Near-infrared branding efficiently correlates light and electron microscopy

Bishop, Derron L.; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

doi:10.1038/nmeth.1622

Cited by 152 publications

(137 citation statements)

References 20 publications

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“…Indeed, even with purely morphological labelling, one important factor in scoring a broken axon is a 'pulling apart' movement of either side of the putative fragmentation point. (3) In studies correlating in vivo with post hoc confocal or electron microscopy 3D reconstructions, gaps in continuity seen in vivo could generally be confirmed by higher contrast and resolution methods 16,34,40 . The rare instances where ambiguities remain were excluded from analysis.…”

Section: Methodsmentioning

confidence: 95%

“…However, in some cases fine processes indicating axonal continuity are not apparent in z-projections, and are only observable in contrast-adjusted image stacks. We have addressed the veracity of axonal fragmentation apparent from in vivo fluorescent microscopic observations in our previous papers 16,34,40 . The main arguments for our interpretation that clearly identifiable gaps in fluorescence contiguity correspond to actual axon discontinuities are: (1) in chronic experiments, where such gaps were identified by us 16 and others 41 , subsequent Wallerian degeneration of the axon segment distal to the gap in fluorescence was observed.…”

Section: Methodsmentioning

confidence: 99%

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A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

et al. 2014

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Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.

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Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

et al. 2014

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“…These were then imaged and a small (30 Â 30 mm 2 ) square was burned with the laser, around the region of interest. This fiducial mark was visible after resin embedding and was used to locate the imaged axon 37,38 .…”

Section: Methodsmentioning

confidence: 99%

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

et al. 2013

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To what extent, how and when axons respond to injury in the highly interconnected grey matter is poorly understood. Here we use two-photon imaging and focused ion beamscanning electron microscopy to explore, at synaptic resolution, the regrowth capacity of several neuronal populations in the intact brain. Time-lapse analysis of 4100 individually ablated axons for periods of up to a year reveals a surprising inability to regenerate even in a glial scar-free environment. However, depending on cell type some axons spontaneously extend for distances unseen in the unlesioned adult cortex and at maximum speeds comparable to peripheral nerve regeneration. Regrowth follows a distinct pattern from developmental axon growth. Remarkably, although never reconnecting to the original targets, axons consistently form new boutons at comparable prelesion synaptic densities, implying the existence of intrinsic homeostatic programmes, which regulate synaptic numbers on regenerating axons. Our results may help guide future clinical investigations to promote functional axon regeneration.

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“…Laser etching can also be used directly on hydrated tissues to precisely locate the ROI. This technology, called near-infrared branding (NIRB) has been developed a few years ago and enables to highlight the position of the ROI, either owing to laser-induced autofluorescence or upon photooxidation of the NIRB mark (Bishop et al, 2011) (Fig. 4B).…”

Section: Ground-breaking In Vitro Clemmentioning

confidence: 99%

“…4B). In that study, by using NIRB on brain vibratome sections, single cortical dendritic spines that had been previously recorded in a living mouse could be dissected at unprecedented resolution (Bishop et al, 2011). Furthermore, NIRB-guided retrieval of the ROI in vibratome sections allows to combine intravital imaging with 3DEM, such as FIB-SEM, which was shown to provide nanoscale isotropic imaging of axons and dendrites .…”

Section: Ground-breaking In Vitro Clemmentioning

confidence: 99%

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Follain

Mercier

Osmani

et al. 2016

Journal of Cell Science

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Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, superresolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

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Near-infrared branding efficiently correlates light and electron microscopy

Cited by 152 publications

References 20 publications

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

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