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Microscopy — pushing the limits

Harini Barath

Anton van Leeuwenhoek, celebrated as the father of microbiology”, was originally a trader of drapes and linens. He became fascinated with lenses, which were commonly used as magnifying glasses to count threads, and soon mastered the art of lens making. He made almost 500 simple microscopes whose magnification far exceeded that of even the compound microscopes his contemporaries could craft. This powerful invention opened up the previously invisible world of cells and microorganisms to the world. Now, about 300 years later microscopes form the spine of many major areas of life science research like cell biology.

Today’s researchers attempt to zoom much further into the cell. They seek to probe and image the wheels and cogs of all living systems — biomolecules. Since these molecules are typically nano-sized, this is no easy feat. Most optical tools are limited by the infamous diffraction limit, which only allows them to discern details larger than half the wavelength of light, almost of the order of microns. Researchers have developed ingenious tools that overcome this constraint and capture the nano-world. One such invaluable technique is fluorescence spectroscopy. Biochemists discovered a whole host of fluorescing molecules that can be chemically coupled to particular proteins. These molecules act as markers that allow researchers to see” proteins as they move and interact.

The fluorescence signal does suffer from fluctuations or noise as a consequence of changes and thermal motions in the tagged system. A smart statistical physics based technique called Fluorescence Correlation Spectroscopy (FCS), uses this very noise to uncover a wealth of quantitative information about the system. The fluctuations in the fluorescence are recorded as a function of time. Several parameters related to the underlying physical properties are extracted from this data. Some examples include the number of molecules (or sample concentration) and their size. FCS works best when a very small volume of solution is in focus, keeping the spotlight on a few molecules diffusing in and out of that volume. The technique and the ideas are old, from the 70s, but we have been able to make instruments in the lab, from scratch, which measure very little volume with incredible sensitivity”, said Dr. Maiti, a professor in the Department of Chemical Sciences at Tata Institute of Fundamental Research, Mumbai.

What’s more, FCS can be combined with a variety of techniques to measure different physical properties. One example is Fluorescent Resonance Energy Transfer (FRET), a popular technique, in which molecules are tagged with two different labels” that talk to each other. The strength of their communication depends on the separation between them and is a way of very accurately measuring distances of the order of nanometers. Using this combination of techniques, Dr. Maiti and his colleagues have made some important discoveries about the anomalous aggregation of the amyloid beta protein, which has been implicated as the cause of Alzheimer’s disease. There is a persistent suspicion that as the aggregation happens, it is actually the folding underneath that is changing, making a stable monomer into an unstable, sticky monomer,” he said. As these sticky monomers clump together, the particle sizes multiply and they become more cumbersome and slow down. FCS picks up this lowered speed, and can monitor the aggregation as it happens. Meanwhile, the change in the protein’s conformation can be inferred by monitoring the distance between its ends using FRET.

One heartening revelation from Dr. Maiti’s experiments is that the process of aggregation, which makes the protein toxic, is actually reversible. His research team found that small aggregates of a few proteins spontaneously unravel into monomers given sufficient time. One can now ask whether this reversal can be speeded up by a catalyst, some possible candidate for a drug. But a recent discovery brings less welcome news. Until now, scientists suspected that toxicity sets in when there are aggregations of 10 or more monomers. However, very recent measurements by Dr. Maiti and his group show that these small aggregates, the dimers and trimers, show a propensity to attach to cells, an ominous sign of increased bio-activity and toxicity. So they may cause harm before they have time to revert back to their docile selves. These are but glimpses of the protein’s mis-behaviour. To effectively arrest their errant ways, we need a clearer picture, an atomic level understanding of their structure. I know already that they fold differently, but I can’t quite visualize that,” said Dr. Maiti. An age-old technique could help do just that.

Raman spectroscopy is a well-established optical method that can be used to investigate a material’s atomic structure and chemical composition from the unique way it scatters light. Unfortunately, it is not a very sensitive technique, especially for biomolecules, many of which scatter light rather weakly. Ten years ago, researchers found that attaching samples to a specially prepared metallic surface, made a huge difference. Nano-sized irregularities on the surface enhance the light’s electric field, which consequently magnifies the output Raman signal by many orders of magnitude, making it possible to probe even single molecules. Dr. Maiti and his collaborators hope to use this technique to map the detailed structure of the wayward protein.

Scientists continue to add new twists to other old technologies, tailoring them to meet current research needs — even to that old workhorse, the microscope. After three centuries of pioneering work that have made it tremendously powerful and sophisticated, there is still, room for improvement. As a physicist, I think there is a huge amount of physics left to be explored and exploited in microscopy,” says Dr. G. V. Pavan Kumar, assistant professor in the Department of Physics at IISER, Pune. His work continues the legacy of physicists before him whose efforts have helped overcome its limitations through the ages.

Biological samples are largely transparent and staining them with contrast agents was one way to make them visible to microscopes. But this required the specimens to be killed and fixed before staining. Was there a way to look at living cells? Transparent samples that don’t affect light amplitude, diffract light and modify its phase, which is imperceptible to our eye. The Dutch mathematician and physicist, Frits Zernike, discovered a method to convert these phase changes into contrasts in intensity, which we can see. By means of a special disk and a phase plate, he separated and increased the phase difference between direct light and light diffracted by the specimen. The subsequent interference of the separated light waves resulted in a visible amplitude contrast.

Until recently, phase-contrast was a qualitative method to see cells and tissues non-invasively. Efforts are now focused on extracting quantitative information from the phase change. Several experimental approaches are being tried. Dr. Pavan Kumar and his colleagues are currently dabbling with one such state-of-the-art technique, which could benefit both material science and biology. They have technology that can tailor a phase pattern into the light shone on the sample. The diffracted light whose phase is modified by the sample, is compared with a reference beam by interference, and phase difference information is extracted, from which a very accurate image of the sample is constructed. The image has information about cellular structures and motions on a nanometer scale. Among other things, this is a method of label-free imaging, which has great advantages in biology,” says Dr. Kumar.

The ongoing quest for nanoscale clarity has many takers. Maybe not drapers any more, but certainly chemists, physicists and engineers among others. Their efforts will go a long way in furthering our understanding of the wonderful mechanisms of biology and hopefully, give us ways to correct those that go wrong. Biology, however, is not merely a muse for innovators. After all, it has been experimenting with the nano-world eons longer than us and has learnt a trick or two, which we can only hope to mimic.