Getting to the bottom of single-molecule spectroscopy
Gary Boas
Single-molecule spectroscopy uncovers information typically obscured
by ensemble averaging in other methods. Widely used single-molecule techniques include
epifluorescence, confocal and total internal reflection microscopy. Each of these
has advantages as well as disadvantages, pertaining specifically to signal-to-background
ratio and parallel detection capabilities.
A group at the University of Bayreuth in Germany
uses each of the techniques in its studies of organic macromolecular systems such
as polymers and proteins. In the May issue of
Journal of Microscopy, the
researchers evaluated the three with respect to single-molecule spectroscopy. “For
our future work, it was advantageous to compare the pros and cons of the three methods
quantitatively,” explained researcher Jürgen Köhler, “taking
into account the trade-off between the parallelization of data acquisition and superior
background suppression.”
Scientists have evaluated three techniques used for single-molecule
spectroscopy, comparing the signal-to-background ratio offered by each. To ensure
identical experimental conditions, they used an optical setup that can accommodate
all three of the techniques. Shown here is a schematic for the part of the setup
used for epifluorescence and confocal microscopy (left) and for total internal reflection
microscopy (right).
Optimization of the signal-to-background
ratio is crucial in single-molecule spectroscopy because the background signal climbs
as excitation intensities rise, after the fluorescence signal has leveled off. Epifluorescence
microscopy optically isolates individual molecules against a relatively large illuminated
area, relying on strong fluorophores to produce a fluorescence signal significantly
higher than the background signal. Confocal microscopy more efficiently reduces
the background signal by limiting the excitation light to a diffraction-limited
volume of less than 1 μm
3, but it is a sequential technique that can probe
only one molecule at a time.
Total internal reflection microscopy
enables parallel detection of multiple molecules, but sometimes at the expense of
background suppression as compared with confocal microscopy. Total internal reflection
microscopy creates an evanescent light field between two dielectric media, limiting
the contributions of sample outside the media to the background signal. Here, though,
violations of the conditions necessary for total internal reflection microscopy
— caused by cracks in the sample, for instance — can lead to significantly
lower signal-to-background ratio.
The investigators compared the signal-to-background
obtained with the three microscopy modes, measuring the same perylene bisimide molecule
under otherwise identical experimental conditions. They used an optical setup that
could accommodate all three modes. The excitation light was provided by a tunable
single-mode dye laser made by Coherent Inc. of Dieburg, Germany, that was pumped
by a separate argon-ion laser.
In epifluorescence and confocal mode,
the light was transmitted into a home-built microscope and then into a cryostat
containing the sample; in total internal reflectance mode, it was directed through
the rear window of the cryostat. The cryostat helps to maintain low temperatures,
where the effects of photobleaching are less of an issue than at higher, ambient
temperatures. Switching between the modes took only a few moments and was achieved
with hardware that moved components into and out of the optical path.
In confocal mode, a 0.90-NA objective
made by Microthek of Hamburg, Germany, focused the light to a 410-nm spot; in epifluorescence
mode, an additional lens defocused the light to an area of roughly 50 x 50 μm.
In all three modes, the objective collected fluorescence emitted by the sample and
sent it to an electron multiplying CCD camera made by Andor Technology of Belfast,
Northern Ireland.
The experiments showed that it is possible
to achieve signal-to-background ratios of ~16 for epifluorescence, ~70
for confocal and ~40 for total internal reflectance microscopy. The researchers
noted that the significantly lower ratio for epifluorescence microscopy was not
surprising because this technique has the largest sample area, which contributes
to the background signal. Total internal reflectance microscopy yielded a higher
ratio, but this is mitigated by the potential for error caused by even slight deviations
from the necessary conditions.
Method of choice
Of the three, confocal microscopy offered a significantly
higher signal-to-background ratio than either of the other modes, prompting the
researchers to describe it as “clearly the method of choice” for sequential,
single-molecule spectroscopy experiments, particularly under cryogenic conditions.
They noted, moreover, that the findings also apply to experiments performed under
ambient conditions. Experiments with several other molecules backed up this assertion.
However, there are still cases in which
researchers will want to use epifluorescence or total internal reflection microscopy,
for example, if they are tracking the movements of molecules. Parallel detection
also will help to increase statistical significance when they are acquiring spectroscopic
data.
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