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 μm3, 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.