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Fluorescence Microscopy Given New Power
Nov 2012
URBANA, Ill., Nov. 5, 2012 —  A new method that studies the critical process of cell transport dynamics at multiple spatial and temporal scales has revealed properties of diffusive and directed motion transport in living cells.

Using dispersion-relation fluorescence spectroscopy (DFS), researchers at the University of Illinois’ Beckman Institute report an approach that labels molecules of interest with a fluorophore whose motion gives rise to spontaneous fluorescence intensity fluctuations that are analyzed to quantify the governing mass transport dynamics. The data are characterized by the effective dispersion relation, they say.

Gabriel Popescu, leader of the university’s Quantitative Light Imaging Laboratory, said the multiplicity of scales the method offers over techniques like fluorescence correlated spectroscopy (FCS) is key.

“It’s like looking from the moon at a highway system in North America and you’re trying to understand the traffic,” he said. “There are so many paths and some cars are moving fast, some slow, some over short distances, some over large distances, and all of these things are happening at the same time. We are actually able to break that information down to these simple pieces that seem to represent a universal behavior for all the cells we measured.

Dispersion-relation fluorescence spectroscopy of mouse embryonic fibroblast (MEF): (a) fluorescence image showing a cell whose actin was labeled with GFP. (b) Dispersion curve measured for the cell in (a). The black and red lines indicate directed motion (along the actin filaments) and diffusion (perpendicular to the actin filaments). Inset shows the dispersion map. Courtesy of Gabriel Popescu.

“With local measurements, it’s actually difficult to measure all these complexities because you only have one point of measurement. That’s why we tried to search for a better way that also uses the spatial information of that traffic. I think we now have solved it,” Popescu said.

“I think that the beauty of this method is that you can use a commercial fluorescence microscope that is found everywhere to collect and analyze data in a very simple way,” said Ru Wang, a researcher in Popescu’s lab. “You don’t need complicated expertise. Everyone can use it.”

The technique relies on taking time-resolved sequential data from fluorescent spectroscopic microscopy images and changing them via Fourier transform. This computational method makes it easier to understand the image data, providing a different representation of the image. It takes advantage of the respective frequency domains of patterns in the data, which is useful for understanding cellular dynamics such as transport.

“It turns out the laws of physics are actually best described in the frequency domain,” Popescu said. “The dispersion relation in all branches of physics connects spatial scales with temporal scales. For example, as things get smaller in space, in length if you like, they tend to move faster. A fly will move faster than an elephant.

“This dispersion relation tells you how much faster. If I make something twice as small, is it going to move twice as fast, or four times or eight times? This relationship basically tells you everything about that dynamic phenomenon. So for the first time we saw this universal transport behavior in a living system: a clear combination of diffusive transport, like Brownian motion, and directed, deterministic transport. As a general trend, we found that diffusion is dominant at short scales and directed transport at large distances.”

They have also used the method to study neurons in work with the Center for Emergent Behaviors of Integrated Cellular Systems (EBICS) at Illinois, a multi-university project aimed at building living, multicellular machines that address real-world problems. The revelations regarding directed versus diffusive transport could be especially useful in reaching that goal.

“The fact that we can tell where the deterministic and the random transport appears is actually very relevant for looking at cells as a machine,” Popescu said. “What makes a cell machine is actually this directed component because you cannot predict with accuracy Brownian motion, but you can predict this directed motion.”

This ability to study the directed and diffusive transport characteristics is more comprehensive than using just fluorescence microscopy. It also provides more information than existing methods such as FCS, which is used in the study of molecular transport and diffusion coefficients at a fixed spatial scale.

The DSF technique enabled the scientists to focus on the cell cytoskeleton subunit actin, where they found that the fluorescently labeled actin cytoskeleton exhibits active transport motion along a direction parallel to the fibers and diffusive on the perpendicular direction. Those results describe at what scale and when directed versus diffusive motion takes place within the cell.

“For the first time, we think we’re able to tell those apart and the spatial scales at which each is dominant,” Popescu said.

“Some traditional methods are good at measuring local transport, and some are good at measuring the larger scales,” Wang said. “Our method gives a fuller view of what happens inside the cell to the patterns of traffic. So we can look at both the local scale and at the larger scales and ask at which scale the motion transitions from random to directed motion.”

The information derived from this method could be valuable for researchers interested in the basic science of cellular dynamics or for those working in biomedical research in areas such as investigating the effects of drugs on the body, Popescu said. It can also be used with current fluorescence microscopy methods.

The technique is described in Physical Review Letters (doi: 10.1103/PhysRevLett.109.188104).

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fluorescence correlation spectroscopy
A powerful method, referred to as FCS, for determining the average diffusion coefficients of fluorescent molecules in solution or membranes. FCS measurements rely on recording the transition of several thousands of molecules through the focal volume. The combination of short measurement times along with free positioning or scanning of the observation spot makes FCS an excellent tool for investigating diffusion heterogeneity over time and space.
fluorescence microscopy
Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction. 
Material that emits fluorescence.
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
AmericasBeckman Institutebiomedical researchbiophotonicscell cytoskeletoncell transport dynamicscellular dynamicsDFSdiffusion coefficientsdiffusive motion transportdirected motion transportdispersion-relation fluorescence spectroscopyenergyFCSfixed spatial scalefluorescence correlation spectroscopyfluorescence microscopyfluorophoreFourier transformGabriel PopescuIllinoisimage dataimagingMicroscopymolecular transportphotonicsResearch & TechnologyRu Wangspatial scalesspontaneous fluorescencetemporal scalestime-resolved sequential dataUniversity of Illinois

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