Novel Technique Opens Door to Super Resolution of Single Molecules in Cells

Researchers report that they have adapted a technique called DNA-PAINT to confocal microscopes to allow them to investigate molecules deep inside cells. They describe DNA-PAINT as a molecular imaging technology to localize fluorescent dyes.

The team says it is now able to visualize a variety of different molecules, including combinations of different proteins, RNAs, and DNA throughout the entire depth of whole cells at high resolution. The study (“Multiplexed 3D Super-Resolution Imaging of Whole Cells Using Spinning Disk Confocal Microscopy and DNA-PAINT”), published in Nature Communications, could open the door for detailed single-molecule localization studies in many areas of cell research, according to the scientists.

“Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells,” write the investigators.

“We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.”

The DNA-PAINT approach attaches a DNA “anchor strand” to the molecule of interest. Then a dye-labeled DNA “imager strand” with a complementary sequence transiently attaches to the anchor and produces a fluorescent signal, which occurs as a defined blinking event at single molecular sites. Because “blinking” is precisely tunable, molecules that are only nanometers apart from each other can be distinguished at the higher resolution end of super-resolution.

“Our new approach, SDC-PAINT, integrates the versatile super-resolution capabilities of DNA-PAINT with the optical sectioning features of confocal microscopes. We thus created the means to explore the entire depth of a cell and to visualize the molecules within it at the nanometer scale,” says Ralf Jungmann, Ph.D., a professor at the Ludwig Maximilian University (LMU) and the Max Planck Institute (MPI) of Biochemistry in Germany.

The team mapped out the presence of different combinations of protein within whole cells and then went beyond that. “By diversifying our labeling approaches, we also visualized different types of individual biomolecules in the chromosome-containing nucleus, including sequences in the DNA, proteins bound to DNA or the membrane that encloses the nucleus, as well as nuclear RNAs,” adds Peng Yin, Ph.D., who is co-leader of the Wyss Institute’s Molecular Robotics Initiative, and professor of systems biology at Harvard Medical School.

Confocal microscopes use so-called pinholes to eliminate unwanted out-of-focus fluorescence from image planes above and below the focal plane. By scanning through the sample, plane after plane, researchers can gather the fluorescence signals emitted from molecule-bound dyes over the entire depth.

Specifically, the MPI/Wyss Institute team developed the technique for “spinning disk confocal” (SDC) microscopes that detect fluorescence signals from an entire plane all at once by sensing them through a rotating disc with multiple pinholes. Moreover, “to achieve 3D super-resolution, we placed an additional lens in the detection path, which allows us to archive subdiffraction-limited resolution in the third dimension,” notes first author Florian Schueder, a grad student working with Dr. Jungmann who also worked with Dr. Yin’s Wyss Institute team as part of his master thesis.

“This addition can be easily customized by manufacturers of SDC microscopes; so, we basically implement super-resolution microscopy without complex hardware changes to microscopes that are generally available to cell biologists from all venues of biomedical research. The approach thus has the potential to democratize super-resolution imaging throughout whole cells and tissues,” says Dr. Jungmann.