The idea behind the key technology of "Fluorescent Nanoscopy based on photoactivatable dyes" is to overcome the limits of optical microscopes by sparsely photoactivating fluorescent molecules. Only a small percentage of molecules of the specimen is activated. This way, molecules that are located far from each other are visible as separate spots. The positions of activated molecules are captured by a high-sensitivity camera. After that the dye fades away and a new portion of molecules is activated. This process (photoactivation, image capturing, search for spot centers and photobleaching) is consecutively repeated until there are enough frames to assemble a high-content image. This allows to research most of biological specimens including cells, proteins, DNA etc.
The second method that we develop is "Fluorescent Nanoscopy based on tracing nanoparticles": the object is dyed with fluorescent nanoparticles, that are resistant to photo-bleaching (e.g. microspheres, phycobiliproteins, quantum dots). Their size is much less than the emitted light wavelength. The intensity of the emitted light is high enough to observe movement of nanoparticles online and to calculate their position with the precision much higher than the spot’s diameter formed by nanoparticle on a video frame. The duration of their fluorescence is very high, which allows these nanoparticles to visit, due to Brownian motion, all the accessible parts of the liquid inside the subject of inquiry. Furthermore, nanoparticles can have an electric charge, which allows use of electrophoresis to maintain an ordered movement of nanoparticles with online observation of their trajectories. Charged particles of different polarities can be used to analyze the distribution of local charges in the object, since they will visit different parts of the object during their movement. There are also nanoparticles sensitive to pH and to some ion concentrations. This allows to research cavities of solid materials such as ceramics, concretes and porous heat insulators which is needed for improvement of quality of these materials and for quality assurance in their manufacture.
2-20 nm spatial resolution for both methods has been predicted by analytical calculations as well as computer modeling of the process and comparison of our data with publications on «single molecule detection». As a result we predict 20 nm spatial resolution, but in many cases even 2 nm spatial resolution is possible for both methods.
We also develop supplies required for the Fluorescent Nanoscopy and numerous improvements to
modern microscopes and other optical imaging systems, such as a new illumination system and video-
detection system, featuring increased qualities and decreased outlays.