FLUORESCENCE IMAGING: Probe permanently marks neurons engaged during specific activity

A new fluorescent protein lets scientists shine a light on an animal's brain to permanently mark neurons that are active at a particular time.1 Called CaMPARI (calcium-modulated photoactivatable ratiometric integrator), it converts from green to red when calcium floods a nerve cell after the cell fires. The permanent mark frees scientists from the need to focus a microscope on the right cells at the right time to observe neuronal activity.

Calcium-sensitive fluorescent molecules called GCaMP can indicate neural activity, and are useful for following the dynamics of neural networks. But their signal is temporary, and if researchers miss it because the microscope is not focused on the right spot, the information is lost. Developed at the Howard Hughes Medical Institute's Janelia Research Campus, CaMPARI enables visualization beyond a microscope's field of view, capturing neural activity across wide swaths of brain tissue. And in many cases it can be used while animals move freely, rather than being confined to a dish or embedded in agar. "The most enabling thing about this technology may be that you don't have to have your organism under a microscope during your experiment," says Loren Looger, a group leader and protein chemist at Janelia.

In this larval zebrafish brain, neurons that were active while the fish was swimming freely were permanently marked in magenta. (Image courtesy of the Looger Lab, HHMI/Janelia)

Eric Schreiter, a senior scientist in Looger's lab, led the development of CaMPARI-working as part of Janelia's Genetically-Encoded Neuronal Indicator and Effector (GENIE) interdisciplinary project team. The team started with a protein called Eos, which fluoresces in green until exposed to violet light—at which point it permanently alters to fluoresce in red. "That conversion from green to red gives us a permanent signal," said Schreiter. "So we just needed a way to couple that conversion to the activity that's going on in the cell." To do that, the scientists incorporated a calcium-sensitive protein known as calmodulin, which makes the color change dependent on the burst of calcium that accompanies neural activity.

To find a useful protein that switches the color of its fluorescence only in the presence of both calcium and violet light, the researchers made and screened tens of thousands of subtly different proteins. "When we finally got one that photoconverted more with calcium than without it, we knew we had a tool. We just needed to make it better to get it to the point where another neuroscientist could sit down and use it," says Ben Fosque, a graduate student in the biochemistry and molecular biophysics program at the University of Chicago.

The use of violet light gives experimenters control over the time period during which neural activity is tracked. "Ideally, we can flip the light switch on while an animal is doing the behavior that we care about, then flip the switch off as soon as the animal stops doing the behavior," Schreiter explains. "Then, we're capturing a snapshot of only the activity that occurs while the animal is doing that behavior."

Various experiments have demonstrated CaMPARI's effectiveness. For instance, the researchers captured a snapshot of neuronal activity over the entire brain volume of a zebrafish during a 10-second period as it swam in a dish. Following the experiment, CaMPARI was red in motor neurons known to be involved in swimming, and other expected sets of neurons—consistent with observations made by other scientists during electrophysiology experiments. The activation patterns changed significantly when the researchers altered the temperature or turbulence of the water.

The scientists expect future versions of CaMPARI will be more sensitive and reliable, but "the idea is probably more powerful than the tool, as it stands right now," Looger says. To get it into the hands of other labs, the team has made the genetic plasmid encoding CaMPARI available through the plasmid repository Addgene, transgenic flies expressing CaMPARI are available through the Bloomington Drosophila Stock Center, and Janelia group leader Misha Ahrens is distributing CaMPARI-expressing zebrafish to researchers. Tools for introducing CaMPARI into mouse cells should be available soon, they say.

1. B. F. Fosque et al., Science, 347, 6223, 755–760 (2015).

Get All the BioOptics World News Delivered to Your Inbox

Subscribe to BioOptics World Magazine or email newsletter today at no cost and receive the latest news and information.

 Subscribe Now
Related Articles

Blood becomes a laser emitter for drug testing, cancer treatment

Combining laser light with an FDA-approved green fluorescent dye can monitor cell structure and activity at the molecular level.

New bioimaging technique offers clear view of nervous system

Scientists at Ludwig-Maximilians University have developed a technique for turning the body of a deceased rodent entirely transparent, revealing the central nervous system in unprecedented clarity....

Fluorescent jellyfish proteins light up unconventional laser

Safer lasers to map your cells could soon be in the offing -- all thanks to the humble jellyfish. Conventional lasers, like the pointer you might use to entertain your cat, produce light by emittin...

Fluorescence microscopy helps provide new insight into how cancer cells metastasize

By using fluorescence microscopy, scientists have discovered an alternate theory on how some cancer cells metastasize.


Neuro15 exhibitors meet exacting demands: Part 2

Increasingly, neuroscientists are working with researchers in disciplines such as chemistry and p...

Why be free?

A successful career contributed to keeping OpticalRayTracer—an optical design software program—fr...

LASER Munich 2015 is bio-bent

LASER World of Photonics 2015 included the European Conferences on Biomedical Optics among its si...

White Papers

Understanding Optical Filters

Optical filters can be used to attenuate or enhance an image, transmit or reflect specific wavele...

How can I find the right digital camera for my microscopy application?

Nowadays, image processing is found in a wide range of optical microscopy applications. Examples ...



Twitter- BioOptics World

Copyright © 2007-2016. PennWell Corporation, Tulsa, OK. All Rights Reserved.PRIVACY POLICY | TERMS AND CONDITIONS