One of the main advantages of fluorescence microscopy is that it can be used to simultaneously detect and identify multiple molecular species in a sample by making use of tags that fluoresce at different wavelengths (colours). However, traditional microscopes, which employ organic dyes or fluorophores, can only detect three to four molecular components (such as proteins or DNA/RNA) at once.

The new metafluorophore, developed by a team led by Ralf Jungmann and Peng Yin of the Wyss Institute for Biologically Inspired Engineering at Harvard University, exploits structural DNA technology to increase this so-called multiplexing factor by a factor of around 30. “This now gives us access to more than 120 virtual colours for microscopic imaging or other analytical methods that can be adapted in the future to visualize multiple molecular players at the same time with ultra-high definition,” says Jungmann.

DNA origami

The researchers constructed their probes using the now well-established DNA origami technique in which a long single-stranded DNA molecule (called the scaffold) is folded into programmable shapes using around 200 short, single-stranded DNA strands (called “staples”). Every staple has a well-defined sequence and specifically binds certain parts of the scaffold together. Once self-assembly is complete, the scaffold is folded into the desired shapes, with the staple strands at pre-defied positions on the final origami.

In their experiments, the researchers used around 200 staples to fold a roughly a 7000-nucelotides-long phage-genome-derived scaffold into predefined shapes and patterns. These “nano-breadboards” can then be used to arrange a large number of dye molecules at precisely defined positions and ratios in a self-assembled manner, explains Jungmann. One such breadboard consists of 24 parallel DNA double helices measuring 90 × 60 nm2 (an area that is substantially smaller than the optical diffraction limit). This structure contains 184 staple strands.

124 fluorescent signals

“By functionalizing specific component strands at the predefined positions of the DNA nanostructure with one of three different fluorescent dyes, we achieve a broad spectrum of up to 124 fluorescent signals with unique colour compositions and intensities," adds Yin. “This DNA nanostructure-based approach can be used like a barcoding system to visually profile the presence of many specific DNA or RNA sequences in a sample.”

The sub-diffraction volume of the composite fluorophores means that they look like traditional organic fluorophores under a diffraction-limited microscope. They thus behave in the same way as these dyes and so can be used in standard microscopes.

High brightness, small size and high multiplexing capacity

“Our origami-based metafluorophores can be thought of as basically a programmable version of conventional dyes and might find applications in in vitro experiments where researchers are interested in detecting a large number of molecular DNA or RNA species,” Jungmann tells “Thanks to their high brightness, small size and high multiplexing capacity, they might also find use in areas such as flow cytometry and fluorescence-activated cell sorting for high-throughput identification of multiple molecular components.”

The team, which also includes researchers from Ludwig Maximilian University in Munich, the Max Planck Institute of Biochemistry, the Massachusetts Institute of Technology and Harvard Medical School, says that they would now like to make their metafluorophores even smaller by using a recently-developed technique called single-stranded tile assembly. “We would also like to trigger intracellular assembly of these fluorophores on site,” adds Jungmann.

The research is detailed in Science Advances DOI: 10.1126/sciadv.1602128.