Daily BulletinDaily Bulletin

The Conversation

  • Written by The Conversation
imageMultiple fluorescent proteins illuminate the cells in a human brainstem.Jeff Lichtman/Harvard University, CC BY-NC-ND

When you look up at the blue sky, where are the stars that you see at night? They’re there but we can’t see them. A firefly flitting across a field is invisible to us during the day, but at night we can easily spot its flashes. Similarly, proteins, viruses, parasites and bacteria inside living cells can’t be seen by the naked eye under normal conditions. But a technique using a fluorescent protein can light up cells' molecular machinations like a microscopic flashlight.

imageThe crystal jellyfish has about 300 photo organs on the bottom edge of the jellyfish’s umbrella.Courtesy Steven Haddock – http://biolum.eemb.ucsb.edu, Author provided

The first fluorescent protein found in nature comes from the crystal jellyfish, Aequorea victoria, where it is responsible for the green light emitted by its photo organs. It’s called green fluorescent protein (GFP). We don’t know why these jellyfish have this lit-up feature.

Fluorescent proteins absorb light with short wavelengths, such as blue light, and immediately return it with a different color light that has a longer wavelength, such as green. In Aequorea victoria, a protein named aequorin produces blue light which GFP converts into the green light emitted by the jellyfish’s photo organs. This visibility under standard conditions is extremely rare; most other organisms have fluorescent proteins that are only visible if they are illuminated by external blue light sources. imageClose up of a few of the photo organs.Courtesy Steven Haddock – http://biolum.eemb.ucsb.edu, Author provided

After the green fluorescent jellyfish protein, many other fluorescent proteins have been both found in nature and created in the lab. We now have a spectrum of fluorescent colors available to us that make previously invisible biological structures and processes visible in blazing fluorescent glory. Many new applications reliant on these colors are being published on a regular basis. imagePetri dish with bacterial colonies expressing differently colored fluorescent proteins. These fluorescent proteins developed by Roger Tsien’s group are called the mFruits and have names like mHoneydew, mTomato, mCherry, mRaspberry, and mPlum.Paul Steinbach and Roger Y. Tsien, University of California, San Diego, CC BY-SA

Shining a light on imaging

Fluorescent protein technology has led to many other interesting developments designed to improve imaging with these glowing molecules.

CaMPARI is one new technique, short for calcium-modulated photoactivatable ratiometric integrator. By exploiting the fact that calcium concentrations change when nerve cells send signals, CaMPARI is able to light up all the neurons that have fired in a living organism. The technique is based on a fluorescent protein called EOS, which changes its fluorescence from green to red. In fruit flies, zebrafish and mice, CaMPARI-genetically-modified neurons fluoresce red if they are active and green if they are less active.

imageCaMPARI fluorescence in a larval zebrafish brain showing active neurons (magenta) that were marked while the fish was swimming freely.Looger Lab (HHMI/Janelia), Science, VOL 347, ISSUE 6223.

Before CaMPARI, all the fluorescent calcium indicators available temporarily lit up when the neuron fired. They couldn’t record the firing history of neurons or indicate whether a neuron had fired in the past. According to Loren Looger, one of the researchers who worked on the development of CaMPARI, “The most enabling thing about this technology may be that you don’t have to have your organism under a microscope during your experiment. So we can now visualize neural activity in fly larvae crawling on a plate or fish swimming in a dish.”

The CLARITY technique removes opaque parts and makes the whole brain transparent.

