982 lines
72 KiB
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72 KiB
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<meta name="description" content="Enhancement of a new DNA-based bioimaging technique… Method allowing near-simultaneous imaging of several thousand neurons in awake behaving mice… Imaging method to overcome biological tissue refractive index inhomogeneity increases large-field-of-view imaging depth… Open-source software package for working with microscopy images boosts high-throughput, single-cell analysis … Improved DNA conjugation method enhances the achievable labeling density and spatial accuracy of highly multiplexed Exchange-PAINT imaging" />
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<h1 class="title"><span class="field field--name-title field--type-string field--label-hidden">BRAIN Publication Roundup – September 2017</span>
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<div class="field field--name-field-simple-date field--type-datetime field--label-hidden field__item"><time datetime="2017-09-22T12:00:00Z" class="datetime">September 22, 2017</time>
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<div class="clearfix text-formatted field field--name-body field--type-text-with-summary field--label-hidden field__item"><p><em>Enhancement of a new DNA-based bioimaging technique… Method allowing near-simultaneous imaging of several thousand neurons in awake behaving mice… Imaging method to overcome biological tissue refractive index inhomogeneity increases large-field-of-view imaging depth… Open-source software package for working with microscopy images boosts high-throughput, single-cell analysis …</em></p>
|
||
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||
<p><strong>Improved DNA conjugation method enhances the achievable labeling density and spatial accuracy of highly multiplexed Exchange-PAINT imaging</strong></p>
|
||
|
||
<p>Recent developments in high-resolution fluorescence imaging methods have overcome the limits of light diffraction. However, these techniques are challenged by their limited multiplexing capability, which hinders researchers’ understanding of multi-protein interactions at the nanoscale level. Exchange-PAINT (i.e., Points Accumulation in Nanoscale Topography), a new DNA-based approach, boosts multiplexing capabilities by sequentially imaging target molecules using orthogonal, dye-labeled DNA strands. Although very promising for bioimaging, the widespread application of this approach has been limited by the availability of DNA-conjugated ligands for protein labeling. At Harvard University, <a href="https://wyss.harvard.edu/team/core-faculty/peng-yin/">Dr. Peng Yin</a> and colleagues have developed <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=28451377">a new labeling platform for Exchange-PAINT</a> that efficiently conjugates DNA oligonucleotides to various labeling probes (e.g., antibodies, nanobodies, and small molecules). By designing and testing the conjugation of 52 oligonucleotides to labeling probes like nanobodies, the group successfully enhanced the achievable labeling density and spatial accuracy of Exchange-PAINT. Finally, they demonstrated high-resolution cellular imaging with their labeling platform. The DNA conjugation method is simple to perform and the group anticipates that this general framework for labeling protein targets will make Exchange-PAINT accessible to a broader scientific community.</p>
|
||
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||
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||
<figcaption><em>High-resolution image of proteins in HeLa cells acquired using nine rounds of Exchange-PAINT. The target proteins were labeled with DNA-conjugated antibodies using direct immunostaining. Complementary DNA strands were sequentially introduced to the sample for imaging. Post-acquisition, a washing buffer with reduced ionic strength was introduced to remove all </em><em>DNA strands. Nine imaging rounds were performed using orthogonal DNA strands conjugated to the same dye.</em></figcaption>
|
||
</figure>
|
||
<strong>Nov</strong><strong>el calcium imaging technique with a two-photon light-sculpting system enables fast volumetric imaging across multiple cortical layers</strong><figure role="group" class="caption caption-drupal-media align-right">
|
||
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|
||
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||
<figcaption><em>Calcium imaging in mouse hippocampus. <strong>(a)</strong> Schematic of the window preparation (red box represents imaging volume). <strong>(b)</strong> Time-averaged image (100μm depth). <strong>(c)</strong> Calcium traces of individual neurons imaged at 158fps in a single plane. <strong>(d)</strong> 3D rendering of time-averaged image (0.5 mm × 0.5 mm × 0.2 mm).</em></figcaption>
|
||
</figure>
|
||
Advancing techniques for imaging the mammalian brain requires developing tools that record the activity of all neurons within a functional network at single-neuron resolution and over physiologically relevant time scales. Despite the recent introduction of various high-speed calcium imaging techniques, it remains a challenge to image the functional dynamics of large-scale neuronal circuits in awake-behaving mammals at high resolution. At the Rockefeller University and the University of Vienna, <a href="http://vaziri.rockefeller.edu/">Dr. Alipasha Vaziri and colleagues</a><a href="http://www.ninds.nih.gov/exit_disclaimer.htm">
|
||
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|
||
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||
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||
<p> reveal a <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=27798612">new calcium imaging method</a> that utilizes a two-photon light-sculpting system. They updated their previously developed methods by tailoring the microscope to view the typical size of neuronal cell bodies in the mouse cortex. These changes allowed for the samplings of larger volumes with minimal numbers of excitation voxels at near-single-cell resolution. The signal-to-noise ratio was maximized using a fiber-based laser amplifier that synchronized pulses to the imaging voxel speed. The overall approach enabled near-simultaneous calcium imaging of several thousand neurons, across cortical layers (0.5 mm × 0.5 mm × 0.5 mm) and in the hippocampus of awake behaving mice. This exciting new method presents the opportunity to test experimentally a variety of theoretical models of information processing in the mammalian neocortex.</p>
