Background
Characteristics of biological molecules can be determined by tagging an RNA or protein molecule on the surface of a microscope slide with fluorophores. These fluorophores emit light upon laser excitation, but by conventional means, background fluorescence can interfere with the desired fluorescent signal. In order to improve the signal-to-background ratio, total internal reflection fluorescence microscopy (TIRFM) was developed here at the University of Michigan by Daniel Axelrod. The sample is illuminated by laser light that undergoes total internal reflection on the microscope slide. While incoming laser light photons bounce off the slide surface, they also have wave properties that create an evanescent field that penetrates the surface and decays exponentially with distance. Therefore, only samples within ~100 nm of the surface are illuminated, greatly reducing background fluorescence. Different techniques have been developed using TIRFM. Two examples that this lab uses regularly are FRET and FIONA.
FRET
Förster resonance energy transfer (aka fluorescence resonance energy transfer or FRET) is a phenomenon that takes place when you have two specific fluorophores in close proximity to one another. Via dipole-dipole coupling, a “donor” fluorophore (ex. Cy3) may excite an “acceptor” fluorophore (ex. Cy5) granted the two are within ~100 Å of each other. Because the amount of energy transfer is dependent on the distance between the fluorophores, FRET may be used as a ruler to give you information on the distance between two fluorophores. This technique is being used in the lab to study ribozymes, a riboswitch, the ribosome, and the spliceosome.
FIONA
Constrained by the Rayleigh criterion, the best optical resolution that we may obtain is ~250 nm. However, the position of the fluorophore can be determined with low nanometer precision by locating the center of the unresolved spot using a 2D Gaussian fit. This “super-resolution” method was first known as fluorescence imaging with one-nanometer accuracy (FIONA), but now has blossomed into a whole bouquet of “pointillism” super-resolution techniques recognized by a Nobel prize in 2014. By using this method over time, a trajectory of the fluorophore’s movement may be monitored making this a useful technique to use for the spider project and other sub-diffraction single particle tracking applications.
Combining these techniques has allowed us to detect and digitally count RNA, DNA and protein molecules at the single molecule level, down to attomolar concentrations in complex biofluids, leading a former student, me and colleagues to found a biotech startup company based on kinetic fingerprinting and digital counting of single biomarker molecules of disease, as illustrated in the following graphic.
