#two-photon imaging

[cstoltzfus]

Here is my first paper! It is all about making fluorescent proteins specifically for two-photon microscopes. To make these new fluorescent proteins we screened thousands of randomly mutated versions of the popular green fluorescent protein, EGFP. The hard part was imaging the two-photon excited fluorescence of these thousands of mutants. We had to build a special imaging setup that used a high-pulse-energy femtosecond laser to image 10 cm Petri dishes. A few hundred colonies of E. coli were grown on each Petri dish we imaged. In each colony of E. coli a different mutated version of EGFP was expressed. This allowed us to look at the two-photon excited fluorescence of about 10,000 mutants a day.

[houseofmind]

How does the brain get rid of its waste?

The current issue of Science features an interesting perspective article titled Garbage Truck of the Brain that talks about mechanisms underlying the clearance of cellular debris. 

The author makes an excellent point:

Essentially all neurodegenerative diseases are associated with misaccumulation of cellular waste products. 

Of these, misfolded or hyperphosphorylated proteins are among the most difficult for the brain to dispose. For example, tau and β-amyloid can accumulate as stable aggregates that are neurotoxic in conditions such as Alzheimer's disease...

To discover underlying mechanisms of waste clearance in the brain, researchers have implanted a window into the skull of mice and used two-photon imaging techniques that allow them to directly observe the movemement of cerebrospinal fluid (CSF) through the living brain! 

The researchers found that the brain itself is surrounded by a membrane called the arachnoid and bathed in cerebral spinal fluid (CSF). CSF flows into the interior of the brain through the same pathways as the arteries that carry blood and is drawn into brain tissue via a system of conduits that are controlled by astrocytes ( a type of glia). The CSF is flushed through the brain tissue at high speeds, which sweeps away excess "garbage" that might be located in the brain. The fluid and waste are exchanged with a similar system that parallels veins which carries the waste out of the brain and down the spine where it is eventually transferred to the lymphatic system and from there to the liver, where it is ultimately broken down.The scientists decided to coin this newly discovered system the "glymphatic system."

Convective glymphatic fluxes of CSF and ISF propel the waste products of neuron metabolism into the paravenous space, from which they are directed into lymphatic vessels and ultimately return to the general circulation for clearance by the kidney and liver.

CREDIT: K. SUTLIFF/SCIENCE

  Source: 

Maiken Nedergaard. (2013). Garbage truck of the brain. Science. 340: 1529-30.