|
||
|
||
<p><strong>Large-field-of-view imaging by multi-pupil adaptive optics allows position-dependent correction of biological tissue optical distortion</strong></p>
|
||
|
||
<p>The refractive index inhomogeneity within biological tissue presents challenges for <em>in vivo </em>optical imaging. Adaptive optics (AO) has corrected some of the distortions caused by this lack of homogeneity. However, the limited field-of-view (FOV) of current methods reduces imaging speed across larger areas, since distortion varies spatially and needs to be corrected accordingly. Approaches that provide simultaneous large-FOV distortion correction, and hence enable imaging of fast dynamics, are needed. At Purdue University, <a href="http://mengtsui.wixsite.com/cuilab">Dr. Meng Cui and colleagues</a> developed <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=28481364">multi-pupil adaptive optics (MPAO)</a>, which enables simultaneous, position-dependent correction over a 450 × 450 μm<sup>2 </sup>FOV and expands the correction area to nine times that of previous methods. In conventional AO, the correction measured from one region is applied to the entire image, improving imaging performance within a limited FOV. In MPAO, the imaging procedure is similar to conventional systems, but independent correction for all regions is achieved. By implementing MPAO with imaging of <em>in vivo</em> mouse microglia dynamics, the group demonstrated improved quality compared with conventional AO. They next performed calcium imaging of neurons and astrocytes at 450 μm depth, achieving high-resolution images with full correction. Compared to typical techniques that provide imaging at 200-300 μm depths, large-FOV imaging at ~650 μm depth is possible using MPAO. Finally, with spatially independent distortion control, MPAO also enables nonplanar microscopy (i.e., brings 3D features at different depths into one imaging plane), which the group demonstrated by imaging 3D neurovasculature dynamics in anesthetized mice. This technique can aid high-spatiotemporal-resolution microscopy in various biological systems.</p>
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<figcaption><em>Calcium imaging at 450 μm depth with MPAO. <strong>(a,b)</strong> Astrocytes at 436-465 μm under the dura with full <strong>(a)</strong> and system <strong>(b)</strong> correction. <strong>(c,d)</strong> Zoomed-in view of the central area in <strong>a </strong>and <strong>b</strong>. <strong>(e,f)</strong> Standard deviation of the time-lapse images of neurons with full <strong>(e)</strong> and system <strong>(f)</strong> correction. The images in <strong>c</strong>–<strong>f </strong>are from the same area. <strong>(g)</strong> Astrocytes (magenta) and neurons (green) at 450 μm depth. <strong>(h)</strong> Regions of interest (ROIs) for computing calcium transients. <strong>(i)</strong> Calcium transients with full and system correction.</em></figcaption>
|
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|
||
<strong>A Python platform for image-guided mass spectrometry profiling facilitates sequential multi-technique analysis of each target in a biological sample</strong><figure role="group" class="caption caption-drupal-media align-right">
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<figcaption><em>Sequential analysis of the same rat cerebellum-derived cell using MS instruments with different capabilities; MALDI-TOF followed by MALDI-FT-ICR. Once a cell is located in the optical image, its location remains fixed through multiple analyses. MALDI-TOF provides high-throughput screening of thousands of cells to highlight rare or representative individuals. FT-ICR provides exact mass measurement for elemental composition analysis. Such a workflow facilitates exhaustive cell population analysis while efficiently utilizing the FT-ICR instrument.</em></figcaption>
|
||
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|
||
Image-guided mass spectrometry (MS) profiling is a methodology for analyzing samples ranging from single cells to tissue sections. The workflow uses whole-slide microscopy to select targets, determine their locations, and perform MS analysis at those locations. This framework provides a link between the spatial dimensions in an image and the physical location of a sample. Single-cell MS has attracted substantial interest due to its sensitivity. Biomolecules within cells are detectible with MS, facilitating discovery of single-cell heterogeneity and enhancing understanding of the relationship between cellular chemical contents and their functions. However, limitations in MS imaging for high-throughput, single-cell analysis have stimulated efforts to develop methods that improve efficiency and resolution. At the University of Illinois at Urbana–Champaign, <a href="http://www.scs.illinois.edu/sweedler/">Dr. Jonathan Sweedler and colleagues</a><a href="http://www.ninds.nih.gov/exit_disclaimer.htm">
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<p> have developed an open-source software package for working with microscopy images called <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=28593377">microMS</a>. The new platform permits effortless sequential analysis, enabling each cell to be scrutinized by multiple techniques. Targets can be automatically located, filtered, and stratified before MS. Specific MS systems are implemented through a novel abstract base class and software architecture, offering impressive simplification of the connection of microMS to new instruments and facilitating more efficientsequential analysis of the same target. The group believes that the ease of extending microMS to a variety of mass spectrometers and other instruments will help advance single-cell profiling.</p></div>
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