Imaging both the position and orientation of single fluorescent molecules attached to DNA. Credit: Maurice Y. Lee / Stanford University
A team from Stanford University (USA) reports developing a new technique that combines super-resolution microscopy with position and orientation measurements of individual molecules. The researchers, led by Nobel Laureate and OSA Fellow William E. Moerner, report obtaining super-resolved images of individual fluorescent dye molecules attached to single DNA strands (Optica, doi: 10.1364/OPTICA.3.000659). The authors say this technique could someday improve scientists’ ability to monitor changes in DNA structure and further their understanding of how these changes can disrupt the cellular processes that affect gene expression.
Improved single-molecule spectroscopy
The enhanced imaging technique is an elaboration on single-molecule spectroscopy, which was developed by Moerner in 1989. Standard single-molecule spectroscopy can be used to image individual DNA strands, but not the orientation or rotational dynamics (i.e., the tilt and wobble) of the fluorescent dye molecules attached to it. This “tilt and wobble” data can be used to characterize how the dye and DNA interact and can provide insight into the DNA strand’s conformation.
Lead author Adam Becker says that he and his colleagues found a way to use “fairly standard tools in a slightly different way” to capture molecular orientation and rotational dynamics. By adding an electro-optic modulator to a single-molecule microscope, the team was able to change the polarization of the fluorescent-dye-activating laser for each camera frame. Molecules that fluctuated between bright and dark in sequential frames were characterized as rigidly constrained to a particular tilt, while molecules whose brightness did not switch on and off in sequential frames were characterized as wobbly.
Enhanced DNA imaging
To demonstrate their approach, the researchers first created super-resolution images of single strands of DNA using two fluorescent dyes: an intercalating dye that attaches between the DNA strand’s base pairs, and a groove-binding dye that consists of an anchor that attaches to the backbone of the DNA strand and a floppy fluorescent tether that hangs off the anchor. By plotting the coordinates of 300,000 single fluorescent-molecule dots detected by the microscope, the researchers were able to reconstruct an image of individual DNA strands with a resolution of 25 nm. (The effect is similar to the pointillism technique used by French post-Impressionist painter Georges Seurat).
Next, the researchers recorded about 30,000 single-molecule orientation measurements that showed that the intercalating dye molecules were oriented perpendicular to the DNA strand’s axis and were rotationally constricted. Conversely, measurements of the groove-binding fluorescent dye molecules were far more wobbly.
The authors say that the combination of super-resolution molecular images with orientation datasets could provide researchers with “unparalleled insight into a multitude of biological and polymeric systems,” as well as a better understanding of the binding modes of different types of DNA fluorescent dyes.