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Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9

Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9 | Imaging | Scoop.it

A new microscopy method could enable scientists to generate snapshots of dozens of different biomolecules at once in a single human cell, a team from the Wyss Institute of Biologically Inspired Engineering at Harvard University reported Sunday in Nature Methods.


Such images could shed light on complex cellular pathways and potentially lead to new ways to diagnose disease, track its prognosis, or monitor the effectiveness of therapies at a cellular level.


Cells often employ dozens or even hundreds of different proteins and RNA molecules to get a complex job done. As a result, cellular job sites can resemble a busy construction site, with many different types of these tiny cellular workers coming and going. Today's methods typically only spot at most three or four types of these tiny workers simultaneously. But to truly understand complex cellular functions, it's important to be able to visualize most or all of those workers at once, said Peng Yin, Ph.D., a Core Faculty member at the Wyss Institute and Assistant Professor of Systems Biology at Harvard Medical School.


To capture ultrasharp images of biomolecules, they had to overcome laws of physics that stymied microscopists for most of the last century. When two objects are closer than about 200 nanometers apart — about one five-hundredth the width of a human hair — they cannot be distinguished using a traditional light microscope: the viewer sees one blurry blob where in reality there are two objects.


Since the mid-1990s, scientists have developed several ways to overcome this problem using combinations of specialized optics, special fluorescent proteins or dyes that tag cellular components.


Ralf Jungmann, Ph.D., now a Postdoctoral Fellow working with Yin at the Wyss Institute and Harvard Medical School, helped develop one of those super-resolution methods, called DNA-PAINT, as a graduate student. DNA-PAINT can create ultrasharp snapshots of up to three cellular workers at once by labeling them with different colored dyes.


To visualize cellular job sites with crews of dozens of cellular workers, Yin's team, including Jungmann, Maier Avendano, M.S., a graduate student at Harvard Medical School, and Johannes Woehrstein, a postgraduate research fellow at the Wyss Institute, modified DNA-PAINT to create a new method called Exchange-PAINT.


Exchange-PAINT relies on the fact that DNA strands with the correct sequence of letters, or nucleotides, bind specifically to partner strands with complementary sequences. The researchers label a biomolecule they want to visualize with a short DNA tag, then add to the solution a partner strand carrying a fluorescent dye that lights up only when the two strands pair up. When that partner strand binds the tagged biomolecule, it lights up, then lets go, causing the biomolecule to "blink" at a precise rate the researchers can control. The researchers use this blinking to obtain ultrasharp images.


To test Exchange-PAINT, the researchers created 10 unique pieces of folded DNA, or DNA origami, that resembled the numerals 0 through 9. These numerals could be resolved with less than 10 nanometers resolution, or one-twentieth of the diffraction limit.


The team was able to use Exchange-PAINT to capture clear images of the 10 different types of miniscule DNA origami structures in one image. They also used the method to capture detailed, ultrasharp images of fixed human cells, with each color tagging an important cellular component — microtubules, mitochondria, Golgi apparatus, or peroxisomes.


Via Dr. Stefan Gruenwald
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Public Domain Collections: Free to Share & Reuse

Public Domain Collections: Free to Share & Reuse | Imaging | Scoop.it
Did you know that more than 180,000 of the items in our Digital Collections are in the public domain? That means everyone has the freedom to enjoy and reuse these materials in almost limitless ways.
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Rescooped by Samantha Lipsky from Stem Cells & Tissue Engineering
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Stem cell injections improve spinal injuries in rats

Stem cell injections improve spinal injuries in rats | Imaging | Scoop.it
Scientists report that a single injection of human neural stem cells produced neuronal regeneration and improvement of function and mobility in rats impaired by an acute spinal cord injury.

