- Multiplying electrons yields multiplexed results
In the past, researchers studying molecular
dynamics could learn a lot about a little but had trouble seeing the big picture.
For example, they could track the flow of molecules but only at about four spots
on a membrane at a time. That made it difficult to fully understand the process.
Now researchers from the National University
of Singapore and from the Genome Institute of Singapore have shown that a system
that uses an electron-multiplying CCD camera can produce good results while allowing
some 200 to 300 measurements in parallel. National University assistant chemistry
professor Thorsten Wohland said that they used the camera because it allows multiplexing
and because its high quantum efficiency and good signal-to-noise ratio produce a
The methods of choice for capturing
molecular dynamics are fluorescence correlation and cross-correlation spectroscopy.
In the former, researchers measure fluorescence intensity fluctuations that can
arise from fluorophore diffusion into and out of a small excitation volume or that
can be caused by chemical reactions involving the fluorophore. Those fluorescence
changes can provide information about diffusion coefficients, molecular interactions,
flow rates and, in biological systems, membrane dynamics.
The fluorescence signal from the focal volume of a laser can be detected
by a 3 x 3-pixel area of the electron-multiplying CCD. With a pixel size of 16 x
16 μm, this area is about the size of a 50-μm-diameter pinhole used in
standard fluorescence correlation spectroscopy under similar conditions. By autocorrelating
the signal from the laser focus (left), one can determine flow speed, and by cross-correlating
the signal from pixels around the laser focus, one can determine flow directions.
The instruments for fluorescence correlation
spectroscopy typically employ avalanche photodetectors or photomultipliers as detectors.
Such devices are fast, with an acquisition time of less than 0.2 ms. However, they
typically have, at most, only four detectors, meaning that measurements can be made
only in four spots simultaneously. Although it is possible to sequentially scan
different points to cover, for example, an entire cell, that method isn’t
ideal because the molecular situation can change during that span.
The big drawback to electron-multiplying
CCDs is their acquisition time, Wohland noted. Fluorescence correlation spectroscopy
requires measurements to be taken about 10 times faster than the slowest process,
and the CCDs have acquisition times in the tens of milliseconds. That’s too
slow for studies of molecular dynamics, but the researchers were able to speed that
up considerably by exploiting aspects of the technology.
As detailed in the May 15 issue of
Analytical Chemistry, they used a camera from Photometrics of Tucson, Ariz.,
which had an 8.2-mm square sensor consisting of a 512 x 512 array of 16-μm
square pixels. The acquisition time for the full array was more than 30 ms, but
the researchers took advantage of the camera’s capabilities to process the
data faster for a smaller region of interest. By reducing the region of interest
to a 20 x 20 array, they decreased the acquisition time to 4 ms, which Wohland noted
was an important threshold.
“The fast acquisition rate of
down to 4ms is just right to allow us to measure membrane diffusion, which is on
the order of tens to hundreds of milliseconds, when using a diffraction-limited
spot,” he said.
The rest of their setup consisted of
a Zeiss inverted epifluorescence microscope, a helium-neon laser from Melles Griot,
and a dichroic mirror and emission filter from Omega. The investigators sent the
543-nm beam from the laser through optics to a focal volume, collected the resulting
fluorescence using the same optics, filtered out the emission and captured it with
the back-illuminated electron-multiplying CCD sensor, which had a better than 90
percent quantum efficiency from 500 to 650 nm. The fluorescent spot covered a 3
x 3-pixel array of the CCD.
Wohland noted that having the focal
volume spanned by multiple detector array elements opened the possibility of extracting
additional information. For example, by autocorrelating the signal, the researchers
could determine flow speed. By doing the same for pixels around that point, they
could measure flow direction and movement, something not possible with conventional
fluorescence correlation spectroscopy instruments because of the symmetry of the
detection volume. The camera also allows for software binning and, thus, the determination
of the correlation for different detection pinhole sizes, all from a single initial
measurement. “This could allow the distinction of normal diffusion versus
anomalous diffusion,” he said.
For their demonstration, the researchers
performed fluorescence correlation spectroscopy on the fluorescent dye Atto565 and
0.01-μm fluorescent polystyrene beads in a highly viscous fluid that was 80
percent glycerol. Both fluorophores had excitation peaks near the 565-nm wavelength
and emission peaks about 30 nm higher. They also performed fluorescence correlation
spectroscopy on Chinese hamster ovary cells, targeting the growth factor receptors
on the surface. They labeled these with a red fluorescent protein.
For flow measurements, they used a
microchannel ~380 μm wide and 300 μm high. By varying the rate of
a syringe pump, they created different flow velocities, which they measured using
For comparison, they used a standard
fluorescence correlation spectroscopy instrument. They did see some differences
in the number of particles detected and in the diffusion time for the fluorescent
dye and beads. These, they explained, were due to differences between the two systems
with regard to laser coupling into the optics, magnification and pinhole size. The
two sets of measurements behaved the same, though, in terms of the effect of changes
in concentration and other parameters.
Using software binning, they demonstrated
multiplexing. Simulations showed that hundreds of detection volumes were possible,
although in practice the resulting acquisition time might be too long and the resulting
temporal resolution too large.
As for cell measurements, the new technique
yielded diffusion times comparable to those obtained in a similar set of previously
measured cells. Both measurement groups, however, exhibited large variations. Wohland
said that these could be caused by differences across the membrane, about which
the new system might be able to reveal more information by simultaneously measuring
diffusion coefficients over the whole cell.
Having demonstrated that the technique
works, the group is applying it to biological samples for diffusion and flow measurements.
There are some aspects of the hardware, however, that Wohland would like to see
improved. He would like to get better time resolution on larger subregions of the
camera, allowing determination of correlation functions for an entire cell at once
and reducing any photodamage problems. He’d also like to speed up data transfer,
with the goal of perhaps eventually allowing online measurements.
When using multiple laser foci,
crosstalk between the observation volumes makes it necessary to space the foci sufficiently
far apart. On the left is an overlay of nine measurements. With a distance of 10
pixels, each binned 3 x 3, crosstalk does not lead to any influence on the measured
diffusion time (blue dashed line represents the fast decay of correlation amplitudes
with increasing distance from the laser focus). However, the amplitude is still
influenced by intensity crosstalk (red solid line), which contributes to the uncorrelated
background. As indicated by the colored squares, the more crosstalk present, the
lower the amplitude. Corner points are least influenced, while the center point
reaches only 40 percent of the expected amplitude.
Finally, he would like to see the crosstalk
between various confocal volumes minimized. The need to space volumes far apart
hinders the efficiency of the setup. “Most of the camera is actually unused,”
Wohland said. “If crosstalk can be reduced, a much larger part of the camera
could be used.”
Contact: Thorsten Wohland, National University of Singapore; e-mail: email@example.com.
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