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The precise modulation of regional cerebral blood flow during neural activation is important for matching local energetic demand and supply and clearing brain metabolites. Here we discuss advances facilitated by high-resolution optical in vivo imaging techniques that for the first time have provided direct visualization of capillary blood flow and its modulation by neural activity. We focus primarily on studies of microvascular flow, mural cell control of vessel diameter, and oxygen level-dependent changes in red blood cell deformability. We also suggest methodological standards for best practices when studying microvascular perfusion, partly motivated by recent controversies about the precise location within the microvascular tree where neurovascular coupling is initiated, and the role of mural cells in the control of vasomotility. In vivo optical imaging studies in rodents over the past two decades have significantly advanced the understanding of cellular mechanisms of neurovascular coupling. Rapid changes in red blood cell deformability and blood rheology due to fluctuations in oxygen levels can increase capillary cell flux prior to microvascular dilation. We review in vivo evidence indicating that activity-dependent changes in regional blood flow are mediated by smooth muscle cell contractility in arterioles but not by pericytes. Technical recommendations are provided for reproducible studies of neurovascular coupling in vivo and proper functional characterization of pericytes and smooth muscle cells. [ABSTRACT FROM AUTHOR]

Aberrant neural hyperactivity has been observed in early stages of Alzheimer's disease (AD) and may be a driving force in the progression of amyloid pathology. Evidence for this includes the findings that neural activity may modulate amyloid (A) peptide secretion and experimental stimulation of neural activity can increase amyloid deposition. However, whether long-term attenuation of neural activity prevents the buildup of amyloid plaques and associated neural pathologies remains unknown. Using viral-mediated delivery of designer receptors exclusively activated by designer drugs (DREADDs), we show in two AD-like mouse models that chronic intermittent increases or reductions of activity have opposite effects on A deposition. Neural activity reduction markedly decreases A aggregation in regions containing axons or dendrites of DREADD-expressing neurons, suggesting the involvement of synaptic and nonsynaptic A release mechanisms. Importantly, activity attenuation is associated with a reduction in axonal dystrophy and synaptic loss around amyloid plaques. Thus, modulation of neural activity could constitute a potential therapeutic strategy for ameliorating amyloid-induced pathology in AD. [ABSTRACT FROM AUTHOR]

Dystrophic neurites are associated with fibrillar amyloid deposition in Alzheimer's disease (AD), but the frequency and types of changes in synaptic structures near amyloid deposits have not been well characterized. Using high-resolution confocal microscopy to image lipophilic dye-labeled dendrites and thioflavin-S-labeled amyloid plaques, we systematically analyzed the structural changes of dendrites associated with amyloid deposition in both a transgenic mouse model of AD (PSAPP) and in human postmortem brain. We found that in PSAPP mice, dendritic branches passing through or within 40 μm from amyloid deposits displayed various dendritic abnormalities such as loss of dendritic spines, shaft atrophy, bending, abrupt branch endings, varicosity formation, and sprouting. Similar structural alterations of dendrites were seen in postmortem human AD tissue, with spine loss as the most common abnormality in both PSAPP mice and human AD brains. These results demonstrate that fibrillar amyloid deposits and their surrounding microenvironment are toxic to dendrites and likely contribute to significant disruption of neuronal circuits in AD. [ABSTRACT FROM AUTHOR]

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury. [ABSTRACT FROM AUTHOR]

Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system. [Copyright &y& Elsevier]

The structural dynamics of synapses probably has a crucial role in the development and plasticity of the nervous system. In the mammalian brain, the vast majority of excitatory axo-dendritic synapses occur on dendritic specializations called ‘spines’. However, little is known about their long-term changes in the intact developing or adult animal. To address this question we developed a transcranial two-photon imaging technique to follow identified spines of layer-5 pyramidal neurons in the primary visual cortex of living transgenic mice expressing yellow fluorescent protein. Here we show that filopodia-like dendritic protrusions, extending and retracting over hours, are abundant in young animals but virtually absent from the adult. In young mice, within the ‘critical period’ for visual cortex development, ∼73% of spines remain stable over a one-month interval; most changes are associated with spine elimination. In contrast, in adult mice, the overwhelming majority of spines (∼96%) remain stable over the same interval with a half-life greater than 13 months. These results indicate that spines, initially plastic during development, become remarkably stable in the adult, providing a potential structural basis for long-term information storage. [ABSTRACT FROM AUTHOR]

Amyloid plaques are a hallmark of Alzheimer disease, but their importance in its pathogenesis is controversial. By neuronal labeling and transcranial two-photon imaging, we show in a transgenic mouse model of Alzheimer disease that dendrites passing through or near fibrillar amyloid deposits undergo spine loss and shaft atrophy, and nearby axons develop large varicosities, together leading to neurite breakage and large- scale, permanent disruption of neuronal connections. Thus, fibrillar amyloid deposition is more detrimental to neuronal circuitry than previously thought, underscoring the importance of prevention and early clearance of plaques. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article about effectiveness of fluorescent dye sulforhodamine 101 in vivo brain imaging.

Recent genetic studies have provided overwhelming evidence of the involvement of microglia-related molecular networks in the pathophysiology of Alzheimer’s disease (AD). However, the precise mechanisms by which microglia alter the course of AD neuropathology remain poorly understood. Here we discuss current evidence of the neuroprotective functions of microglia with a focus on optical imaging studies that have revealed a role of these cells in the encapsulation of amyloid deposits (“microglia barrier”). This barrier modulates the degree of plaque compaction, amyloid fibril surface area, and insulation from adjacent axons thereby reducing neurotoxicity. We discuss findings implicating genetic variants of the microglia receptor, triggering receptor expressed on myeloid cells 2, in the increased risk of late onset AD. We provide evidence that increased AD risk may be at least partly mediated by deficient microglia polarization toward amyloid deposits, resulting in ineffective plaque encapsulation and reduced plaque compaction, which is associated with worsened axonal pathology. Finally, we propose possible avenues for therapeutic targeting of plaque-associated microglia with the goal of enhancing the microglia barrier and potentially reducing disease progression. [ABSTRACT FROM AUTHOR]

We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans. [ABSTRACT FROM AUTHOR]

During the neonatal period, activity-dependent neural-circuit remodelling coincides with growth and refinement of the cerebral microvasculature. Whether neural activity also influences the patterning of the vascular bed is not known. Here we show in neonatal mice, that neither reduction of sensory input through whisker trimming nor moderately increased activity by environmental enrichment affects cortical microvascular development. Unexpectedly, chronic stimulation by repetitive sounds, whisker deflection or motor activity led to a near arrest of angiogenesis in barrel, auditory and motor cortices, respectively. Chemically induced seizures also caused robust reductions in microvascular density. However, altering neural activity in adult mice did not affect the vasculature. Histological analysis and time-lapse in vivo two-photon microscopy revealed that hyperactivity did not lead to cell death or pruning of existing vessels but rather to reduced endothelial proliferation and vessel sprouting. This anti-angiogenic effect was prevented by administration of the nitric oxide synthase (NOS) inhibitor L-NAME and in mice with neuronal and inducible NOS deficiency, suggesting that excessive nitric oxide released from hyperactive interneurons and glia inhibited vessel growth. Vascular deficits persisted long after cessation of hyperstimulation, providing evidence for a critical period after which proper microvascular patterning cannot be re-established. Reduced microvascular density diminished the ability of the brain to compensate for hypoxic challenges, leading to dendritic spine loss in regions distant from capillaries. Therefore, excessive sensorimotor stimulation and repetitive neural activation during early childhood may cause lifelong deficits in microvascular reserve, which could have important consequences for brain development, function and pathology. [ABSTRACT FROM AUTHOR]

Layer formation in the developing cerebral cortex requires the movement of neurons from their site of origin to their final laminar position. We demonstrate, using time-lapse imaging of acute cortical slices, that two distinct forms of cell movement, locomotion and somal translocation, are responsible for the radial migration of cortical neurons. These modes are distinguished by their dynamic properties and morphological features. Locomotion and translocation are not cell-type specific; although at early ages some cells may move by translocation only, locomoting cells also translocate once their leading process reaches the marginal zone. The existence of two modes of radial migration may account for the differential effects of certain genetic mutations on cortical development. [ABSTRACT FROM AUTHOR]

The article explores the development of a photochemical and viral methodologies to induce death in single cells and combined this with intravital optical imaging. It mentions the glial cell's role in the astrocyte processes rapidly polarized and engulfed numerous small dendritic apoptotic bodies, while microglia migrated and engulfed the soma and apical dendrites.

Objective: Cortical spreading depression (CSD) has long been implicated in migraine attacks with aura. The process by which CSD, a cortical event that occurs within the blood-brain barrier (BBB), results in nociceptor activation outside the BBB is likely mediated by multiple molecules and cells. The objective of this study was to determine whether CSD activates immune cells inside the BBB (pia), outside the BBB (dura), or in both, and if so, when.Methods: Investigating cellular events in the meninges shortly after CSD, we used in vivo two-photon imaging to identify changes in macrophages and dendritic cells (DCs) that reside in the pia, arachnoid, and dura and their anatomical relationship to TRPV1 axons.Results: We found that activated meningeal macrophages retract their processes and become circular, and that activated meningeal DCs stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid, and dural DCs 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DCs can appear in close proximity to TRPV1-positive axons.Interpretation: The findings suggest that activation of pial macrophages may be more relevant to cases where aura and migraine begin simultaneously, that activation of dural macrophages may be more relevant to cases where headache begins 20 to 30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DCs in the subarachnoid space and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and DCs supports a role for these immune cells in the modulation of head pain. Ann Neurol 2018;83:508-521. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that has a progression that is closely associated with oxidative stress. It has long been speculated that the reactive oxygen species (ROS) level in AD brains is much higher than that in healthy brains. However, evidence from living beings is scarce. Inspired by the "chemistry of glow stick," we designed a near-IR fluorescence (NIRF) imaging probe, termed CRANAD-61, for sensing ROS to provide evidence at micro- and macrolevels. In CRANAD-61, an oxalate moiety was utilized to react with ROS and to consequentially produce wavelength shifting. Our in vitro data showed that CRANAD-61 was highly sensitive and rapidly responsive to various ROS. On reacting with ROS, its excitation and emission wavelengths significantly shifted to short wavelengths, and this shifting could be harnessed for dual-color two-photon imaging and transformative NIRF imaging. In this report, we showed that CRANAD-61 could be used to identify "active" amyloid beta (Aβ) plaques and cerebral amyloid angiopathy (CAA) surrounded by high ROS levels with twophoton imaging (microlevel) and to provide relative total ROS concentrations in AD brains via whole-brain NIRF imaging (macrolevel). Lastly, we showed that age-related increases in ROS levels in AD brains could be monitored with our NIRF imaging method. We believe that our imaging with CRANAD-61 could provide evidence of ROS at micro- and macrolevels and could be used for monitoring ROS changes under various AD pathological conditions and during drug treatment. [ABSTRACT FROM AUTHOR]

Summary Haplodeficiency of the microglia gene TREM2 increases risk for late-onset Alzheimer’s disease (AD) but the mechanisms remain uncertain. To investigate this, we used high-resolution confocal and super-resolution (STORM) microscopy in AD-like mice and human AD tissue. We found that microglia processes, rich in TREM2, tightly surround early amyloid fibrils and plaques promoting their compaction and insulation. In Trem2 - or DAP12 -haplodeficient mice and in humans with R47H TREM2 mutations, microglia had a markedly reduced ability to envelop amyloid deposits. This led to an increase in less compact plaques with longer and branched amyloid fibrils resulting in greater surface exposure to adjacent neurites. This was associated with more severe neuritic tau hyperphosphorylation and axonal dystrophy around amyloid deposits. Thus, TREM2 deficiency may disrupt the formation of a neuroprotective microglia barrier that regulates amyloid compaction and insulation. Pharmacological modulation of this barrier could be a novel therapeutic strategy for AD. [ABSTRACT FROM AUTHOR]

Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wildtype brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology. [ABSTRACT FROM AUTHOR]

Summary The precise regulation of cerebral blood flow is critical for normal brain function, and its disruption underlies many neuropathologies. The extent to which smooth muscle-covered arterioles or pericyte-covered capillaries control vasomotion during neurovascular coupling remains controversial. We found that capillary pericytes in mice and humans do not express smooth muscle actin and are morphologically and functionally distinct from adjacent precapillary smooth muscle cells (SMCs). Using optical imaging we investigated blood flow regulation at various sites on the vascular tree in living mice. Optogenetic, whisker stimulation, or cortical spreading depolarization caused microvascular diameter or flow changes in SMC but not pericyte-covered microvessels. During early stages of brain ischemia, transient SMC but not pericyte constrictions were a major cause of hypoperfusion leading to thrombosis and distal microvascular occlusions. Thus, capillary pericytes are not contractile, and regulation of cerebral blood flow in physiological and pathological conditions is mediated by arteriolar SMCs. [ABSTRACT FROM AUTHOR]

