Many biological and pathological processes are not strictly controlled by the presence, absence or function of biomolecules such as proteins or nucleic acids but rather by subtle changes in their numbers at specific locations within cells. However, despite the recent revolution of optical imaging technologies that has enabled the distinction of molecular targets residing less than 200 nm apart from each other, modern super-resolution techniques still face the challenge to accurately and precisely count the number of biomolecules at cellular locations.

A new analytical tool developed by a team at the Wyss Institute for Biologically Inspired Engineering solves this problem. The team led by Peng Yin, Ph.D., a Core Faculty member at the Wyss Institute and Professor of Systems Biology at Harvard Medical School, has forged ahead with its previously developed DNA-PAINT and Exchange-PAINT super-resolution microscopy platform to now count different molecular species in biological samples with high accuracy and precision. DNA-PAINT affords higher resolution than costly super-resolution microscopes and Exchange-PAINT can survey multiple different molecules in the same biological sample. The method is reported in the March 28 issue of Nature Methods.


"We now have enhanced our DNA-powered super-resolution microscopy methods with a highly quantitative analytical tool kit. qPAINT, as we named it, can accurately count the actual numbers of specific molecules at specific locations inside the cell," said Yin. "Introducing this quantitative power has crucially extended the spectrum of imaging capabilities of this comprehensive and inexpensive technology so that it can be applied in many areas of biological and clinical research."

Key to the DNA-driven imaging technology is the transient interaction of two short strands of DNA, one called the "docking strand" that is attached to the molecular target to be visualized and the other, called the "imager strand", which carries a light-emitting dye.

"We can precisely program the time interval for which the two complementary DNA strands transiently interact with each other so that when the pair of strands goes through binding and dissociation, the dye will blink at a specific frequency. From an increase of this frequency, we can then deduce with qPAINT analysis how many targets exactly are located at a specific cellular location without spatially resolving each target," said Ralf Jungmann, Ph.D., one of the two co-first authors of the study, a former Postdoctoral Fellow in Yin’s lab and now a Group Leader at the Max Planck Institute of Biochemistry at the Ludwig Maximilian University in Munich, Germany.

Applying this kind of binding analysis to DNA-PAINT and Exchange-PAINT allows the Wyss team to disregard common problems that fluorescent dyes pose for achieving truly quantitative potential in super-resolution microscopy, like their hard-to-model photophysical properties and tendency to wane under the influence of light, a phenomenon known as photobleaching.

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