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Peering “under the hood” of a live cell

Jan 2013

URBANA, Ill. – A fluorescence extension of a spectroscopy method originally developed for label-free imaging is providing valuable information about the machinery of a live cell.

The new technique, dispersion-relation fluorescence spectroscopy (DFS), is an extension of dispersion-relation phase spectroscopy (DPS).

“DPS relies on quantitative phase imaging, which is an emerging approach for studying cells and tissues and, unlike fluorescence microscopy, uses no contrast agents,” Gabriel Popescu of the University of Illinois’ Beckman Institute told BioPhotonics. DPS is label-free, but “DFS uses fluorescence, which can provide information that is specific only to the structure that fluoresces.”

The new method studies the critical process of cell transport dynamics at multiple spatial and temporal scales.

“DFS allows us to distinguish between random (diffusive) and deterministic (active) transport and the spatial and temporal scales at which each is dominant,” said Popescu, leader of the university’s Quantitative Light Imaging Laboratory. “For example, studying the machinery of a live cell, one must understand the balance between these two components.”

Dispersion-relation fluorescence spectroscopy of mouse embryonic fibroblast: (a) fluorescence image showing a cell whose actin was labeled with green fluorescent protein; (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). The inset shows the dispersion map. The DFS method, developed at the Beckman Institute, is proving valuable for studying the mechanics of a live cell. Courtesy of Gabriel Popescu

The main advantage of DFS over fluorescence correlation spectroscopy, an established method for studying transport based on information obtained at a single point, is that DFS “studies transport at both small and large spatial scales from fractions of microns to hundreds of microns,” Popescu said. “It is precisely this ability that allows us to differentiate between the two types of transport.

Ru Wang, a researcher in Popescu’s lab, added that, in terms of temporal resolution, the method “is able to study both fast dynamics – for example, red blood cell membrane fluctuations – which is in the range of milliseconds, and slow dynamics,” which, in the case of neuron network formation, would take weeks.

“Imagine that we set a small drop of ink in a cup of water: Due to thermal diffusion, the ink tends to disperse uniformly in water. The time for the particles to reach the cup wall is relatively long,” Popescu explained. “Now imagine that a cell is trying instead to capture all the ink particles, one by one, and send them along strict paths that reach the edge fast. Instead of a continuous cloud of particles, the cell creates distinct ‘highways.’

“This form of transport is much more efficient, yet the process of organizing transport is exhausting. The cell must continuously produce energy to accomplish this task,” he said. Neurons experience a similar process, with organelles and vesicles permanently carried from the cell body to axons and dendrites and vice versa.

“Remarkably, we measured that diffusion is always subdominant in this case; i.e., neurons do not have time for such a disorganized form of transport,” Popescu said. “They prefer to consume energy and get the job done faster.”

The method relies on taking time-resolved sequential data from fluorescent spectroscopic microscopy images and changing them via Fourier transform. This computational technique 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.

The researchers used DFS to focus on the cell cytoskeleton subunit actin, measuring its motions.

“Actin and microtubules are two critical components of cytoskeleton,” Popescu said. “Both are fiber structures in the cell, along which cargo can slide, much as cars along highways. We found that the highways themselves are in continuous motion and that there is an active type of motion along the fibers and random across the fibers.”

The information gained could be valuable for basic cellular dynamics science, drug research and studies of Alzheimer’s disease. It also can be used with current fluorescence microscopy methods.

The technique is described in Physical Review Letters (doi: 10.1103/PhysRevLett.109,188104.

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...
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