Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Development of a FRET sensor for real-time imaging of intracellular redox dynamics

08.06.2011
In work published in the June 2011 issue of Experimental Biology and Medicine, Kolossov, Spring and their co-investigators - a multidisciplinary team within the Institute for Genomic Biology at the University of Illinois - have transferred the concept of redox-sensitive Green Fluorescent Proteins (GFPs) to a quantitative Förster resonance energy transfer (FRET) imaging platform.

For the FRET-based sensors, a change in redox induces a conformational change in a redox-sensitive switch that links two fluorescent proteins (the donor and acceptor), changing their distance, which in turn causes a detectable change in FRET efficiency.

In its oxidized state the wavelength spectrum of the sensor's fluorescence emission is red-shifted (due to increased acceptor fluorescence), independent of variations in the local sensor concentration or in the intensity of the excitation light. As explained by Robert Clegg, a pioneer in the development of novel applications of optical microscopy in the biological sciences and key collaborator on the study, "FRET-based sensors circumvent the complications associated with imaging methods based on fluorescence intensity, since the increase in the FRET acceptor molecule's fluorescence can only take place if there is a change in the efficiency of energy transfer. This specific and discriminatory feature of FRET is one of the driving motives behind our development of a FRET-based assay rather than relying only on changes in the fluorescent intensity of a single component."

The current publication builds on the authors' previous work, where they reported a series of first-generation redox-sensitive linkers flanked by FRET donor and acceptor GFP-variants. As summarized by co-author Vladimir Kolossov, "The major advance in the current study is an improved dynamic range of the spectroscopic signal; in other words, a greater difference between fully reduced and oxidized states. Increasing the dynamic range leads to better discrimination between the redox states of the probe in complex biological specimens. Furthermore, the highly oxidative midpoint potential of the novel probe is ideal for measuring glutathione redox potentials in oxidative compartments of mammalian cells."

Recently, a different innovative ratiometric probe - a redox-sensitive GFP (roGFP) - has been developed in another lab. The measurement with the roGFP sensor involves the ratio of intensities of two sequential images, acquired at two different excitation wavelengths. Two thiol groups form/break a disulfide bond that modulates the peak excitation wavelength of the roGFP chromophore in response to the redox environment. Bryan Spring, a co-author, notes, "The roGFP and the FRET-based sensors have contrasting characteristics. The FRET-based sensor may prove advantageous for intravital microscopy studies, because only a single laser line is required. In contrast, roGFP requires sequential scanning of two laser lines, which slows the frame rate of image acquisition; also, the images must be compensated for the different laser intensities in order to correct for wavelength-dependent tissue scattering, and the measurement relies on the optical alignment of two excitation light beams. However, the roGFP probe is sensitive to a different range of oxidation-reduction potentials than our FRET probe, possibly leading to complementary applications." Spring adds, "We look forward to further exciting innovations for optimizing the performance of oxidation-reduction-based sensors."

Dr. Rex Gaskins, who led the project remarked, "Distinct advantages of the FRET-based approach include: (1) the ability to quantify the change in redox state; (2) independence of sensor concentration; and (3) modularity, the ability to precisely tune the redox sensitivity and range by exchange of the switch or the fluorophore modules in the probe. We expect that newly developed redox-sensitive probes could potentially be critical to a better understanding of the pharmacologic and toxicological actions of chemotherapeutic drugs and oxidants."

Dr. Steven R. Goodman, Editor-in-Chief of Experimental Biology and Medicine, said "This multidisciplinary group has developed a novel FRET-based biosensor which is a major advance in the measurement of oxidative stress in living cells in real-time. This will allow the measurement of intraorganellar glutathione potentials in living cells".

Experimental Biology and Medicine is a journal dedicated to the publication of multidisciplinary and interdisciplinary research in the biomedical sciences. The journal was first established in 1903.

Experimental Biology and Medicine is the journal of the Society of Experimental Biology and Medicine. To learn about the benefits of society membership visit www.sebm.org. If you are interested in publishing in the journal please visit http://ebm.rsmjournals.com.

Vladimir L. Kolossov | EurekAlert!
Further information:
http://www.illinois.edu

More articles from Life Sciences:

nachricht Rainbow colors reveal cell history: Uncovering β-cell heterogeneity
22.09.2017 | DFG-Forschungszentrum für Regenerative Therapien TU Dresden

nachricht The pyrenoid is a carbon-fixing liquid droplet
22.09.2017 | Max-Planck-Institut für Biochemie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: The pyrenoid is a carbon-fixing liquid droplet

Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.

A warming planet

Im Focus: Highly precise wiring in the Cerebral Cortex

Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.

The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...

Im Focus: Tiny lasers from a gallery of whispers

New technique promises tunable laser devices

Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...

Im Focus: Ultrafast snapshots of relaxing electrons in solids

Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!

When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...

Im Focus: Quantum Sensors Decipher Magnetic Ordering in a New Semiconducting Material

For the first time, physicists have successfully imaged spiral magnetic ordering in a multiferroic material. These materials are considered highly promising candidates for future data storage media. The researchers were able to prove their findings using unique quantum sensors that were developed at Basel University and that can analyze electromagnetic fields on the nanometer scale. The results – obtained by scientists from the University of Basel’s Department of Physics, the Swiss Nanoscience Institute, the University of Montpellier and several laboratories from University Paris-Saclay – were recently published in the journal Nature.

Multiferroics are materials that simultaneously react to electric and magnetic fields. These two properties are rarely found together, and their combined...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

“Lasers in Composites Symposium” in Aachen – from Science to Application

19.09.2017 | Event News

I-ESA 2018 – Call for Papers

12.09.2017 | Event News

EMBO at Basel Life, a new conference on current and emerging life science research

06.09.2017 | Event News

 
Latest News

Rainbow colors reveal cell history: Uncovering β-cell heterogeneity

22.09.2017 | Life Sciences

Penn first in world to treat patient with new radiation technology

22.09.2017 | Medical Engineering

Calculating quietness

22.09.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>