Multispectral tracing in densely labeled mouse brain with nTracer

SUMMARY: This note describes nTracer, an ImageJ plug-in for user-guided, semi-automated tracing of multispectral fluorescent tissue samples. This approach allows for rapid and accurate reconstruction of whole cell morphology of large neuronal populations in densely labeled brains. AVAILABILITY AND IMPLEMENTATION: nTracer was written as a plug-in for the open source image processing software ImageJ. The software, instructional documentation, tutorial videos, sample image and sample tracing results are available at https://www.cai-lab.org/ntracer-tutorial. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.United States. National Institutes of Health (1R01GM104948)United States. National Institutes of Health (1DP1NS087724)United States. National Institutes of Health ( NIH 1R01EY023173)United States. National Institutes of Health (1U01MH106011

Consensual Uncertainty

Abstract included in tex

Growth cones advance by forward translocation of the C-domain and axonal framework <i>in</i><i>vitro</i>.

<p>(<b>A</b>) Phase and (<b>B</b>) fluorescent images over 1 h of MitoTracker labelled <i>Drosophila</i> neurons grown on poly-ornithine. Kymographs of the phase images (<b>C</b>) and fluorescent images (<b>D</b>) show the position of the growth cone and mitochondria over time. (<b>E</b>) Green arrows overlaid on the kymograph illustrate the movement of docked mitochondria and the blue arrows show the tracks of fast transported mitochondria. The corresponding images from a neuron grown on DECM are shown in panels (<b>F</b>-<b>J</b>). Time arrow = 30 min and scale bar is 10 µm for both the time-lapse images and kymographs. (<b>K</b>) Quantitative analysis of the velocity of docked mitochondria plotted against distance from the growth cone. Errors bars are 95% confidence intervals. The numbers at the base of the bars denote the number of mitochondria analyzed in each bin. The growth cone is defined as the first 5 µm of axon. </p

Growth cones advance by forward translocation of the C-domain and axonal framework <i>in</i><i>vivo</i>.

<p>(<b>A</b>) A 3D reconstruction of late stage 16 embryo expressing the membrane marker myr-tdTomato in the nervous system via <i>elav-Gal4</i>. After the intersegmental axon of the aCC neuron passes the point where the RP2 axon forms a synapse on muscle 2, it is in a region free of other axons and the cell bodies of surrounding sensory neurons. The box indicates the region of the aCC motor axon that was used for 3D analysis of mitochondrion advance. (<b>B</b> - <b>D</b>) Time-lapse series of an elongating <i>Drosophila</i> aCC motor neuron in stage 16 embryo of the genotype +/elav-Gal4;;<i>UAS-mtGFP</i>, <i>dmiro</i>^B682/ <i>IVS-10XUAS-myr-tdTom</i>, shown at 2 min intervals. (<b>B</b>) myr-tdTomato (red in <b>D</b>) labels neuronal plasma membranes. (<b>C</b>) mitoGFP (green in <b>D</b>) labels mitochondria. The arrow shows a mitochondrion in growth cone. In the last half of the series a mitochondrion docks in the distal axon (triangle in <b>B</b>) and advances. (<b>E</b>) Average velocity of docked mitochondria in the growth cone, defined as the last five µm of the axon, and in binned regions along the distal axon. Because the RP2 axon is fasciculated with the aCC axon (<b>A</b>), only mitochondria in the last 25 µm of the aCC axon were analyzed. Error bars show the 95% confidence intervals. The number at the base of the bar is the number of docked mitochondria that were analyzed. Scale bars = 10 µm.</p