Company > 16 - November - 2015

How Fluorescent Proteins Have Advanced Bio-Imaging Techniques


In this issue:

How Fluorescent Proteins Have Advanced Bio-Imaging Techniques

The 'Journal of Biomedical Optics' recently celebrated the accomplishments of Nobel Laureate Osamu Shimomura, and his contribution to the field of Bio-imaging. His experiments in the 60's lead to the discovery of GFP- Green Fluorescent Protein- in jellyfish, and paved the way for many applications of such proteins in microscopy and live-tissue imaging. His work also provided inspiration to use light-protein interaction in the field of Optogenetics.

Fluorescent proteins are used to tag structures in live cells and tissues, and report on characteristics such as pH and temperature. They allow investigators to apply molecular cloning methods by tagging fluorophores to present enzymes, and then observe cellular activity in living systems using optical microscopy. With technological advancements in wide-field fluorescence and confocal microscopy, including ultrafast digital cameras and multi-tracking laser control systems, the GFP and its color-shifted derivatives have been used in thousands of Bio-imaging experiments. They have enabled researchers to observe processes that were previously invisible - such as the development of nerve cells in the brain, the spread of cancer cells, the spread of Alzheimer's disease in nerve cells and how insulin producing cells are created in the pancreas of an embryo.

Shimomura shared the Nobel Prize in Chemistry 2008 with Martin Chalfie, who successfully introduced the gene for GFP into the DNA of other live organisms, allowing GFP to be used as a luminous genetic tag, and Roger Tsien, who determined how GFP's structure produces green fluorescence, then tweaked the structure to produce molecules that emit light at slightly different wavelengths, creating tags of different colors.

Since then, many fluorescent protein variants have been developed that show fluorescence emission in different colors. The original GFP from jellyfish has variants in blue and yellow that are widely used as in-vivo biological markers. Longer wavelengths emitting proteins in yellow, orange and red spectrum have been developed from other marine species. A major development was the creation of red-emission fluorescent proteins. Since red light penetrates tissue more effectively than green, these proteins can be used to image entire organs.

Key attributes of a usable red fluorescent protein (RFP) are brightness, photo-stability and utility in fusions. The first RFP was DsRed, which was characterized and cloned from sea anemone. Improvements of DsRed variants over the years lead to the formation of mRFP1- the very first monomeric red fluorescent protein, and the 'mFruit' series - a group of six new monomeric RFPs exhibiting emission maxima ranging from 540 nm to 610 nm. These new FPs were named mHoneydew, mBanana, mOrange, mTangerine, mStrawberry, and mCherry (the 'm' referring to monomer), referencing the common fruits that bear colors similar to their respective emission profiles. These were direct derivatives of mRFP1, and have contributing significantly to multi-color bio-imaging techniques. mCherry exhibits high intrinsic brightness and photostability, so it is the preferred choice for cellular imaging.

The series has since expanded to include mRaspberry and mPlum- derivatives reached through a process known as Somatic Hypermutation (SHM). At present, there are over 70 different derivatives of the original proteins- each carefully crafted for a particular function.

Researchers continue their efforts to develop proteins that would function as reliable biomarkers and subsequently aid physicians to track progression of disease and effectiveness of medical treatments.

Sources:

1. 'Protein Photonics special section in Journal of Biomedical Optics honors Nobel Laureate Osamu Shimomura' - SPIE: Online Link-SPIE

2. 'The fluorescent protein palette: tools for cellular imaging'- Richard N Day and Michael Davidson: Online Link-NCBI Resources

3. 'Introduction to Fluorescent Proteins'- Nikon MicroscopyU: Online Link-Microscopy U

4. 'mCherry Fluorescent Proteins' - Clontech: Online Link-Clontech


Raman Spectroscopy Enables Early Detection of Dengue & West-Nile Viruses

Researchers at University of Southern Mississippi and Jackson State University have demonstrated a probe that is capable of highly sensitive detection of the Dengue and West-Nile viruses in a sample. The probe is created using principles of Surface Enhanced Raman Spectroscopy (SERS probe) and is bio-conjugated with gold nanoparticles. Laserglow's 671 nm LabSpec-DPSS laser was used as an excitation source.

Dengue fever and West-Nile Virus disease collectively account for over a thousand deaths annually in the world. The single-stranded RNA viruses of dengue and West-Nile virus that are transmitted by mosquitoes belong to the 'Flavivirus' family. 4G2 is a type of Anti-Flavivirus antibody, and is conjugated to the gold nanoparticles (AuNP) in the setup of the SERS probe. The probe can now be used as a Raman fingerprint for the detection of these viruses in a biological sample.

Researchers observed that due to a phenomenon known as plasmonic coupling, the probe was able to detect quantities as low as 10 plaque-forming units of DENV-2 and WNV per mL of sample. To ensure the probe works only for the 2 specified viruses, a strain of chikunguniya (CHIKV) was used as a negative control. The huge SERS intensity observed was attributed to strong electric field enhancement once the probe was introduced to the viruses. The researchers are confident that AuNP-4G2-based SERS probe will have great potential in screening viral particles in clinics and diagnostic centers around the world.

The research was first published in The Journal of Physical Chemistry on Sept 22, 2015. To learn more about the experimental setup, read the full article on: Journal of Physical Chemistry Full Paper


Lasers Can Make Materials Hotter Than the Sun

New research suggests that lasers can heat up materials to temperatures higher than the center of the sun in only 20 quadrillionth of a second. This could provide new avenues of research in thermonuclear fusion energy, as scientists work to replicate the sun's ability to create clean energy.

The method, proposed by Theoretical Physicists at Imperial College, London, directly heats up the ions in certain materials. This is different from usual laser-heating setups where electrons are first to be excited by a laser and hence, take longer as the energy passes from electrons to ions-which make up the bulk of the matter. The researchers discovered that when a high-intensity laser was fired at a certain type of material, it created an electrostatic shockwave that heated up the ions directly. Due to the way ions are arranged in the material, the shockwave caused them to accelerate at different speeds - this caused friction, which in turn complimented the process to gain heat rapidly.

The technique could prove to be the fastest heating rate ever demonstrated in a lab for a large number of particles. To learn more, go to: Phys.org full article

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