Nobel Laureate unveils new super-resolution microscopy method
New method images position and orientation of single fluorescent molecules attached to DNA. [Maurice Y. Lee, Stanford University]
Stanford University researchers, led by Professor William Moerner, have enhanced super-resolution microscopy to show the orientation of individual fluorescent molecules, probing the structure of single DNA strands to 25 nm resolution.
The simple, high-throughput method measures azimuthal orientations and rotational dynamics of single fluorescent molecules, which is compatible with localisation microscopy.
Crucially, Moerner and colleagues have used the method to obtain super-resolved images of thousands of single fluorescent dye molecules attached to DNA strands, studying the structure of the strand while characterising dye-DNA interactions.
“You can think of these new measurements as providing little double-headed arrows that show the orientation of the molecules attached along the DNA strand,” explains Moerner.
“This orientation information reports on the local structure of the DNA bases because they constrain the molecule," he adds. "If we didn’t have this orientation information the image would just be a spot.”
To understand how a fluorescent dye molecule attaches to the DNA strand, Moerner's colleague, Adam Backer, developed a simple way to obtain orientation and rotational dynamics from thousands of single molecules in parallel.
The method is based on a well-studied technique that adds an electro-optic modulator to the single-molecule microscope; for each camera frame, this device changes the polarisation of the laser light used to illuminate all the fluorescent dyes.
Fluorescent dye molecules with orientations closely aligned with the laser light’s polarisation will appear brightest, so measuring the molecule brightness in each camera frame allows the researchers to quantify orientation and rotational dynamics.
Molecules that switched between bright and dark in sequential frames were rigidly constrained at a particular orientation while those that appeared bright for sequential frames were not rigidly holding their orientation.
“If someone has a single-molecule microscope, they can perform our technique pretty easily by adding the electro-optic modulator,” highlights Backer. “We’ve used fairly standard tools in a slightly different way and analysed the data in a new way to gain additional biological and physical insight.”
The new technique offers more detailed information than today’s so-called “ensemble” methods, which average the orientations for a group of molecules.
What's more, it is much faster than confocal microscopy techniques, which analyse one molecule at a time, and can even be applied to relatively dim molecules.
The researchers used the enhanced DNA imaging technique to analyse an intercalating dye; a fluorescent dye that slides into the areas between DNA bases.
In a typical imaging experiment, they acquired up to 300,000 single molecule locations and 30,000 single-molecule orientation measurements in a little more than 13 minutes.
Analyses revealed that the individual dye molecules were oriented perpendicular to the DNA strand’s axis and that while molecules tended to orient in this perpendicular direction, they also moved around within a constrained cone.
The investigators next performed a similar analysis using a different type of fluorescent dye that consists of two parts: one part that attaches to the side of the DNA and a fluorescent part that is connected via a floppy tether.
The enhanced DNA imaging technique detected this 'floppiness', showing that the method could help researchers understand, on a molecule by molecule basis, whether different labels attach to DNA in a mobile or fixed way.
The researchers demonstrated a spatial resolution of around 25 nm and single-molecule orientation measurements with an accuracy of around 5 degrees.
They also measured the rotational dynamics, or floppiness, of single-molecules with an accuracy of about 20 degrees.
The researchers reckon the new method could be useful for monitoring DNA conformation changes or damage to a particular region of the DNA, which would show up as changes in the orientation of dye molecules.
It could also be used to monitor interactions between DNA and proteins, which drive many cellular processes.
Research is published in Optica.