons in the circuit lie in a column of morphologically similar cells that is one of a series of such columns that form a developmental and functional ground plan for building hindbrain networks. Repetition within the columns of the network motif we defined may therefore lie at the foundation of other lateralized behavioral choices.