A research conducted by Graduate Student Yifei Jiang and Chief Investigator Jason McNeill of Clemson University’s department of chemistry now makes it possible to clearly visualize biological cells under a microscope.
With the assistance of Rhonda Powell and Terri Bruce of Clemson’s Light Imaging Facility, the team was able to create a nanoparticle “switch” that fluoresces to perfect the resolution of microscopic images that portray small cellular structures. As recently reported in Nano Letters, this switch builds upon an imaging technique that won the 2014 Nobel Prize in Chemistry.
Since cellular structures discharge light at wavelengths smaller than 400-700 nm on the electromagnetic spectrum, they mostly look blurred through a light microscope. This constraint is termed as the diffraction limit, and it happens because of the wave properties of light. As light travels around structures within biological cells, it bends, or diffracts, to a point that light microscopes cannot plainly resolve. The 2014 prize-winning imaging technique – single molecule localization microscopy – was invented to overcome this drawback.
Single molecule localization microscopy is based on molecular ‘photoswitches’ – fluorescent molecules that you can turn on and off, like a light switch, to beat the diffraction limit. With this imaging method, the sample is imaged one fluorescent molecule at a time and a computer is used to construct an image that is much sharper than what you could get with a regular light microscope.
Jason McNeill, Chief Investigator, Clemson University
The issue, however, is that the fluorescence produced by photoswitches is dim at best, with just a slight enhancement in image resolution. Single molecule localization microscopy also needs specialized equipment that can be costly to acquire.
The solution lies in the “buckyswitch” – the Clemson Researchers’ improved version of a photoswitch. This new type of nanoparticle preserves the photoswitch’s on-off capability, but is easier to use and 10 times brighter. It also allows microscopes to obtain images up to the terapixel level. (That is the corresponding to one trillion pixels, or one million megapixels.)
These nanoparticles are the first photoswitches to achieve precision down to approximately 1 nanometer, which greatly improves the resolution of super-resolution imaging. Also, our method only requires one excitation light source, where conventional super-resolution techniques require two lasers; thus, we have simplified the microscope setup.
Yifei Jiang, Graduate Student, Clemson University
Jiang put together the buckyswitch using a fluorescent, semiconducting conjugated polymer complexed with a chemical derivative of buckminsterfullerene: a soccer-ball-shaped form of carbon.
“The hard part of making a fluorescent nanoparticle that you can turn on and off is that there are lots of areas emitting fluorescence at once,” McNeill said. “In the case of fluorescent conjugated polymer, there are dozens or hundreds of chain segments. You can try to make a lot of little switches for each segment, but it’s hard to get them all to switch off at the same time. You can’t get them synchronized.”
By incorporating the derivative of buckminsterfullerene, called PCBM, to the fabrication of buckyswitches, a “master switch” is created that controls the atomic charge of the polymer’s segments, thereby synchronizing fluorescence. PCBM can capture electrons from the polymer segment, offering the segment a complete positive charge. This positive charge decreases the fluorescence of adjacent segments, which has a domino effect that switches off fluorescence in the whole nanoparticle.
Bruce – whose background spans the topics of chemical engineering, cell biology, applied biology and experience in teaching and industry – compares this imaging technique to the view of a suspension bridge at night.
“The wires of the bridge are often illuminated, and when you are standing far away from the bridge, the lights look like one continuous ‘rope’ of light, instead of individual bulbs. However, if you can make the bulbs blink – such that only every other bulb is ‘on’ at any time – your eyes can discern the individual bulbs from far away,” Bruce said. “The basis for super-resolution microscopy lies in the ability to make fluorescent labels ‘blink’ just like the lights on the bridge. The work that Dr. McNeill’s lab is doing is vital for the advancement of this technology because it focuses on making those individual blinks much brighter, so that our current photon detectors can actually see the blinks. If we can see the blinks with a camera or other photon detector, we can map where the blink occurs, and create an image where we can discern two points of light that are within 10-20 nanometers of one another.”
Once the buckyswitch was created, Jiang examined it in E. coli, but only after developing a unique growth media for the bacteria. Normally, E. coli is grown in media that is autofluorescent, meaning that it naturally discharges light. Without the appropriate media, the buckyswitch’s fluorescence would be hidden by background light, something that Powell emphasized.
“A study like the one Yifei conducted required very little background fluorescence, so I researched media components that would be less likely to be autofluorescent and prepared a ‘recipe’ for a non-conventional, less autofluorescent nutrient media for bacteria culture,” said Powell, who researched both microbiology and biological sciences at Clemson before becoming the research lab manager of the Clemson Light Imaging Facility. Powell and Bruce also helped to provide Jiang with the E. coli for the research.
After all of the essential components were gathered together, Jiang attached the nanoparticle buckyswitches to the surface of E. coli. As anticipated, the buckyswitches released small flashes of light, which enabled the Researchers to establish their precise positions. They then patched together each flash of light to rebuild the shape of the E. coli, producing a super-resolution image.
“We hope this breakthrough will eventually be able to help researchers tackle difficult problems in biology, leading to breakthroughs in the understanding and treatment of disease,” the Clemson team said.
The team engineered the buckyswitches to function with standard fluorescent microscopes and free software that is available online, making the technology economical and accessible for labs around the world.
Their publication, titled “Improved Superresolution Imaging Using Telegraph Noise in Organic Semiconductor Nanoparticles,” has been featured in the June 14th issue of Nano Letters. The research is supported by the National Science Foundation.