nano bio / nano characterization

Seeing nanoscale with optical microscope; simple idea that took 2014 Nobel chemistry prize

Limits are made to be broken. Every year, handful of people who pushed the limits of mankind’s hardest of problems to the point that they give away, will be offered with the most distinguished honor a man can receive : noble prize. In the year 2014, noble prize for chemistry was offered to group of scientists who broke the most fundamental limit of microscopy using an ingenious technique. These scientists pioneered an optical microscopy technique that is so powerful, it can even image a single molecule.

The diffraction limit

For many long years, scientists and engineers believed that optical microscopy cannot be used to resolve nanoscale dimensions. This understanding primarily came due to the work by a physicist named Ernst Abbe, who published a theoretical limitation in imaging with light waves in 1873. He calculated that resolution of optical microscopy technique is limited by the diffraction. This led most of the scientists to believe that optical microscopy would never be able to use in imaging things smaller than 200 nm. However, most of the nanoparticles, nanostructures and nanoscale assemblies-both natural and manmade- exist well below this size range.

Molecular optical imaging

The nobel price of 2014, rewarded three great scientists for their work on finding an ingenious ways to work around the theoretical limitation explained by Ernst Abbe. The decorated scientists are Eric Betzig, Stefan W. Hell and William E. Moerner who developed two unique optical microscopy techniques for imaging objects with much higher resolution than 200 nm.  It’s important to note that the diffraction limitation in microscopy still holds and always will. But these scientists took clever use of fluorescence behavior of molecules for the development of the images. Fluorescence is a phenomenon seen in some molecules which produce a glow when irradiated with a light from a certain wavelength. Fluorescence microscopes, can excite an object with a suitable wavelength of light to make it glow. Fluorescence microscopes are not a new thing and they are also bound by the diffraction limitation in optical imaging. The ingenuity of their findings came from how they played with fluorescence microscopes to extract the fine details of the imaged objects.

Noble Prize winners of 2014 for chemistry

Two separate techniques were rewarded in the 2014 Nobel prize for chemistry; Simulated Emission Depletion Microscopy (STED) and single molecule microscopy.

Simulated Emission Depletion Microscopy

STED technique was pioneered by Stefan W. Hell inn year 2000. In this technique, two concentric laser beams are utilized for imaging in contrast to a single beam of laser in conventional fluorescence microscopy. The innermost laser beam is designed to have nanometer sized volume and used to excite the fluorescent molecules. The outermost laser beam is design to cancel out any fluorescence coming out from the sample except the bit coming from the inner beam. This optical assembly scans over the sample, picking up much weaker signals of light to yield an image having better resolution than Abbe’s limit. This is how the microscope was converted in to a nanoscope in the first method.

STED microscopy technique is presented

Single molecule microscopy

Other two scientists, Eric Betzig and William Moerner developed so called single molecule microscopy separately using a completely different technique. This method is based on a technique called photo switching where scientists can switch on and off fluorescence of individual molecules on the object. Then they take multiple images of the same area, making only few molecules which are interspersed to grow each time the image is recorded. Then the images of each of these glowing molecules are superimposed using a computer algorithm to produce an image with very high resolution which is beyond the theoretical diffraction limit.

single molecule microscopy at ninithi.

These nanoscopy techniques are used worldwide today primarily f orbiological applications. Nanoscopic imaging has proven its advantages specially in live cell imaging as no other technique offers resolution as low as these techniques offer with the constrains associated with the live cells. These techniques can routinely achieve resolutions in 70-90 nm range making structural imaging of complex bio structures a rather easy business.

Further reading


  2. Hell, Stefan W., and Jan Wichmann. “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.”Optics letters 19, no. 11 (1994): 780-782.

  3. Hell, Stefan W., and M. Kroug. “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit.” Applied Physics B 60, no. 5 (1995): 495-497.

  4. Betzig, Eric, George H. Patterson, Rachid Sougrat, O. Wolf Lindwasser, Scott Olenych, Juan S. Bonifacino, Michael W. Davidson, Jennifer Lippincott-Schwartz, and Harald F. Hess. “Imaging intracellular fluorescent proteins at nanometer resolution.” Science 313, no. 5793 (2006): 1642-1645.

  5. Rittweger, E., B. R. Rankin, V. Westphal, and S. W. Hell. “Fluorescence depletion mechanisms in super-resolving STED microscopy.” Chemical physics letters 442, no. 4 (2007): 483-487.


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