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Super-resolution Imaging: Exploring the strange world inside a living cell.

Humans have always been curious to explore deeper into nature’s creations. Considering the fact that humans are themselves among the most intelligent beings created by nature, looking deep into our body structure one finds that Cells are our building blocks. We are able to see skin with oureyes, but not the cells of which it is made up. Why? If we take a slice of skin and look at it under a simple microscope, skin cells may become visible. What do microscopes do? We may now be able to see the cells, but not its structure. Why? We can continue to ask; is there any limit after which we cannot look deeper? In this article I will try to address these questions. In order to understand them you need to understand the concept of Resolution Limit.

Text: Rajwinder Singh, Dept. of Physics and Technology, UiT-The Arctic University of Norway, TromsØ

Resolution:

Imagine that you are looking into the sky and seeing an object which looks as if it were a star. But Hubble’s telescope (a giant telescope which is stationed outside the Earth’s atmosphere) confirms that it is actually a galaxy i.e. collection of stars which appear as one. Our eyes were not able to resolve the stars lying close together. Hence our eyes have some limit such that if we start bringing two objects closer, there will be a point after which our eyes will not be able to identify whether it is one object or more. This limit is known as Resolution Limit of our eye. Fig 1 (a) shows single object, 1(b) show two objects which are just resolved and 1(c) shows two objects which can’t be resolved and appears to be as one.

Wikimedia Commons
Photo: Wikimedia Commons

This limit depends on the wavelength of light which is reflected from the objects. More is the wavelength of light, less is the resolution. As an example, it might happen that two objects which are lying close together appear to be one in red light but can be seen as two if we use blue or ultraviolet light. Blue and ultraviolet light have a smaller wavelength than red light. Looking at cells through a simple microscope may help us resolving up to the limit where we can distinguish the cells which our eyes were unable to. Its resolution is not high enough to see the sub-cellular structure. Hence the resolution power of a microscope is larger than our eye but we will have to do more in order to see the structure inside.

Magnification doesn’t increase resolution:

At this point one might think that a microscope magnified the skin slice, which is true but at the same time it also increased resolution. If you do not trust me, try taking a photo of your hand with your mobile camera and then zooming-in. I am pretty sure that you will not be able to see skin cells even if you magnify it 100 times! This kind of magnification is called Empty Magnification where we are unable to get extra information after magnifying further. This means that your camera doesn’t have high enough resolution!  One must not confuse magnification with resolution. Magnification helps us to easily see the information provided by the high resolution of the system. Higher resolution means more details. Fig 2 show difference between high and low resolution images.

Super-resolution:

If we use green light to see the objects, no matter how strong  our microscope, we will not be able to distinguish the objects lying closer than 200 nanometer (nm) approximately. To get an idea, this is one millionth of the width of human hair. But the organelles (specialised structures in a living cell) are about 10 times smaller than the resolution limit of our best microscope. Resolving the objects beyond the resolution limit of conventional light microscopes is known as Super-resolution. If we achieve super-resolution, we may be able to see the sub-cellular structure. There are different methods to do it, but I will discuss one which is easy to understand.

Super-resolution Imaging: From Microscopy to Nanoscopy

Cells are made up of macromolecules like Proteins, DNA, RNA etc. There are some natural/synthetic molecules which start glowing when we shine some light on them. These molecules are known as fluorescent molecules. Fig. 2 show Jelly fish glowing in light as it contain Green Fluorescent Proteins(GFP) inside. Now the fluorescent molecules are attached to protein molecules inside the cell one on one. More scientifically we say that protein molecules are tagged with fluorescent molecules. This process is known as labelling.  Cell which is to be imaged is labelled very densely (  molecules/μm²). If we now see it under the strong microscope, the cell will appear to be coloured. We will not be able to see the structure yet! Now the fluorescent molecules are made to turn on and off such that at a given time only few molecules lying at a distance greater than resolution limit generally glow. Images are taken one by one at different instants of time. In each frame different fluorescent molecules are glowing. We know the coordinates of these glowing molecules on which a Gaussian curve is fitted. In simple language, a point is marked on those coordinate positions in each frame. After taking tens of thousands of images, all the point marked frames are overlapped on top of each other to give us the clear structure inside a cell. This technique is known as Stochastic Optical Reconstruction Microscopy (STORM).

Photo: Wikimedia Commons
Photo: Wikimedia Commons

These microscopes are known as Nanoscopes. The study of developing nanoscopes and their application is known as Optical Nanoscopy. The development of these nanoscopes is done by Physicists and Electrical Engineers, development of fluorophors for labelling is done by Chemists and these nanoscopes are generally used by Biologists. Hence it is a multidisciplinary field. It is worth pointing out that 2014 Nobel Prize in Chemistry was awarded to Betzig, Hell and Moerner for developing super resolution microscopy techniques. In general we can achieve resolution up to 20-30 nm with these nanoscopes which is about 10 times better than resolution of a conventional microscopes. There are some other techniques by which we can achieve super-resolution like Structured Illumination Microscopy(SIM), Stimulated Depletion Emission(STED) Microscopy, etc. If you’re more interested, you can look it up online.

Applications of super-resolution imaging:

Major application is in the field of Biology. It allows us to see much greater details inside a living system, from a molecule to an entire microorganism. We can see the cellular mechanisms at a molecular level. An example in Neuroscience, scientists are looking at the molecular mechanisms of proteins which go wrong and do not function properly in brain cells causing Parkinson’s disease using nanoscopes. Now with this we have possibility to see whether we can use drugs to inhibit this process. We can also track optically the drug-cell interaction with these nanoscopes. We can see the pathogen-cell, or intercellular interactions.

STED_Confocal_Comparison_50nm_HWFM

Research in both development as well as application of nanoscopes is carried out at our own University of TromsØ very actively. Assoc. Prof. Balpreet Ahluwalia’s project on “High-speed chip-based nanoscopy to discover real-time sub-cellular dynamics” was awarded with European Research Council prestigious ERC Starting Grant in 2013. The project aims to develop a novel optical nanoscopy with high resolution (50-100 nm) and fast imaging speed (25 Hz). At the same time applied research in this field is done jointly by Dept. of Physics, Dept. of Medical Biology and Dept. of Fisheries. Main focus is on live cell imaging using SIM which is available at Dept. of Physics.

Concluding Remarks:

Although still in its infancy, super-resolution fluorescence microscopy has shown great promise for studying biological structures and processes at macromolecular scale. This is one of the fastest growing fields with a bright future ahead. In recent years, the invention of various super-resolution techniques has broken the resolution limit. Images obtained from these super-resolution approaches enable scientists to directly visualize biological samples at the nanometer scale, significantly expanding our understanding of molecular interactions and dynamic processes in living systems.