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

Polarization Controlled Coherent Anti-Stokes Raman Scattering Microscopy for Determination of Structural Order of the Myelin Sheath

Coherent Anti-Stokes Raman Scattering (CARS) is a laser-scanning microscopy technique that generates a strong label-free signal in lipid. The myelin sheath surrounding nerves is up to 80% lipid, and therefore is an ideal candidate for CARS imaging. The long lipid chains forming the myelin wraps have a directional preference when the myelin is healthy, with CH2 bonds aligned parallel to the axis of the nerve. The optical polarization dependence of the CARS signal can be used to probe the orientation of CH2 bonds in the sheath and determine their nanoscopic orientation. As myelin becomes unhealthy the organization of the lipids begins to loosen from their native organized packing structure. In the early stages this is not visible by conventional microscopy, however polarization-resolved CARS can measure the increasing disorder in the arrangement of the bonds before obvious morphological changes can occur. The degree of disorder is also measured to provide a metric of myelin disorganization in disease models

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

Previous research based on theoretical simulations has shown the potential of the wavelet transform to detect damage in a beam by analysing the time-deflection response due to a constant moving load. However, its application to identify damage from the response of a bridge to a vehicle raises a number of questions. Firstly, it may be difficult to record the difference in the deflection signal between a healthy and a slightly damaged structure to the required level of accuracy and high scanning frequencies in the field. Secondly, the bridge is going to have a road profile and it will be loaded by a sprung vehicle and time-varying forces rather than a constant load. Therefore, an algorithm based on a plot of wavelet coefficients versus time to detect damage (a singularity in the plot) appears to be very sensitive to noise. This paper addresses these questions by: (a) using the acceleration signal, instead of the deflection signal, (b) employing a vehicle-bridge finite element interaction model, and (c) developing a novel wavelet-based approach using wavelet energy content at each bridge section, which proves to be more sensitive to damage than a wavelet coefficient line plot at a given scale as employed by others.    European Commission - Seventh Framework Programme (FP7)7th European Framework Project ASSETInclude in School of Civil Engineering Collection and also Earth Institute Collectio

Axonal and myelinic pathology in 5xFAD Alzheimers 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 Alzheimers research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimers 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 Alzheimers pathology and disease progression.Funding Agencies|Dr. Frank Leblanc Chair for Spinal Cord Research; Canadian Institutes of Health Research (CIHR); Canada Research Chairs (CRC); Alberta Prion Research Institute (APRI)</p

Threads may cause damage to axons.

<p>(A) Many APP-positive axonal spheroids (red) can be seen in a representative cross-section from a 27 week old 5xFAD mouse. 6E10-positive threads (green) are often seen co-labeling with APP staining (arrow) at this age. (B) Z-stack images (0. 5μm optical sections) from the spheroid indicated by the arrow in A reveal that a thread lies on the outside of the spheroid rather than inside it. (C) Similarly, a sagittal section showed a 6E10-labeled (green) thread at the tip of an APP-positive (red) spheroid. (D) Sagittal section stained with the aminergic fibre marker, tyrosine hydroxylase (red), and 6E10 (green) shows a thread, extending from a spheroid at the end of an aminergic fibre, that forms a coil about 30μm caudal to the spheroid. Scale bar in A = 50μm; B-C = 10μm and D = 15μm.</p

Primary antibodies used in the study.

<p>Primary antibodies used in the study.</p