Expanding and transparent brains

Even with the help of light emitted by fluorescent proteins, it’s difficult to image neurons tangled deep within the brain. Ed Boyden, a neuroscientist from MIT, has created a method to expand brains to make fluorescent neurons deep within the brain more visible. He uses acrylate, which forms a dense mesh to hold the brain in place and expand in the presence of water thereby inflating the brain equally by about 4.5 times in each direction. It’s a lot like a diaper expanding when it gets wet. Boyden thinks that this “expansion microscopy may provide a key tool for comprehensive, precise, circuit-wide, brain mapping.”

imageIntact adult mouse brain before and after the CLARITY process.The Deisseroth Lab

One of the reasons expansion microscopy is so useful is that the brain can be made see-through before it is blown up several sizes larger. In 2013 Karl Deisseroth and Viviana Gradinaru at Stanford published a method called CLARITY that removes opaque molecules such as fats and makes the brain transparent without changing its shape. According to Thomas Insel, director of the US National Institute of Mental Health, “This is probably one of the most important advances for doing neuroanatomy in decades.” Since developing CLARITY for brains, Gradinaru has extended the method to all other organs including an entire mouse.

Both of these methods can be applied to brains that have been genetically modified with fluorescent proteins, therefore allowing for the visualization of neurons deep within the brain.

imageMouse neurons labeled by GFPs.Wellcome Images, CC BY-NC-ND

In 2008, the three scientists responsible for taking GFP from the jellyfish and making it a common tool used in over a million experiments all over the world were awarded the 100th Nobel Prize in chemistry. And in 2014 three other scientists were awarded the Nobel Prize for using fluorescent protein to increase the resolution of light microscopes.

imageE. coli with GFPs glowing in their petri dishes.Carlos de Paz, CC BY-NC-SA

Revolutionary and resilient

I’ve been researching the photochemistry and photophysics of fluorescent proteins since they were first used in imaging technology in 1994, I’ve written two books on them, and still I’m stunned by the many different ways in which this fairly simple protein can be used. Perhaps I shouldn’t be surprised that plasmid DNA molecules coding for GFP have survived space flight – not inside the rocket, but on the outside where they were exposed to 1800F (1000C) temperatures and mad friction. 53% of the DNA intentionally placed inside the screw heads in the TEXUS-49 rocket mission expressed fully fluorescent GFP when inserted into cells upon return to earth.

Like stars at night, fluorescent proteins have been lighting up science for the last 20 years. And it won’t be long before they’re guiding surgeons to tumorous growths during surgery and allowing researchers to switch on and off selected biomolecular processes.

Marc Zimmer receives funding from NIH.

Authors: The Conversation

Read more http://theconversation.com/fluorescent-proteins-light-up-science-by-making-the-invisible-visible-39272

Australia's smallest fish among 22 at risk of extinction within two decades


A 3-decade 'moving picture' of young Australians' study, work and life, thanks to LSAY


In COVID's shadow, global terrorism goes quiet. But we have seen this before, and should be wary


The Conversation


Did BLM Really Change the US Police Work?

The Black Lives Matter (BLM) movement has proven that the power of the state rests in the hands of the people it governs. Following the death of 46-year-old black American George Floyd in a case of ...

a Guest Writer - avatar a Guest Writer

Scott Morrison: the right man at the right time

Australia is not at war with another nation or ideology in August 2020 but the nation is in conflict. There are serious threats from China and there are many challenges flowing from the pandemic tha...

Greg Rogers - avatar Greg Rogers

Prime Minister National Cabinet Statement

The National Cabinet met today to discuss Australia’s COVID-19 response, the Victoria outbreak, easing restrictions, helping Australians prepare to go back to work in a COVID-safe environment an...

Scott Morrison - avatar Scott Morrison

Business News

Link Building Secrets - Comprehensive Guide

Link building has proven to be an effective approach when it comes to promoting your online website. Let's analyze the topic of developing an effective link building strategy for site promotion ...

Julia Smith - avatar Julia Smith

What to Expect from Your NDIS Verification & Certification Audit

The National Disability Insurance Agency administers NDIS (National Disability Insurance Scheme) in Australia. The NDIS Quality and Safeguards Commission governs it. As a welfare support scheme of...

Sarah Williams - avatar Sarah Williams

Why You May Need A Tower Scaffold Hire

When constructing a building, or even a multilevel structure, you must use a tower scaffold to get you into position. What is unique about this type of scaffolding is that you can build it highe...

News Company - avatar News Company

News Company Media Core

Content & Technology Connecting Global Audiences

More Information - Less Opinion