Antibodies are large Y-shaped proteins used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. That makes them valuable tools for the analysis of biomolecules in research, diagnostics and therapy. However, antibodies are huge (150 kDa) biomolecules and are not functional within a living cell due to the reductive environment of the cytoplasm. Normally, antibodies are used to detect antigens on fixed an permeabilized cells (in other words: dead cells). But neither does that provide any information about the dynamic changes of the antigen within different stages of the cell cycle, nor about its overall mobility. A research group at the University of Munich has now succeeded in developing much smaller molecules for antigen detection in living cells.
Objects a thousand times smaller than the wavelength of infrared light (10 micrometers) are undetectable by standard far-field optical infrared microscopy since the weak nanoparticle signals would be buried far below the background level. To overcome this drawback and to achieve nanoscale spatial resolution researchers in Germany illuminate the nanoparticles by a highly intensive nanoscale infrared light spot. It is generated in the nano-gap between a laser-illuminated scanning metal tip and the substrate supporting the particles. The simple but very efficient trick finally allowing detection of sub-10 nm particles is the use of highly reflecting substrates instead of glass slides typically used as a sample carrier in optical microscopy.
Cantilever based back-illuminated full body glass tips with thin metal layers can be used as apertureless optical near-field probes with single molecule sensitivity and optical resolutions down to 15 nm exceeding by far the classical diffraction limit of Abbe.
Single molecule Raman spectroscopy of molecules on metal surfaces was achieved by means of optical field-enhancement from a scanning probe tip. This was made possible by the optical antenna configuration formed by optical coupling between metallic tip and the substrate.