Researchers at Columbia University have made a significant step toward visualizing small biomolecules inside living biological systems with minimum disturbance, a longstanding goal in the scientific community.
In a study published March 2nd in Nature Methods, Assistant Professor of Chemistry Wei Min’s research team has developed a general method to image a broad spectrum of small biomolecules, such as small molecular drugs and nucleic acids, amino acids, lipids for determining where they are localized and how they function inside cells.
When studying biological functions of a molecule in complex and mysterious cells, researchers typically label the molecules of interest with fluorophores, a kind of molecules that glow when illuminated.
Using a fluorescence microscope, common in research labs, the fluorophore-tagged molecules can be located and tracked with high precision. The invention of green fluorescent protein (GFP), in 1994, compatible with imaging inside live cells and animals, has since made fluorescence microscopy even more popular.
However, when it comes to small biomolecules, fluorophore tagging is problematic, because the fluorophores are almost always larger or comparable in size to the small molecules of interest. As a result, they often disturb the normal functions of these small molecules with crucial biological roles.
Raman scattering imaging at a unique frequency
To address this problem, Min and his team departed from the conventional paradigm of fluorescence imaging of fluorophores, and pursued a novel combination of physics and chemistry. Specifically, they coupled an emerging laser-based technique called stimulated Raman scattering (SRS) microscopy with a small but highly vibrant alkyne tag (that is, C=C, carbon-carbon triple bond), a chemical bond that, when it stretches, produces a strong Raman scattering signal at a unique “frequency” (different from natural molecules inside cells).
This new technique, labeling the small molecules with this tiny alkyne tag, avoids perturbation that occurs with large fluorescent tags, while obtaining high detection specificity and sensitivity by SRS imaging. By tuning the laser colors to the alkyne frequency and quickly scanning the focused laser beam across the sample, point-by-point, SRS microscopy can pick up the unique stretching motion of the C=C bond carried by the small molecules and produce a three-dimensional map of the molecules inside living cells and animals.
In this way, Min’s team demonstrated tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors in living cells.
“The major advantages of our technique lie in the superb sensitivity, specificity and biocompatibility with dynamics of live cells and animals for small molecule imaging,” says the lead author Lu Wei, a Ph.D. candidate in chemistry.
Next, Min’s team will apply this new technique to biomedical questions, such as detecting tumor cells and probing drug pharmacokinetics in animal models. They are also creating other alkyne-labeled biologically active molecules for more versatile imaging applications.
“Our new technique will open up numerous otherwise difficult studies on small biomolecules in live cells and animals”, says Min. “In addition to basic research, our technique could also contribute greatly to translational applications. I believe SRS imaging of alkyne tags could do for small biomolecules what fluorescence imaging of fluorophores such as GFP has done for larger species.”
Abstract of Nature Methods paper
Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors.
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Researchers at the the Institute of Photonic Sciences (ICFO) in Catalonia have invented nano-optical tweezers capable of trapping and moving an individual nano-object in three dimensions using the force of light.
“This technique could revolutionize the field of nanoscience since, for the first time, we have shown that it is possible to trap, 3D-manipulate, and release a single nano-object without exerting any mechanical contact or other invasive action,” said Romain Quidant, ICREA Professor and leader at ICFO of the Plasmon Nano-Optics research group.
Invented in Bell Labs in the 80′s, optical trapping demonstrated the capability to trap and manipulate small objects of micrometer-size dimensions using laser light. By shining a laser light through a lens, it is possible to focus light in a tiny spot, creating an attractive force due to the gradient of the light intensity of the laser and thus attracting an object/specimen and maintaining it in the spot/focus.
However, optical tweezers have not been able directly trap objects smaller than a few hundreds of nanometers, such as proteins or nanoparticles, without overheating and damaging the specimen.
A few years ago, ICFO researchers demonstrated that, by focusing light on a very small gold nano-structure lying on a glass surface that acts as a nano-lens, one can trap a specimen at the vicinity of the metal where the light is concentrated. This proof of concept was limited to demonstrate the mechanism but did not enable any 3D manipulation needed for practical applications.
Now researchers at ICFO have taken this a crucial step further by implementing plasmonic nano-tweezers at the send of a mobile optical fiber that is nano-engineered with a bowtie-like gold aperture. Using this approach, they have demonstrated trapping and 3D displacement of specimens as small as a few tens of nanometers using extremely small, non-invasive laser intensity.
Both trapping and monitoring of the trapped specimen can be done through the optical fiber, performing the manipulation of nano-objects in a simple and manageable way outside of the physics research lab.
This technique opens up new research directions requiring non-invasive manipulation of objects at the single molecule/virus level, the researchers say. It is potentially attractive in the field of medicine as a tool to further understand the biological mechanisms behind the development of diseases. It also holds promise for assembling future miniaturized devices, among other potential applications.
“The next steps are to apply our nano tool to concrete problems,” Quidant told KurzweilAI. “We are willing to share our technology with anybody interested in using it for advancing his research. We hope this new technique becomes an universal tool for different kinds of scientists who would need to manipulate nano-objects in a non invasive way.”
This research was supported by the European Research Council through the grant Plasmolight no. 259196 and Fundació privada CELLEX.
Abstract of Nature Nanotechnology paper
Recent advances in nanotechnologies have prompted the need for tools to accurately and non-invasively manipulate individual nano-objects1. Among the possible strategies, optical forces have been predicted to provide researchers with nano-optical tweezers capable of trapping a specimen and moving it in three dimensions2, 3, 4. In practice, however, the combination of weak optical forces and photothermal issues has thus far prevented their experimental realization. Here, we demonstrate the first three-dimensional optical manipulation of single 50 nm dielectric objects with near-field nanotweezers. The nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities. This non-invasive approach is foreseen to open new horizons in nanosciences by offering an unprecedented level of control of nanosized objects, including heat-sensitive biospecimens.