Microscopy for recognition of individual biomolecules

One frontier challenge in microscopy and analytical chemistry is the analys is of soft matter at the single molecule level with biological systems as m ost complex examples. Towards this goal we have developed two novel microsc opy methods. Both employ highly specific molecular recognition schemes used by nature-the recognition of specific protein sites by antibodies and liga nds. One method uses fluorescence labeled ligands for detecting single mole cules in fluid systems like membranes (Fig. 1B). Unitary signals are reliab ly resolved even for millisecond illumination periods. The knowledge of the unitary signal from single molecules permits the determination of stoichio metries of component association (Fig. 3). Direct imaging of the diffusiona l path of single molecules became possible for the first time (Fig. 4). Usi ng linear polarized excitation, the angular orientation of single molecules can be analyzed single molecule linear dichroism, (Fig. 5), which opens a new perspective for detecting conformational changes of single biomolecules . In the other method, an antibody is flexibly linked to the tip of an atom ic-force microscope. This permits the identification of receptors in multi- component systems. Molecular mapping of biosurfaces and the study of molecu lar dynamics in the ms to s range become possible with atomic force microsc opy. (C) 1999 Wiley-Liss, Inc.