A biologist's guide to planning and performing quantitative bioimaging experiments.

Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.

Functional ionotropic glutamate receptors on peripheral axons and myelin.

INTRODUCTION: Neurotransmitter-dependent signaling is traditionally restricted to axon terminals. However, receptors are present on myelinating glia, suggesting that chemical transmission may also occur along axons. METHODS: Confocal microscopy and Ca(2+) -imaging using an axonally expressed FRET-based reporter was used to measure Ca(2+) changes and morphological alterations in myelin in response to stimulation of glutamate receptors. RESULTS: Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors induced a Ca(2+) increase in axon cylinders. However, only the latter caused structural alterations in axons, despite similar Ca(2+) increases. Myelin morphology was significantly altered by NMDA receptor activation, but not by AMPA receptors. Cu(2+) ions influenced the NMDA receptor-dependent response, suggesting that this metal modulates axonal receptors. Glutamate increased ribosomal signal in Schwann cell cytoplasm. CONCLUSIONS: Axon cylinders and myelin of peripheral nervous system axons respond to glutamate, with a consequence being an increase in Schwann cell ribosomes. This may have implications for nerve pathology and regeneration. Muscle Nerve 54: 451-459, 2016.

High-resolution fluorescence microscopy of myelin without exogenous probes.

Myelin is a critical element of the central and peripheral nervous systems of all higher vertebrates. Any disturbance in the integrity of the myelin sheath interferes with the axon's ability to conduct action potentials. Thus, the study of myelin structure and biochemistry is critically important. Accurate and even staining of myelin is often difficult because of its lipid-rich nature and multiple tight membrane wraps, hindering penetration of immunoprobes. Here we show a method of visualizing myelin that is fast, inexpensive and reliable using the cross-linking fixative glutaraldehyde that produces strong, broad-spectrum auto-fluorescence in fixed tissue. Traditionally, effort is generally aimed at eliminating this auto-fluorescence. However, we show that this intrinsic signal, which is very photostable and particularly strong in glutaraldehyde-fixed myelin, can be exploited to visualize this structure to produce very detailed images of myelin morphology. We imaged fixed rodent tissues from the central and peripheral nervous systems using spectral confocal microscopy to acquire high-resolution 3-dimensional images spanning the visual range of wavelengths (400-750 nm). Mathematical post-processing allows accurate and unequivocal separation of broadband auto-fluorescence from exogenous fluorescent probes such as DAPI and fluorescently-tagged secondary antibodies. We additionally show the feasibility of immunohistochemistry with antigen retrieval, which allows co-localization of proteins of interest together with detailed myelin morphology. The lysolecithin model of de- and remyelination is shown as an example of a practical application of this technique, which can be routinely applied when high-resolution microscopy of central or peripheral myelinated tracts is required.

Functional remodeling of presynaptic voltage-gated calcium channels in superficial layers of the dorsal horn during neuropathic pain.

N- and P/Q-type voltage-gated Ca2+ channels are critical for synaptic transmission. While their expression is increased in the dorsal root ganglion (DRG) neuron cell bodies during neuropathic pain conditions, less is known about their synaptic remodeling. Here, we combined genetic tools with 2-photon Ca2+ imaging to explore the functional remodeling that occurs in central presynaptic terminals of DRG neurons during neuropathic pain. We imaged GCaMP6s fluorescence responses in an ex vivo spinal cord preparation from mice expressing GCaMP6s in Trpv1-Cre lineage nociceptors. We show that Ca2+ transient amplitude is increased in central terminals of these neurons after spared nerve injury, and that this increase is mediated by both N- and P/Q-type channels. We found that GABA-B receptor-dependent inhibition of Ca2+ transients was potentiated in the superficial layer of the dorsal horn. Our results provide direct evidence toward nerve injury-induced functional remodeling of presynaptic Ca2+ channels in Trpv1-lineage nociceptor terminals.

Portable, miniaturized, fibre delivered, multimodal CARS exoscope.

We demonstrate for the first time, a portable multimodal coherent anti-Stokes Raman scattering microscope (exoscope) for minimally invasive in-vivo imaging of tissues. This device is based around a micro-electromechanical system scanning mirror and miniaturized optics with light delivery accomplished by a photonic crystal fibre. A single Ti:sapphire femtosecond pulsed laser is used as the light source to produce CARS, two photon excitation fluorescence and second harmonic generation images. The high resolution and distortion-free images obtained from various resolution and bio-samples, particularly in backward direction (epi) successfully demonstrate proof of concept, and pave the path towards future non or minimally-invasive in vivo imaging.

Spectral focusing-based stimulated Raman scattering microscopy using compact glass blocks for adjustable dispersion.

Spectral focusing is a well-established technique for increasing spectral resolution in coherent Raman scattering microscopy. However, current methods for tuning optical chirp in setups using spectral focusing, such as glass rods, gratings, and prisms, are very cumbersome, time-consuming to use, and difficult to align, all of which limit more widespread use of the spectral focusing technique. Here, we report a stimulated Raman scattering (SRS) configuration which can rapidly tune optical chirp by utilizing compact adjustable-dispersion TIH53 glass blocks. By varying the height of the blocks, the number of bounces in the blocks and therefore path length of the pulses through the glass can be quickly modulated, allowing for a convenient method of adjusting chirp with almost no necessary realignment. To demonstrate the flexibility of this configuration, we characterize our system's signal-to-noise ratio and spectral resolution at different chirp values and perform imaging in both the carbon-hydrogen stretching region (MCF-7 cells) and fingerprint region (prostate cores). Our findings show that adjustable-dispersion glass blocks allow the user to effortlessly modify their optical system to suit their imaging requirements. These blocks can be used to significantly simplify and miniaturize experimental configurations utilizing spectral focusing.

Cuprizone-induced Demyelination in Mouse Brain is not due to Depletion of Copper.

SUMMARY STATEMENT: The demyelinating effects of CPZ are not due to Cu deficiency but are instead consistent with acute toxicity of a CPZ + Cu complex.

Label-free assessment of myelin status using birefringence microscopy.

BACKGROUND: Label-free methods for quantifying myelination can reduce expense, time, and variability in results when examining tissue white matter pathology. NEW METHOD: We sought to determine whether the optical birefringent properties of myelin could be exploited to determine myelination status of white matter in tissue sections. Sections of forebrains of mice (normal, and treated with cuprizone to cause demyelination) were examined by birefringence using a birefringence imaging system (Thorlabs LCC7201), and results compared with sections stained using Luxol Fast Blue. RESULTS: Quantitative birefringence analysis of myelin was not only reliable in detecting demyelination, but also showed abnormalities that preceded myelin loss in cuprizone-treated mice. COMPARISON WITH EXISTING METHODS: Subtle myelin pathology visible with electron microscopy but not with conventional histopathological staining was readily detected with birefringence microscopy. CONCLUSIONS: Birefringence imaging provides a rapid, label-free method of analyzing the myelin content and nanostructural status in longitudinal white matter structures, being sensitive to subtle myelin changes that precede overt pathological damage.

Miniaturized multimodal CARS microscope based on MEMS scanning and a single laser source.

We demonstrate a novel miniaturized multimodal coherent anti-Stokes Raman scattering (CARS) microscope based on microelectromechanical systems (MEMS) scanning mirrors and custom miniature optics. A single Ti:sapphire femtosecond pulsed laser is used as the light source to produce the CARS, two photon excitation fluorescence (TPEF) and second harmonic generation (SHG) images using this miniaturized microscope. The high resolution and distortion-free images obtained from various samples such as a USAF target, fluorescent and polystyrene microspheres and biological tissue successfully demonstrate proof of concept, and pave the path towards future integration of parts into a handheld multimodal CARS probe for non- or minimally-invasive in vivo imaging.

Spectral Characterization of Stem Cell-Derived Myelination within the Injured Adult PNS Using the Solvatochromic Dye Nile Red.

BACKGROUND: Myelin is an essential component of the peripheral and central nervous system, enabling fast axonal conduction and supporting axonal integrity; limited tools exist for analysis of myelin composition in-vivo. OBJECTIVE: To demonstrate that the photophysical properties of myelin-incorporated solvatochromic dyes can be exploited to probe the biochemical composition of living peripheral nerve myelin at high spatial resolution. METHODS: Using the myelin-incorporated fluorescent dye Nile Red we sequentially analyzed the spectral characteristics of remyelinating myelin membranes both in-vitro and in-vivo, including in living rats. RESULTS: We demonstrated a consistent bi-phasic evolution of emission spectra during early remyelination, and visually report the reliable biochemical flux of myelin membrane composition in-vitro and in-vivo. CONCLUSIONS: Solvatochromic spectroscopy enables the analysis of myelin membrane maturity during remyelination, and can be performed in-vivo. As the formation of myelin during early-to-late remyelination likely incorporates fluctuating fractions of lipophilic components and changes in lateral membrane mobility, we propose that our spectrochemical data reflects the observation of these biochemical processes.

Excitation parameters optimized for coherent anti-Stokes Raman scattering imaging of myelinated tissue.

Coherent anti-Stokes Raman scattering (CARS) generates a strong label-free signal in the long wavenumber C─H stretching region. Lipid-rich myelinated tissues, such as brain and spinal cord, would appear to be ideal subjects for imaging with CARS laser-scanning microscopy. However, the highly ordered, biochemically complex, and highly scattering nature of such tissues complicate the use of the technique. A CARS microscopy approach is presented that overcomes the challenges of imaging myelinated tissue to achieve chemically and orientationally sensitive high-resolution images.

A novel approach to 32-channel peripheral nervous system myelin imaging in vivo, with single axon resolution.

OBJECTIVE Intravital spectral imaging of the large, deeply situated nerves in the rat peripheral nervous system (PNS) has not been well described. Here, the authors have developed a highly stable platform for performing imaging of the tibial nerve in live rodents, thus allowing the capture of high-resolution, high-magnification spectral images requiring long acquisition times. By further exploiting the qualities of the topically applied myelin dye Nile red, this technique is capable of visualizing the detailed microenvironment of peripheral nerve demyelination injury and recovery, while allowing us to obtain images of exogenous Schwann cell myelination in a living animal. METHODS The authors caused doxorubicin-induced focal demyelination in the tibial nerves of 25 Thy-1 GFP rats, of which 2 subsets (n = 10 each) received either BFP-labeled SKP-SCs or SCs to the zone of injury. Prior to acquiring images of myelin recovery in these nerves, a tibial nerve window was constructed using a silicone hemitube, a fast drying silicone polymer, and a small coverslip. This construct was then affixed to a 3D-printed nerve stage, which in turn was affixed to an external fixation/microscope stage device. Myelin visualization was facilitated by the topical application of Nile red. RESULTS The authors reliably demonstrated intravital peripheral nerve myelin imaging with micron-level resolution and magnification, and minimal movement artifact. The detailed microenvironment of nerve remyelination can be vividly observed, while exogenously applied Schwann cells and skin-derived precursor Schwann cells can be seen myelinating axons. CONCLUSIONS Topically applied Nile red enables intravital study of myelin in the living rat PNS. Furthermore, the use of a tibial nerve window facilitates stable intravital peripheral nerve imaging, making possible high-definition spectral imaging with long acquisition times.

Coherent anti-Stokes Raman scattering microscopy using photonic crystal fiber with two closely lying zero dispersion wavelengths.

We demonstrate coherent anti-Stokes Raman scattering (CARS) microscopy of lipid-rich structures using a single unamplified femtosecond Ti:sapphire laser and a photonic crystal fiber (PCF) with two closely lying zero dispersion wavelengths (ZDW) for the Stokes source. The primary enabling factor for the fast data acquisition (84 micros per pixel) in the proof-of-principle CARS images, is the low noise supercontinuum (SC) generated in this type of PCF, in contrast to SC generated in a PCF with one ZDW. The dependence of the Stokes pulse on average input power, pump wavelength, pulse duration and polarization is experimentally characterized. We show that it is possible to control the spectral shape of the SC by tuning the pump wavelength of the input pulse and the consequence for CARS microscopy is discussed.

Effects of laser polarization on responses of the fluorescent Ca2+ indicator X-Rhod-1 in neurons and myelin.

Laser-scanning optical microscopes generally do not control the polarization of the exciting laser field. We show that laser polarization and imaging mode (confocal versus two photon) exert a profound influence on the ability to detect [Formula: see text] changes in both cultured neurons and living myelin. With two-photon excitation, increasing ellipticity resulted in a [Formula: see text] reduction in resting X-Rhod-1 fluorescence in homogeneous bulk solution, cell cytoplasm, and myelin. In contrast, varying the angle of a linearly polarized laser field only had appreciable effects on dyes that partitioned into myelin in an ordered manner. During injury-induced [Formula: see text] increases, larger ellipticities resulted in a significantly greater injury-induced signal increase in neurons, and particularly in myelin. Indeed, the traditional method of measuring [Formula: see text] changes using one-photon confocal mode with linearly polarized continuous wave laser illumination produced no appreciable X-Rhod-1 signal increase in ischemic myelin, compared to a robust [Formula: see text] fluorescence increase with two-photon excitation and optimized ellipticity with the identical injury paradigm. This underscores the differences in one- versus two-photon excitation and, in particular, the under-appreciated effects of laser polarization on the behavior of certain [Formula: see text] reporters, which may lead to substantial underestimates of the real [Formula: see text] fluctuations in various cellular compartments.

Axonal and myelinic pathology in 5xFAD Alzheimer's mouse spinal cord.

As an extension of the brain, the spinal cord has unique properties which could allow us to gain a better understanding of CNS pathology. The brain and cord share the same cellular components, yet the latter is simpler in cytoarchitecture and connectivity. In Alzheimer's research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimer's mouse models accumulate beta amyloid plaques in spinal cord, suggesting that the cord possesses the same molecular machinery and conditions for plaque formation. Here we report a spatial-temporal map of plaque load in 5xFAD mouse spinal cord. We found that plaques started to appear at 11 weeks, then exhibited a time dependent increase and differential distribution along the cord. More plaques were found in cervical than other spinal levels at all time points examined. Despite heavy plaque load at 6 months, the number of cervical motor neurons in 5xFAD mice is comparable to wild type littermates. On detailed microscopic examination, fine beta amyloid-containing and beta sheet-rich thread-like structures were found in the peri-axonal space of many axons. Importantly, these novel structures appear before any plaque deposits are visible in young mice spinal cord and they co-localize with axonal swellings at later stages, suggesting that these thread-like structures might represent the initial stages of plaque formation, and could play a role in axonal damage. Additionally, we were able to demonstrate increasing myelinopathy in aged 5xFAD mouse spinal cord using the lipid probe Nile Red with high resolution. Collectively, we found significant amyloid pathology in grey and white matter of the 5xFAD mouse spinal cord which indicates that this structure maybe a useful platform to study mechanisms of Alzheimer's pathology and disease progression.

Fluorescent Phosphorus Dendrimer as a Spectral Nanosensor for Macrophage Polarization and Fate Tracking in Spinal Cord Injury.

Dendrimers and dendriplexes, highly branched synthetic macromolecules, have gained popularity as new tools for a variety of nanomedicine strategies due to their unique structure and properties. We show that fluorescent phosphorus dendrimers are well retained by bone marrow-derived macrophages and exhibit robust spectral shift in its emission in response to polarization conditions. Fluorescence properties of this marker can also assist in identifying macrophage presence and phenotype status at different time points after spinal cord injury. Potential use of a single dendrimer compound as a drug/siRNA carrier and phenotype-specific cell tracer offers new avenues for enhanced cell therapies combined with monitoring of cell fate and function in spinal cord injury.

Fast diagnosis with sensors of uncertain quality.

This correspondence presents an approach to the detection and isolation of component failures in large-scale systems. In the case of sensors that report at rates of 1 Hz or less, the algorithm can be considered real time. The input is a set of observed test results from multiple sensors, and the algorithm's main task is to deal with sensor errors. The sensors are assumed to be of threshold test (pass/fail) type, but to be vulnerable to noise, in that occasionally true failures are missed, and likewise, there can be false alarms. These errors are further assumed to be independent conditioned on the system's diagnostic state. Their probabilities, of missed detection and of false alarm, are not known a priori and must be estimated (ideally along with the accuracies of these estimates) online, within the inference engine. Further, recognizing a practical concern in most real systems, a sparsely instantiated observation vector must not be a problem. The key ingredients to our solution include the multiple-hypothesis tracking philosophy to complexity management, a Beta prior distribution on the sensor errors, and a quickest detection overlay to detect changes in these error rates when the prior is violated. We provide results illustrating performance in terms of both computational needs and error rate, and show its application both as a filter (i.e., used to "clean" sensor reports) and as a standalone state estimator.