Photonics Spectra BioPhotonics Vision Spectra Photonics Showcase Photonics Buyers' Guide Photonics Handbook Photonics Dictionary Newsletters Bookstore
Latest News Latest Products Features All Things Photonics Podcast
Marketplace Supplier Search Product Search Career Center
Webinars Photonics Media Virtual Events Industry Events Calendar
White Papers Videos Contribute an Article Suggest a Webinar Submit a Press Release Subscribe Advertise Become a Member


Fluorescent Label Aids Whole-Brain Imaging In Vivo

A new permanent fluorescent label allows researchers to study complex neural activity in wide swaths of brain tissue in moving animals.

Developed at the Howard Hughes Medical Institute, the label frees scientists from the need to use a microscope to observe neuronal activity. Called CaMPARI, for calcium-modulated photoactivatable ratiometric integrator, the label has been tested in living mice, fruit flies and zebrafish.

“The most enabling thing about this technology may be that you don’t have to have your organism under a microscope during your experiment,” said Dr. Loren Looger, a group leader and protein chemist at HHMI’s Janelia Research Campus. “So we can now visualize neural activity in fly larvae crawling on a plate or fish swimming in a dish.”


CaMPARI fluorescence in a larval zebrafish brain showing active neurons (magenta) that were marked while the fish was freely swimming. Courtesy of the Looger Lab, HHMI/Janelia.


To make CaMPARI, the team started with a fluorescent protein called Eos, which fluoresces in green until it is exposed to violet light, which permanently alters it to fluoresce in red.

“That was the perfect starting place,” said senior scientist Dr. Eric Schreiter. “That conversion from green to red gives us a permanent signal. 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 the calcium-sensitive protein calmodulin, which makes the color change dependent on the burst of calcium that accompanies neural activity.

The researchers made and screened tens of thousands of subtly different proteins to find one that switched color only in the presence of both calcium and violet light.

“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,” said University of Chicago graduate student Ben Fosque, the first author of a paper on the findings published in Science (doi: 10.1126/science.126092).

Using violet light to trigger the red fluorescence 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 said. “Then we’re capturing a snapshot of only the activity that occurs while the animal is doing that behavior.”

The scientists conducted a series of experiments to demonstrate CaMPARI’s effectiveness. In one set of experiments, they captured a snapshot of neuronal activity over the entire brain volume of a zebrafish during a 10-s period as it swam in a dish. Following the experiment, CaMPARI was red in motor neurons known to be involved in swimming, as well as 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.

In fruit flies, the team used CaMPARI to identify neurons that were activated in response to specific odors. Here too, the observations were as expected based on previous experiments: CaMPARI indicated that different odors activated distinct sets of neurons in the flies’ antennal lobes.

The researchers also experimentally activated the neurons that directly responded to the odors, then looked for neurons elsewhere in the brain that subsequently turned red. Those experiments revealed neurons that Schreiter said may be secondary, tertiary or even quaternary components to the olfactory circuits.

The Janelia teams is now sharing CaMPARI with other researchers to see how its use can be expanded and improved.

For more information, visit www.janelia.org.

Explore related content from Photonics Media




LATEST NEWS

Terms & Conditions Privacy Policy About Us Contact Us

©2024 Photonics Media