Search Menu
Photonics Media Photonics Buyers' Guide Photonics EDU Photonics Spectra BioPhotonics EuroPhotonics Vision Spectra Photonics Showcase Photonics ProdSpec Photonics Handbook
More News

Light plus flow make gradients grow

Facebook Twitter LinkedIn Email Comments
Michael J. Lander

To understand how cancer cells spread or how white blood cells track down invaders, scientists must figure out how they respond to chemical stimuli. If the concentration of a compound is stronger at one end of a cell than at the other, for example, within seconds its machinery may initiate movement into or out of the chemical gradient. But determining the exact nature and timing of a cell’s reaction can be challenging because gradients can take minutes to establish in the lab. What researchers would require for more accurate assessment is a system that can switch a defined gradient on and off very quickly.

Carsten Beta, Eberhard Bodenschatz and colleagues at Max Planck Institute for Dynamics and Self-Organization in Göttingen, Germany, at Cornell University in Ithaca, N.Y., and at the University of California, San Diego, have designed a cell stimulation device in which caged molecules flowing through a chamber are released by light, spreading out to form a gradient and then making contact with cells attached to the bottom of the chamber downstream.

Researchers illuminated a triangular region (red) of a microfluidic channel to release caged fluorescent molecules flowing through it and measured intensity across the profile downstream (yellow line), as seen in the top image. Plots generated from the measurements, on the bottom, represent concentration profiles at low (red) and high (blue) flow speeds. Courtesy of Carsten Beta, Max Planck Institute for Dynamics and Self-Organization.

As a foundation for the setup, the researchers used a long microfluidic channel produced with a soft lithographic technique. Inlet and outlet holes were punched in the trough, which was sealed with a glass coverslip.

Into the chamber, the scientists introduced a suspension of Dictyostelium discoideum cells and allowed them to attach to the channel floor. The cells had GFP bound to their cytosolic regulator of adenylyl cyclase. When such cells are exposed to cyclic adenosine monophosphate (cAMP), the regulators are recruited to the plasma membrane, resulting in an increase of fluorescence at the membrane and a decrease of it in the cytosol.

Next, the scientists flowed a solution of photosensitive caged cAMP molecules through the chamber. To measure the cells’ response, they used an inverted confocal laser scanning microscope from Olympus Corp. of Tokyo equipped with an additional scanning unit. The scanning unit exposed a small circular area of the channel upstream of the cells to a beam from a 405-nm solid-state laser diode. This induced photochemical release of a portion of the caged molecules, making them biologically active. With the additional scanner, photoactivation could be initiated inside the field of view but independent of the imaging lasers.

Images revealed that sections of the cells located downstream of wide regions of laser exposure fluoresced most strongly. By comparison, parts of the cells aligned with illuminated zones that were less extended in the direction of the flow glowed dimly. This indicated that the laser-scanned area’s form related closely to the distribution of the released chemical.

To better characterize the gradient, the researchers flowed fluorescein carrying the same photosensitive caging group through the channel in an analogous experiment. Fluorescence intensity measurements taken across the channel downstream of the circular laser-excited region showed that a steady gradient formed within 0.8 s.

By comparing the intensity values with the fluorescence from a solution in which all fluorescein had been uncaged, the scientists estimated the molecule’s relative concentration across the gradient and found that it assumed a Gaussian distribution. This allowed them to approximate cAMP’s concentration profile in the cell experiment as well, because the molecules diffuse at a similar rate.

Moving the illuminated region enabled the researchers to shift the gradient profile completely in less than a second. By changing the scanned pattern’s shape to a triangle, they created a linear profile. In fact, they found that areas of many forms could be illuminated to create exponential and other gradient profiles. Increasing the intensity of the light source or the length of the illuminated area in the direction of flow permitted up to 100 percent uncaging.

Although systems that use diffusion-based techniques can produce gradients with similar profiles, they lack temporal resolution that is on the order of seconds. As Bodenschatz noted, this means that the device can deliver signals to the cell at a faster time scale than its internal responses. The fluorescent signal detected at the cells’ surface, for example, appeared and decayed within 30 s. Constant flow in the system also prevents the buildup of cellular waste.

“Many cellular mechanisms are conserved between Dictyostelium and higher organisms,” Beta explained, meaning that the results of this and further studies could apply to humans as well. Using the system to image the response of cancer cells to chemical gradients could help explain cancer metastasis. And although the availability of photoactivatable compounds limits the technology’s range of applications, improved preparation processes could make a larger selection of the chemicals available in the near future.

Analytical Chemistry, May 15, 2007, pp. 3940-3944.

Jun 2007
Basic ScienceBiophotonicscellschemicalsenergymicrofluidic channelMicroscopymoleculesNews & Features

back to top
Facebook Twitter Instagram LinkedIn YouTube RSS
©2019 Photonics Media, 100 West St., Pittsfield, MA, 01201 USA,

Photonics Media, Laurin Publishing
x Subscribe to BioPhotonics magazine - FREE!
We use cookies to improve user experience and analyze our website traffic as stated in our Privacy Policy. By using this website, you agree to the use of cookies unless you have disabled them.