MIT super-resolution platform views structures in subatomic detail

Researchers at MIT and the Broad Institute have developed a new variant of super-resolution microscopy that should offer new views of small-scale biological processes.

The technique is based on stochastic optical reconstruction microscopy or STORM, an established wide-field fluorescence super-resolution process which introduces photoswitchable fluorophores to a target and uses a low-power activation laser to ensure that any particular fluorophore has only a finite chance of being photoactivated. This simplifies the optical set-up needed to achieve super-resolution results.

Described in Nature Nanotechnology, the new upconversion-enabled STORM (U-STORM) method achieves simultaneous multicolour imaging with a single excitation source and a single imaging round, by employing a new class of compositionally engineered upconverting nanoparticles (UCNPs) that blink spontaneously and indefinitely. 

For decades the scientific community widely considered upconverting nanoparticles to be completely photostable and non-blinking, commented MIT. Because localization-based super-resolution microscopy techniques like STORM rely entirely on the stochastic "blinking," ie. the switching between on and off states of light emitters, to distinguish closely packed molecules, UCNPs were historically deemed unsuitable for this type of imaging.

"Our laboratory has long been interested in overcoming these limitations," said MIT's Sam Peng. "Our work began with a question: Can we develop a super-resolution imaging platform that is simultaneously long-term, multicolor, simple to operate, and capable of achieving extremely high localization precision without using imaging buffers or additional optical control?"

The Peng Lab engineered a new class of UCNPs based around lanthanide compounds and each 10 nanometers in size, containing tuned amounts of sensitizer and emitter ions. The particles exhibit spontaneous and indefinite blinking under continuous near-infrared excitation without optical modulation, photobleaching or statistical aging problems.

Multiple colors from one laser excitation

Further adjustments to the UCNPs composition allowed the project to create red- and blue-emitting particles, to allow simultaneous multicolour super-resolution imaging. 

This means that unlike conventional multicolor super-resolution approaches needing multiple lasers and meticulous optical alignment, U-STORM can operate with just one near-infared laser simultaneously exciting nanoparticles emitting different colors. This results in a drastic reduction of an experiment's complexity, noted MIT.

"To obtain images with multiple colors, rather than capturing images sequentially over multiple rounds U-STORM captures multiple colors simultaneously," commented MIT. "Researchers have successfully demonstrated this by mapping epidermal growth factor receptor dimers and multimers in biological samples under physiological conditions without any specialized imaging buffers."

The MIT team is now working to expand the color palette, make the particles even smaller and brighter, and deploy U-STORM to investigate complex nanoscale protein organizations and cellular signaling pathways.

"This demonstrates multicolour super-resolution microscopy in which the probe itself - not the instrument or additional reagents - provides the switching mechanism," wrote the project. "U-STORM promises to provide an accessible, easy-to-implement, yet incredibly powerful route toward high-precision molecular imaging."