By Ben Mezick and Eric Drier, Mad City Labs Inc.
Many advanced microscopy techniques are vulnerable to experimental and measurement parameters impacting their quantitative sensitivity. For example, complex ratiometric measurements, such as single molecule fluorescence (or Förstner) resonance energy transfer (smFRET), are dependent on many factors — encompassing chemistry, instrumentation, and analysis choices — that can affect sensitivity and, by extension, the ability to discern meaningful quantitative information from the data.
In this article we examine, via the example of smFRET, the role of excitation volume and signal-to-noise ratio (SNR) and their impact on advancing this technique’s quantitative ability. We also discuss prudent instrumentation choices that can aid in this task. smFRET is used “to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules,” revealing information about the structural dynamics of proteins, DNA, and lipid membranes.
There are two methods of delivering TIR excitation in the context of fluorescence microscopy: prism-based and objective lens-based. Mad City Labs’ RM21 MicroMirror TIRF Microscope uses an objective based method. In this instance, rather than the typical dichroic filter intervening at the back plane of the objective lens to deliver the desired excitation light, it employs micromirrors to deliver the excitation light. This method gives the user control over both angle and focus of each individual excitation wavelength, which is essential to achieving uniform excitation volume and, ultimately, leads to more quantitative control over all these measurements.
A distinguishing feature of this method is that the excitation light is spatially separated from the emission, or detected, light. This has implications for improving the signal-to-noise ratio of the measurements.