Advances in microscopy have allowed cell biologists to catalogue many subcellular processes in great detail. Optical microscopes are even capable of imaging and tracking single molecules. However, the weak signals of single molecules and complications like autofluorescence, out-of-focus fluorescence, or secondary excitation can make discerning these individual molecules from the background impossible.

Total Internal Reflection Fluorescence (TIRF) microscopy is a form of sophisticated light microscopy that makes it possible to see very faint individual molecules by greatly reducing this background, and Dr. Steven Magennis’ group at the University of Glasgow has been working to push the limits of the technology even further.1 Dr. Magennis’ lab specializes in single molecule techniques — probing the chemical or physical properties of individual molecules, such as in DNA and proteins.2,3 Among a number of interdisciplinary tools, they’ve used TIRF microscopy to enhance their ability to see molecular dynamics.

How TIRF works

Figure 1: In TIRF microscopy, light is delivered to the glass/sample interface at the critical angle, which causes light to be refracted away from the sample in a way that illuminates only the surface of what is being imaged. Dr. Magennis and colleagues refined way the light is delivered, creating a precise way of visualizing single molecules.


In traditional fluorescence microscopy, fluorescence emitters above and below the focal plane are excited, resulting in a hazy background of out-of-focus fluorescence that obscures the feature of interest.

However, with TIRF, light is directed at the sample in such a way that excitation light is confined to an evanescent field that only illuminates the ~100nm of sample nearest to the cover slip. This creates very little background from the surrounding solution, and almost all the fluorescence collected is in focus and usable.

Combining TIRF with multiphoton laser excitation leads to further potential improvements, notably further enhancements in signal-to-background, reduced photo bleaching, and simultaneous non-resonant excitation of multiple fluorophores.

However, delivering the 10–15 fs pulses required for efficient multiphoton excitation is difficult in TIRF setups due to dispersion of the light as it transits through the various components in the optical path.

That’s where Dr. Magennis and his group have made major improvements. They used “pulse shaping” of a Titanium:Sapphire laser to precisely deliver photons in time and space to the samples for imaging. To demonstrate efficacy, they imaged quantum dots with and without the pulse shaping. The quantum dots were placed on a slide that included both aggregates and single quantum dots. Without the pulse shaping the aggregates could be visualized, but not the individual dots. However, with the pulse shaping enhancement, single quantum dots could be visualized. This delivery method enhanced signal by nearly an order of magnitude.1

Increased signal means that previously unresolved particles or molecules can now be detected and resolved at higher resolution. Pulse shaping provides researchers with a powerful tool to understand cellular mechanisms, create new treatments, and test therapeutics in action.

As the high-resolution data is created, it’s equally important to have the right system capable of collecting single photon data. The Magennis group selected a Photometrics EMCCD camera (Evolve 512) and a dual-channel imaging device (DualView) based on their ability to reliably detect the single molecule signal. The Evolve 512 is critical to the efficacy of the imaging system, as its extreme sensitivity is necessary for discerning the weak signal that can be one of the limiting factors in high-speed single-molecule imaging.

In combination with total internal reflection fluorescence (TIRF) microscopy, the Evolve 512 has allowed us to routinely detect and analyze the fluorescence from single molecules, providing long-time dynamic information on immobilized molecules and particles.” (Dr. Magennis).4

Magennis’ refined multiphoton TIRF signal is akin to precisely shining a flashlight on only those molecules that are of interest in a cell. This allows researchers to visualize molecular interactions in real time with the Photometrics Evolve camera without a hazy background obscuring the process.

Although Dr. Magennis and his group have previously used conventional TIRF for studying DNA and enzyme dynamics, multiphoton TIRF microscopy could offer an improved method to study the mechanisms and dynamics of a large variety of biomolecular reactions at the single molecule level.

1. Lane, R.S.K, Macpherson, A.N., and Magennis, S.W. (2012) Signal enhancement in multiphoton TIRF microscopy by shaping of broadband femtosecond pulses. Optics Express20(23):25948–59.
2. Sabir, T., Toulmin, A., Ma, L., Jones, A.C., McGlynn, P., Schröder, G. F., and Magennis, S. W. (2012) Branchpoint expansion in a fully-complementary three-way DNA junction, J. Am. Chem. Soc. 134, 6280-5.
3. Pudney, C. R., Lane, R. S. K., Fielding, A. J., Magennis, S. W., Hay, S., and Scrutton, N. S. (2013) Enzymatic single-molecule kinetic isotope effectsJ. Am. Chem. Soc. 135:3855–64.
4. Dr. Steven Magennis. (2014) Single Molecule and TIRF. Photometrics Customer Reference.