Via Jacob Blumenthal
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Jacob Blumenthal's curator insight, May 28, 2013 1:43 AM

Link to the paper "

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation"

http://stemcellres.com/content/4/5/57/abstract

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Super Resolution Microscopy: Improve Your Imaging Don’t Reinvent It

Super Resolution Microscopy: Improve Your Imaging Don’t Reinvent It | Imaging | Scoop.it
Learn how super resolution microscopy can improve your imaging in this webinar.
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The British Library Puts Over 1,000,000 Images in the Public Domain: A Deeper Dive Into the Collection

The British Library Puts Over 1,000,000 Images in the Public Domain: A Deeper Dive Into the Collection | Imaging | Scoop.it
The British Library’s Flickr Commons project presents over 1,000,000 images from the 17th, 18th, and 19th centuries. Microsoft digitized the books represented here, and then donated them to the Library for release into the Public Domain.
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FIONA and other super-high resolution microscopy techniques

FIONA and other super-high resolution microscopy techniques | Imaging | Scoop.it

Methodology designed to circumnavigate the classical Abbe diffraction barrier in optical microscopy is rapidly advancing using both ensemble and single-molecule techniques.

 

Over the past several decades, fluorescence microscopy has become an essential tool for examining a wide variety of biological molecules, pathways, and dynamics in living cells, tissues, and whole animals. In contrast to other techniques (such as electron microscopy), fluorescence imaging is compatible with cells that are being maintained in culture, which enables minimally invasive optical-based observation of events occurring on a large span of timescales. In terms of spatial resolution, several techniques including positron-emission tomography, magnetic resonance imaging, and optical coherence tomography can generate images of animal and human subjects at resolutions between 10 centimeters and 10 micrometers, whereas electron microscopy and scanning probe techniques feature the highest spatial resolution, often approaching the molecular and atomic levels (see Figure ). Between these two extremes in resolving power lies optical microscopy. Aside from the benefits derived from being able to image living cells, the most significant drawback to all forms of fluorescence microscopy (including widefield, laser scanning, spinning disk, multiphoton, and total internal reflection) are the limits to spatial resolution that were first elucidated and described by Ernst Abbe in the late 1800s.

 

The Abbe diffraction limit (or at least the recognition of this limit) stood for almost a century before inventive microscopists began to examine how their instruments could be improved to circumvent the physical barriers in order to achieve higher resolution. Due to the fact that axial resolution is far lower than lateral resolution (by at least a factor of two), much of the work conducted in the latter part of the twentieth century addressed improvements to performance in the axial dimension. Researchers discovered that laser scanning confocal instruments produced very modest increases in resolution at the cost of signal-to-noise, and that other associated technologies (including multiphoton, structured illumination, and spinning disk) could be used for optical sectioning, but without significant improvement in axial resolution. An important concept to note, and one of the most underappreciated facts associated with optical imaging in biology, is that the achieved microscope resolution often does not reach the physical limit imposed by diffraction. This is due to the fact that optical inhomogeneities in the specimen can distort the phase of the excitation beam, leading to a focal volume that is significantly larger than the diffraction-limited ideal. Furthermore, resolution can also be compromised by improper alignment of the microscope, the use of incompatible immersion oil, coverslips having a thickness outside the optimum range, and improperly adjusted correction collars.

 

The most significant advances in superresolution imaging have been achieved in what is termed far-field microscopy and involve either spatially or temporally modulating the transition between two molecular states of a fluorophore (such as switching between a dark and bright state) or by physically reducing the size of the point-spread function used in the excitation illumination. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion; from the Stefan Hell laboratory) and SSIM (saturated structured illumination microscopy; pioneered by Mats Gustafsson). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivated localization microscopy; introduced by Eric Betzig and Harald Hess) and STORM (stochastic optical reconstruction microscopy; first reported by Xiaowei Zhang). As will be discussed, there are many variations on these techniques, as well as advanced methods that can combine or even improve the performance of existing imaging schemes. Even more importantly, new superresolution techniques are being introduced with almost breathtaking speed (relative to traditional advances in microscopy) and it is not unreasonable to suggest that at some point in the near future, resolution of a single nanometer may well be attainable in commercial instruments.


Via Dr. Stefan Gruenwald
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sonia ramos's curator insight, January 9, 2013 2:31 AM

Introducción a la Microscopía de Alta Resolución y técnicas de ultra-alta resolución