A team of Harvard chemists led by X. Sunney Xie has developed a new microscopic technique for seeing, in color, molecules with undetectable fluorescence. The room-temperature technique allows researchers to identify previously unseen molecules in living organisms and offers broad applications in biomedical imaging and research.
The scientists' results are published in the Oct. 22 issue of Nature. Partial funding for the project was provided by the National Science Foundation (NSF).
Fluorescence is a phenomenon in which an electron in a molecule absorbs energy from light and moves to a higher energy level or excited state. The energy of the light is contained in a unit called a photon.
After a very brief stay at the excited state, the electron returns to its previous energy level, or ground state, by emitting a new photon. The energy of the released photon is discharged in wavelengths of detectable visible light lasting only a few billionths of a second.
Many biologically important colored molecules such as hemoglobin--an oxygen-transport protein in red blood cells--absorb light but do not fluoresce. Instead, the electrons in these molecules release their additional but transient energy by converting it to heat.
"Since these molecules do not fluoresce, they have literally been overlooked by modern optical microscopes," Xie said.
To detect non-fluorescent molecules in biological systems, Xie and his team developed a new type of microscopy based on stimulated emission.
Stimulated emission was first described by Albert Einstein in 1917, and was the basis for today's lasers. In a nutshell, it is a process by which an excited-state electron, perturbed by a photon having the correct energy, drops to its ground state producing an additional photon.
Xie's new microscopic technique generates and records a stimulated emission signal by using two carefully timed input and output pulse trains. In the input pulse train, a modulator switches the intensity of the excitation beam on and off at five megahertz, or MHz. The modulation creates a stimulated emission signal at the same frequency. Each train has an incredibly short pulse duration of approximately 200 femtoseconds. A femtosecond is equal to one billionth of one millionth of a second or 10-15 seconds.
The signal produced by the non-fluorescent molecules provides a highly sensitive image of previously "invisible" molecules.
One of the several possible applications of the scientists' invention is mapping in color the delivery of non-fluorescent drugs to their target cells. Another possible use is imaging tiny structures such as blood vessels including individual red blood cells and single capillaries (see images).
The structure and hemoglobin-dynamics of blood vessels play a major role in many biomedical processes. Two example processes are the transition of tumors from a dormant to malignant state and oxygen delivery in the brain.
Current established imaging technologies like MRIs and CT scans either lack the spatial resolution needed to resolve individual capillaries or require external contrast agents.
Fluorescent labels such as the green fluorescent protein, or GFP, are extensively used for observing the activity of biomolecules and distinguishing target molecules in a cell. The GFP labeling technique provides well-defined images. However, the bulky protein can disturb delicate biological pathways, especially when it is larger than the biomolecules it is illuminating.
Xie's team mapped the delivery of a non-fluorescent drug molecule and imaged blood vessels without fluorescent labels.
Their new technique is also capable of imaging non-fluorescent proteins in cells of live Escherichia coli bacteria.
"While earlier studies made use of similar pump-probe experiments to provide images of fluorescent molecules with spatial resolution comparable to that of confocal fluorescence microscopy and high temporal resolution, this study, for the first time, makes use of stimulated emission microscopy to image non-fluorescent molecules," said Zeev Rosenzweig, a program director in the NSF Division of Chemistry.
Although potential photo-damage, and the complexity and cost of the system still need to be addressed for the technique to gain wide applicability, "there is no doubt that the study provides a unique way to image a wide range of molecules currently inaccessible to today's state-of-the-art optical microscopes," notes Rosenzweig.
"This is just the beginning," added Xie. "Many interesting applications of this new imaging modality are forth coming."
Additional authors of the Nature paper include Wei Min, Sijia Lu, Shasha Chong, Rahul Roy and Gary R. Holtom. Min and Roy are postdoctoral fellows; Lu and Chong are graduate students; and Holtom is a research scientist. All authors are members of Xie's research group.
Funding for the research was provided by the NSF Division of Chemistry, award number CHE-0634788, and the U.S. Department of Energy's Basic Energy Sciences Program, award number DE-FG02-07ER15875.
Jennifer A. Grasswick | EurekAlert!
Closing the carbon loop
08.12.2016 | University of Pittsburgh
Newly discovered bacteria-binding protein in the intestine
08.12.2016 | University of Gothenburg
In recent years, lasers with ultrashort pulses (USP) down to the femtosecond range have become established on an industrial scale. They could advance some applications with the much-lauded “cold ablation” – if that meant they would then achieve more throughput. A new generation of process engineering that will address this issue in particular will be discussed at the “4th UKP Workshop – Ultrafast Laser Technology” in April 2017.
Even back in the 1990s, scientists were comparing materials processing with nanosecond, picosecond and femtosesecond pulses. The result was surprising:...
Have you ever wondered how you see the world? Vision is about photons of light, which are packets of energy, interacting with the atoms or molecules in what...
A multi-institutional research collaboration has created a novel approach for fabricating three-dimensional micro-optics through the shape-defined formation of porous silicon (PSi), with broad impacts in integrated optoelectronics, imaging, and photovoltaics.
Working with colleagues at Stanford and The Dow Chemical Company, researchers at the University of Illinois at Urbana-Champaign fabricated 3-D birefringent...
In experiments with magnetic atoms conducted at extremely low temperatures, scientists have demonstrated a unique phase of matter: The atoms form a new type of quantum liquid or quantum droplet state. These so called quantum droplets may preserve their form in absence of external confinement because of quantum effects. The joint team of experimental physicists from Innsbruck and theoretical physicists from Hannover report on their findings in the journal Physical Review X.
“Our Quantum droplets are in the gas phase but they still drop like a rock,” explains experimental physicist Francesca Ferlaino when talking about the...
The Max Planck Institute for Physics (MPP) is opening up a new research field. A workshop from November 21 - 22, 2016 will mark the start of activities for an innovative axion experiment. Axions are still only purely hypothetical particles. Their detection could solve two fundamental problems in particle physics: What dark matter consists of and why it has not yet been possible to directly observe a CP violation for the strong interaction.
The “MADMAX” project is the MPP’s commitment to axion research. Axions are so far only a theoretical prediction and are difficult to detect: on the one hand,...
16.11.2016 | Event News
01.11.2016 | Event News
14.10.2016 | Event News
08.12.2016 | Life Sciences
08.12.2016 | Physics and Astronomy
08.12.2016 | Materials Sciences