Researchers working at Heidelberg University's Institute of Physical Chemistry and members of the Cluster of Excellence, CellNetworks have developed a method to replace light-dependent processes with chemical reactions to form cellular structures for high-resolution optical microscopy.
This development allows for new applications in fluorescence microscopy. The research paper has been published in the online journal Angewandte Chemie International Edition.
Fluorescence microscopy helps study cellular structures. But the diffraction limit hampers a detailed study. Objects separated by less than 0.3 ¼m cannot be studied. Recent research shows that it is possible to overcome this limit with stochastic optical reconstruction microscopy (STORM). In this process, the cell structures are tagged with fluorescent dyes reacting to fluorescent emission in the presence of light of a certain wavelength. High resolution of 0.02 ¼m is possible when most of the dyes are turned out and only a small number is still left on. The light from the surrounding dyes is not allowed to affect. This switching on or off of the dyes is also controlled by light. The dyes left on are individually resolved, their positions being measured with mathematical analysis to a very high accuracy of 0.003 ¼m. Replicating this procedure gives accurate data on the position of the dyes, allowing for a high-resolution build up of the cell structures being examined.
This puts stress on the microscope and light sources being used. Switching of the dyes involves varying laser lines (excitation wavelength) or high light intensities causing problems in the study of living cells. The researchers led by chemist Dr. Dirk-Peter Herten have used a light-independent process instead of the switching of dyes via laser light. They used a chemical probe to track copper ions in a manner that the probe and fluorescent properties mark the cellular structures. Fluorescence is suppressed when copper (II) binds itself to the probe. This is reversible, restoring the fluorescence of the probe. The microscopic examination of cell structures is monitored by a reversible chemical reaction.