Loss- or gain-of-function mutations in ATP-sensitive potassium channel (K-ATP)-encoding genes, KCNJ8 and ABCC9 , cause human central nervous system disorders with unknown pathogenesis. Here, using mice, zebrafish, and cell culture models, we investigated cellular and molecular causes of brain dysfunctions derived from altered K-ATP channel function. We show that genetic/chemical inhibition or activation of KCNJ8/ABCC9-containing K-ATP channel function leads to brain-selective suppression or promotion of arterial/arteriolar vascular smooth muscle cell (VSMC) differentiation, respectively. We further show that brain VSMCs develop from KCNJ8/ABCC9-containing K-ATP channel-expressing mural cell progenitor and that K-ATP channel cell autonomously regulates VSMC differentiation through modulation of intracellular Ca2+ oscillation via voltage-dependent calcium channels. Consistent with defective VSMC development, Kcnj8 knockout mice showed deficiency in vasoconstrictive capacity and neuronal-evoked vasodilation leading to local hyperemia. Our results demonstrate a role for KCNJ8/ABCC9-containing K-ATP channels in the differentiation of brain VSMC, which in turn is necessary for fine-tuning of cerebral blood flow. [Display omitted] • Pericyte-enriched K-ATP channel cell autonomously promotes arterial VSMC development • K-ATP channel controls VSMC differentiation by modulating Ca2+ oscillation via VDCC • K-ATP channel dysfunction leads to defective VSMC differentiation • VSMC defects evoked by K-ATP channel dysfunction leads to neurovascular uncoupling Ando et al. show that an ATP-sensitive potassium (K-ATP) channel composed of Kir6.1 (KCNJ8) and Sur2B (ABCC9) is essential for brain functional hyperemic responses and that this reflects a critical and brain-specific role of K-ATP channels during vascular smooth muscle cell (VSMC) differentiation from mural cell progenitors. [ABSTRACT FROM AUTHOR]

Oligodendrocytes in the mammalian brain are continuously generated from NG2 cells throughout postnatal life. However, it is unclear when the decision is made for NG2 cells to self-renew or differentiate into oligodendrocytes after cell division. Using a combination of in vivo and ex vivo imaging and fate analysis of proliferated NG2 cells in fixed tissue, we demonstrate that in the postnatal developing mouse brain, the majority of divided NG2 cells differentiate into oligodendrocytes during a critical age-specific temporal window of 3-8 d. Notably, within this time period, damage to myelin and oligodendrocytes accelerated oligodendrocyte differentiation from divided cells, and whisker removal decreased the survival of divided cells in the deprived somatosensory cortex. These findings indicate that during the critical temporal window of plasticity, the fate of divided NG2 cells is sensitive to modulation by external signals. [ABSTRACT FROM AUTHOR]

In this report, we designed a highly bright bifunctional curcumin analogue CRANAD-28. In vivo two-photon imaging suggested that CRANAD-28 could penetrate the blood brain barrier (BBB) and label plaques and cerebral amyloid angiopathies (CAAs). We also demonstrated that this imaging probe could inhibit the crosslinking of amyloid beta induced either by copper or by natural conditions. [ABSTRACT FROM AUTHOR]

Cerebral function and viability are critically dependent on efficient delivery of oxygen and glucose through the microvasculature. Here, we studied individual microvessels in the intact brain using high-resolution confocal imaging and long-term time-lapse two-photon microscopy across the lifetime of a mouse. In the first postnatal month, we found large-scale sprouting but to our surprise the majority of sprouts underwent pruning and only a small fraction became perfused capillaries. After the first month, microvessel formation and elimination decreased and the net number of vessels stabilized. Although vascular stability was the hallmark of the adult brain, some vessel formation and elimination continued throughout life. In young adult mice, vessel formation was markedly increased after exposure to hypoxia; however, upon return to normoxia, no vessel elimination was observed, suggesting that new vessels constitute a long-term adaptive response to metabolic challenges. This plasticity was markedly reduced in older adults and aging where hypoxia-induced angiogenesis was absent. Our study describes, for the first time in vivo patterns of cerebral microvascular remodeling throughout life. Disruption of the observed balance between baseline turnover and vascular stability may underlie a variety of developmental and age-related degenerative neurological disorders. [ABSTRACT FROM AUTHOR]

