Leaders in Pharmaceutical Business Intelligence (LPBI) Group

New method for neuron visualization

Reporter: Danut Dragoi, PhD

A new method for visualization of neurons shows promises for neuroscientists and cell biologists. The method uses a spectral confocal microscope to image tissues impregnated with silver or gold. As we know, the optical microscope takes a micro-image from a flat surface, like a plan biological sample confined between two transparent glass as usual. For micro-objects that are distributed in space, not even close to be in a plan surface, the optical microscope is less usable. Since neurons are distributed in 3D space, a normal optical microscope has to be adapted to the new situation. A confocal microscope in combination with a fluorescent surface is considered to be a good way to visualize micro-objects that are not in-plan distributed. The schematic diagram of a conventional confocal microscope is described here , where rotating mirrors scan the laser beam on the sample. The moving beam on surface is needed to cover the entire surface of the specimen and to avoid fluorescence saturation. More details about the technique for visualization of neurons from a grasshopper utilizing the confocal microscope and a fluorescent sample is shown here. The main idea of neuron visualization is to use Ag or Au nanoparticles deposited on the surface of the neurons and use the plasmon surface resonance effect to receive light from them, not from the incident beam that usually scatters the light, but from an emitted light from the nanoparticles, After collecting the light energy emitted from vibrating surface plasmons in the spectral LSCM, the team obtained spectacular three-dimensional computer images of silver and gold-impregnated neurons. This holds enormous potential for stimulating a re-examination of archived preparations, including Golgi-stained and cobalt/silver-labelled nervous systems. Additionally, by using a number of different metal-based cell-labeling techniques in combination with the new LSCM protocols, tissue and cell specimens can be generated and imaged with ease and in great three-dimensional detail. Changes in even small structural details of neurons can be identified, which are often important indicators of neurological disease, learning and memory, and brain development. It is important to mention that this method is not applicable in-vivo. Because neurons have poor light scattering properties, we expect that new physical effects to be considered. As an example by using stimulated emission scientists can quench fluorescent molecules. They direct a laser beam at the molecules that immediately lose their energy and become dark. In 1994, Stefan Hell, Nobel prize winer 2014, published an article outlining a new method for a performant microscope. In the proposed method, so-called stimulated emission depletion (STED), a light pulse excites all the fluorescent molecules, while another light pulse quenches fluorescence from all molecules except those in a nanometre-sized volume. Only this volume is then registered. By sweeping along the sample and continuously measuring light levels, it is possible to get a comprehensive image. The smaller the volume allowed to fluoresce at a single moment, the higher the resolution of the final image. Hence, there is, in principle, no longer any limit to the resolution of optical microscopes. Since the light emitters are not in the same plan, and assuming the method can be applied to neurons, a new instrument can be devised using among other key elements a confocal setup microscope.