(Image caption: Engineers have developed a new microscopy method that uses a fine needle or cannula and an LED light to make 3-D images. They hope this new microscope technology, shown here, can be implanted into the brains of mice to show images of cells. Credit: Ganghun Kim, University of Utah)

3-D Microscope Method to Look Inside Brains

A University of Utah team discovered a method for turning a small, $40 needle into a 3-D microscope capable of taking images up to 70 times smaller than the width of a human hair. This new method not only produces high-quality images comparable to expensive microscopes, but may be implanted into the brains of living mice for imaging at the cellular level.

The study appears in the Aug. 18 issue of the journal Applied Physics Letters.

Designed by Rajesh Menon, an associate professor of electrical and computer engineering, and graduate student Ganghun Kim, the microscope technique works when an LED light is illuminated and guided through a fiberoptic needle or cannula. Returned pictures are reconstructed into 3-D images using algorithms developed by Menon and Kim.

“Unlike miniature microscopes, our approach does not use optics,” Menon says. “It’s primarily computational.”

He says this approach will allow researchers not only to take images far smaller than those taken by current miniature microscopes, but do it for a fraction of the cost.

“We can get approximately 1-micron-resolution images that only $250,000 and higher microscopes are capable of generating,” Menon says. “Miniature microscopes are limited to the few tens of microns.”

Menon hopes to extend the technology in the future so it can see details down to submicron resolutions, compared with the current 1.4 microns. (A micron is a millionth of a meter. A human hair is about 100 microns wide.)

The microscope was originally designed for the lab of Nobel Prize-winning U human genetics professor, Mario R. Capecchi, whose team will use it to observe the brains of living mice to gain insight into how certain proteins in the brain react to various stimuli. Because the microscope can be assembled so inexpensively and easily go into hard-to-reach places, Menon and Kim expect many other uses for the device.

“This microscope will open up new avenues of research,” Menon says. “Its low-cost, small-size, large field-of-view and implantable features will allow researchers to use this in fields ranging from biochemistry to mining.”

Neuroimaging: Live from inside the cell

A novel imaging technique provides insights into the role of redox signaling and reactive oxygen species in living neurons, in real time. Scientists of the Technische Universität München (TUM) and the Ludwig-Maximilians-Universität München (LMU) have developed a new optical microscopy technique to unravel the role of “oxidative stress” in healthy as well as injured nervous systems. The work is reported in the latest issue of Nature Medicine.

Reactive oxygen species are important intracellular signaling molecules, but their mode of action is complex: In low concentrations they regulate key aspects of cellular function and behavior, while at high concentrations they can cause “oxidative stress”, which damages organelles, membranes and DNA. To analyze how redox signaling unfolds in single cells and organelles in real-time, an innovative optical microscopy technique has been developed jointly by the teams of LMU Professor Martin Kerschensteiner and TUM Professor Thomas Misgeld, both investigators of the Munich Cluster for Systems Neurology (SyNergy).

“Our new optical approach allows us to visualize the redox state of important cellular organelles, mitochondria, in real time in living tissue” Kerschensteiner says. Mitochondria are the cell’s power plants, which convert nutrients into usable energy. In earlier studies, Kerschensteiner and Misgeld had obtained evidence that oxidative damage of mitochondria might contribute to the destruction of axons in inflammatory diseases such as multiple sclerosis.

The new method allows them to record the oxidation states of individual mitochondria with high spatial and temporal resolution. Kerschensteiner explains the motivation behind the development of the technique: “Redox signals have important physiological functions, but can also cause damage, for example when present in high concentrations around immune cells.”

First surprises
Kerschensteiner and Misgeld used redox-sensitive variants of the Green Fluorescent Protein (GFP) as visualization tools. “By combining these with other biosensors and vital dyes, we were able to establish an approach that permits us to simultaneously monitor redox signals together with mitochondrial calcium currents, as well as changes in the electrical potential and the proton (pH) gradient across the mitochondrial membrane,” says Thomas Misgeld.

The researchers have applied the technique to two experimental models, and have arrived at some unexpected insights. On the one hand, they have been able, for the first time, to study redox signal induction in response to neural damage – in this case, spinal cord injury – in the mammalian nervous system. The observations revealed that severance of an axon results in a wave of oxidation of the mitochondria, which begins at the site of damage and is propagated along the fiber. Furthermore, an influx of calcium at the site of axonal resection was shown to be essential for the ensuing functional damage to mitochondria.

Perhaps the most surprising outcome of the new study was that the study’s first author, graduate student Michael Breckwoldt, was able to image, also for the first time, spontaneous contractions of mitochondria that are accompanied by a rapid shift in the redox state of the organelle. As Misgeld explains, “This appears to be a fail-safe system that is activated in response to stress and temporarily attenuates mitochondrial activity. Under pathological conditions, the contractions are more prolonged and may become irreversible, and this can ultimately result in irreparable damage to the nerve process.”

Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies.

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The gears that help cells divide are coming into clearer focus. Researchers have used a new type of super-resolution microscopy to zoom in on centrosomes, which anchor the fibers that enable chromosomes to separate during cell division. Centrosomes have intrigued scientists since their discovery in the late 1800s, in part because cancer cells often amass extra copies of the structures. But they’re so tiny that they’re barely visible through traditional light microscopes, and researchers haven’t nailed down how they form and what role they play in cancer. So cell biologist David Glover of the University of Cambridge in the United Kingdom and his postdoc Jingyan Fu turned to three-dimensional structured illumination microscopy to provide sharper portraits of centrosomes and to pinpoint several proteins they harbor. Each centrosome consists of two cylindrical components called centrioles shrouded by a molecular cloud, which balloons when cells start the process of division. As the team reveals online today in Open Biology, many of the cloud proteins first gather on the centrioles, moving into the cloud once division begins. That’s the case with the protein Cnn (green), shown above close to the cylindrical centriole (top) and dispersed in the cloud (bottom, inset). With further research, scientists might be able to determine how different proteins interact to construct centrosomes. “We can put the molecular jigsaw together,” Glover says.