In Alzheimer's disease (AD), amyloid-β (Aβ) deposits are frequently surrounded by activated microglia but the precise role of these cells in disease progression remains unclear. The chemokine receptor CX3CR1 is selectively expressed in microglia and is thought to modulate their activity. To study the specific effects of microglia activation on amyloid pathology in vivo, we crossbred mice lacking CX3CR1 with the Alzheimer's mouse model CRND8. Surprisingly, we found that CX3CR1-deficient mice had lower brain levels of Aβ40 and Aβ42 and reduced amyloid deposits. Quantification of Aβ within microglia and time-lapse two-photon microscopy in live mice revealed that these cells were highly effective at the uptake of protofibrillar amyloid but were incapable of phagocytosis of fibrillar congophilic Aβ. CX3CR1 deletion was associated with increased phagocytic ability, which led to greater amyloid content within microglial phagolysosomes. Furthermore, CX3CR1-deficient mice had an increased number of microglia around individual plaques because of higher proliferative rates, which likely contributed to an overall greater phagocytic capacity. CX3CR1 deletion did not affect the degree of neuronal or synaptic damage around plaques despite increased microglia density. Our results demonstrate that microglia can regulate brain Aβ levels and plaque deposition via selective protofibrillar Aβ phagocytosis. Modulation of microglia activity and proliferation by CX3CR1 signaling may represent a therapeutic strategy for AD. [ABSTRACT FROM AUTHOR]

Cerebral microvascular occlusion is a common phenomenon throughout life that might require greater recognition as a mechanism of brain pathology. Failure to recanalize microvessels promptly may lead to the disruption of brain circuits and significant functional deficits. Haemodynamic forces and the fibrinolytic system are considered to be the principal mechanisms responsible for recanalization of occluded cerebral capillaries and terminal arterioles. Here we identify a previously unrecognized cellular mechanism that may also be critical for this recanalization. By using high-resolution fixed-tissue microscopy and two-photon imaging in living mice we observed that a large fraction of microemboli infused through the internal carotid artery failed to be lysed or washed out within 48 h. Instead, emboli were found to translocate outside the vessel lumen within 2–7 days, leading to complete re-establishment of blood flow and sparing of the vessel. Recanalization occurred by a previously unknown mechanism of microvascular plasticity involving the rapid envelopment of emboli by endothelial membrane projections that subsequently form a new vessel wall. This was followed by the formation of an endothelial opening through which emboli translocated into the perivascular parenchyma. The rate of embolus extravasation was significantly decreased by pharmacological inhibition of matrix metalloproteinase 2/9 activity. In aged mice, extravasation was markedly delayed, resulting in persistent tissue hypoxia, synaptic damage and cell death. Alterations in the efficiency of the protective mechanism that we have identified may have important implications in microvascular pathology, stroke recovery and age-related cognitive decline. [ABSTRACT FROM AUTHOR]

Imaging is a dominant strategy for data collection in neuroscience, yielding stacks of images that often scale to gigabytes of data for a single experiment. Machine learning algorithms from computer vision can serve as a pair of virtual eyes that tirelessly processes these images, automatically detecting and identifying microstructures. Unlike learning methods, our Flexible Learning-free Reconstruction of Imaged Neural volumes (FLoRIN) pipeline exploits structure-specific contextual clues and requires no training. This approach generalizes across different modalities, including serially-sectioned scanning electron microscopy (sSEM) of genetically labeled and contrast enhanced processes, spectral confocal reflectance (SCoRe) microscopy, and high-energy synchrotron X-ray microtomography (μCT) of large tissue volumes. We deploy the FLoRIN pipeline on newly published and novel mouse datasets, demonstrating the high biological fidelity of the pipeline’s reconstructions. FLoRIN reconstructions are of sufficient quality for preliminary biological study, for example examining the distribution and morphology of cells or extracting single axons from functional data. Compared to existing supervised learning methods, FLoRIN is one to two orders of magnitude faster and produces high-quality reconstructions that are tolerant to noise and artifacts, as is shown qualitatively and quantitatively. [ABSTRACT FROM AUTHOR]