Physical conditions tend to restrict the sharpness of a light microscope, for example, structures that are closer together than 0.2 thousandths of a millimeter will blur into one another and will no longer be differentiated from each other.
Diffraction is responsible for this blurring: To put this in simple terms, diffraction ensures that light rays cannot be bundled with random precision. Hence, each point-shaped object is displayed as a “blurry spot,” and not as a point.
Through mathematical techniques, it is still possible to radically enhance the resolution. While one would compute its precise center from the brightness distribution of the “blurry spot,” it works only when the object’s two closely adjacent points are initially not concurrently but subsequently evident, and are later combined in the image processing. Superimposition of the “blurry spot” is prevented by this temporal decoupling. For some years, scientists in life sciences have been applying this tricky technique for super high-resolution light microscopy of cells.
The research team of Prof. Dr Markus Sauer at the University of Würzburg developed one type of this technique—direct stochastic optical reconstruction microscopy, or dSTORM. This robust SMLM method is capable of providing a lateral resolution of approximately 20 nm. For this reason, specific structures, for instance, a pore of a cell nucleus, are stained using fluorescent dyes. Each dye molecule blinks at random intervals and denotes a part of the pore. As a result, the image of the entire nuclear pores is not visible at first, but becomes apparent after the image is processed by the superposition of several thousand images.
With the help of the dSTORM method, the resolution of a traditional light microscope can be increased by a factor of 10. “It allows, for example, to visualise the architecture of a cell down to its molecular level,” explained researcher Hannah Heil. She is doing her doctorate at the Rudolf Virchow Center of the University of Würzburg in the group of Prof. Katrin Heinze.
Conversely, a virtual resolution limit in resolution is defined by the photon statistics itself. In order to deal with this problem, Katrin Heinze came up with an idea to apply comparatively simple biocompatible nanocoatings to improve the signal. In a joint effort with Markus Sauer and coworkers from the faculty of Physics, Hannah Heil engineered and developed metal-dielectric nanocoatings that act similar to a tunable mirror. They nearly increase the resolution by two-fold.
Mirror, mirror on the wall: Which image is the sharpest of them all?
The researchers then vapor-deposited a coverslip, on which the cells were positioned at the time of observation, with a thin reflective nanocoating composed of transparent silicon nitrite and silver. Since the coating is biocompatible, it keeps the cell intact. Through this technique, both groups were able to attain two effects: the mirror reflected the light emitted to the microscope, which boosted the fluorescence signal’s brightness and thus effectively increased the image sharpness.
A second phenomenon is also there: the reflected and emitted light waves are superimposed, producing the so-called interference. Based on the distance to the mirror, the light is attenuated or amplified. “In this way, we primarily see structures in a certain image plane,” stated Heil.
“Everything that is above or below and could possibly disturb the image is, on the other hand, hidden.” To make sure that the actual parts of the image become perceptible, it is important to properly select the thickness of the transparent layer applied to the mirror. Among other things, Heil and Heinze apply computer simulations to customize the coating in accordance with the object.
On the whole, the method proved to be unexpectedly easy to use, stated Hannah Heil. “That’s what I really like about our approach.”
Except for the cheap metal-dielectric coated coverslip there is no need of any additional microscope hardware or software to boost the localization precision, and thus is a fantastic add-on in advanced microscopy.
Dr Katrin Heinze, Professor, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg.