Microscopy goes nano

Nanoscopy wins Nobel

Manu Forero Sheldon

Departamento of Physics, Biophysical Group

Universidad de los Andes

Email: anforero@uniandes.edu.co

Last October 8 2014, the Nobel Prize in chemistry was announced, and the prize was awarded to three researchers who helped create the field of nanoscopy: microscopy at the nanoscale [1]. To understand the importance of these developments we need to go back a couple hundred years and understand why microscopy became so important in the life sciences.
The first microscopes in the sixteenth century were just upside-down telescopes, with which you could see smaller animals like fleas. These improved over time and eventually allowed the observation of much smaller objects. One of the key milestones of microscopy was the development of a high magnification microscope by Anton van Leeuwenhoek, a draper during the first industrial revolution in the seventeenth century. The main use of microscopes at the time was to count the number of threads in a fabric, in order to estimate its quality. Van Leeuwenhoek decided to make his own microscopes, very differently from the traditional technique, and this allowed him to achieve magnifications of up to 270x, impossible with other technologies at the time. With these microscopes he was able to deduce that cells were the fundamental component of living organisms and that sperm was a special type of cell that mediated reproduction. He was also the first to see individual red blood cells and described single-celled microorganisms like bacteria and protists for the first time. These fundamental discoveries marked the birth of microbiology and opened a new world for scientists of all kinds.

Figure 1. Drawing of microscopes owned by Antonie van Leeuwenhoek. Author: Henry Baker. Public domain image.

Optical microscopes continued to improve in magnification and image quality, but with a fundamental limitation that scientist Ernst Abbe discovered in the nineteenth century: It is not possible to distinguish (“resolve” in scientific terms) two structures that are closer than a distance related to the wavelength (size) of visible light photons, which is between 400 and 700 nanometers (a nanometer is a millionth of a millimeter, or about 10 atoms in a row). This barrier is a significant hurdle for understanding how cells work because much of the key components that make cells work including so called organelles as the energy making mitochondria such as well as most proteins have structures that cannot be discerned. Although electron microscopes can see the structures of many of these components, they can only do so in dead cells. These new methods of optical nanoscopy promise to reveal the inner workings of live cells, which is why they will probably contribute significantly to our understanding of life, in particular how different parts of the cell interact with each other over time, especially in the context of diseases for which there is no cure such as most cancers.

Figure 2. Ernst Abbe (1840-1905). Public domain image.

For a century and a half, the diffraction barrier was thought to be insurmountable, but in the last two decades, scientists found several ingenious ways to overcome it. The first step to one of the tricks to cheat the diffraction barrier was taken when W.E. Moerner (one of the three laureates) developed methods for observing fluorescent molecules individually [2]. Fluorescent molecules absorb light of a particular color, and reemit light of a different color, and can be seen in some money bills using an ultraviolet light. The ability to follow a single molecule at a time is important because there are cases when averages do not necessarily reflect what goes on at the individual level. For example, if there is a barrier on a road and there are molecules passing through both sides of it, the average of the paths will be in the middle of the barrier, which clearly does not reflect what is happening.

By observing isolated single molecules, researchers realized that it was possible to find their positions with accuracy in the order of nanometers [3], but when the molecules came too close, this ability was lost due to the diffraction barrier. This problem was solved by Eric Betzig (another of the three laureates), taking advantage of another key discovery: Fluorescent ´blinking´ molecules that can be turned on and off [4]. Betzig figured that by turning on only a few isolated molecules at a time, he could locate their position and then turn them off again. By repeating this process several cycles he could accurately locate molecules that would have been too close to resolve had they been turned on at the same time, and by superimposing those positions he was able to get images that beat Abbe´s limit [5].

Figure 3. Optical nanoscopy. View of a nucleus of a bone cancer cell: using normal high resolution fluorescence microscopy (image on the left). Using the two Color Localization Microscopy 2CLM (image on the right) it is possible to localize 70,000 histone molecules (red: RFP-H2A) and 50,000 chromatin remodeling proteins (green: GPF-Snf2H) in a field of view of 470 µm2 with an optical depth of 600 nm.  Author: Andy Nestl.  Lic: CC BY-SA 3.0

In short, Betzig took the opportunity to locate individual isolated molecules accurately, to circumvent the problem of resolution between neighboring molecules.

In the biophysics laboratory at Universidad de los Andes, we have achieved images with a resolution of about 60 nanometers based on techniques derived from that of Betzig, but with equipment that cost a fraction of what is normally used for this. We are doing this in order to study the distribution of important molecules of the parasite Tripanosoma cruzi, which causes Chagas disease. The figure shows the distribution of an antibody against a peptide called KMP-11 in a trypanosome, in a traditional image (left) and a super resolution image (right). This image is the result of a collaboration with the group of Basic Medical Sciences at the university and was taken and analyzed by undergraduate student Jorge Madrid.

A second super-resolution technique, developed by Stefan Hell (the third laureate), is also based on the ability to turn on and off fluorescent molecules. In this case the molecules of a small volume the size of the diffraction limit are excited using a light pulse, and two tricks are used: The first is to turn off some of the molecules using a second pulse. This second pulse has a shape so that it does not turn off the molecules at the very center of the diffraction-limited volume, but the ones around that center. You might think of the diffraction volume that is turned on as a bread bun, and of the inactivating volume as a donut. The initial illumination creates the illuminated bun, and the second light pulse ´eats´ a donut, and we are left with a donut hole much smaller than the bun or the donut. By scanning the sample point by point in this manner, it is also possible to distinguish details beyond the diffraction barrier [6].

Figure 4. Distribution of an antibody against a peptide called KMP-11 in a trypanosome, a) in a traditional image and b)  a super resolution image.

The same way that the van Leeuwenhoek microscope made it possible to discover new structures and organisms through its resolution, the new possibilities of these new super-resolution microscopes will surely bring important discoveries. The last decade has seen a revolution in microscopy. This revolution is not limited to nanoscopy, but includes a new technique called light sheet microscopy [7]. Light sheet microscopy has enabled studies of whole live animals in three dimensions, and is strongly contributing in key areas such as developmental biology and neurosciences. It is possible that in some years, another Nobel will be given for this other technique that the biophysics lab at Uniandes is also using in several studies.

References

[1]  The Nobel Prize in Chemistry 2014 – Press Release. (2014). at <http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2014/press.html>

[2] Ambrose, W. P., Basché, T. & Moerner, W. E. Detection and spectroscopy of single pentacene molecules in ap-terphenyl crystal by means of fluorescence excitation. J. Chem. Phys. 95, 7150–7163 (1991).

[3] Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

[4] Patterson, G. H. & Lippincott-Schwartz, J. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 297, 1873–1877 (2002).

[5] Betzig, E. et al. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 313, 1642 –1645 (2006).

[6] Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. 97, 8206–8210 (2000).

[7] Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007 –1009 (2004).