Forbidden light detection from single molecules

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to mor e than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to similar to 10(-1 7) L. This allows for an exceptional discrimination of bulk-generated again st surface generated fluorescence, which may be of great value when surface -binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determin ing antigen concentrations down to 5 fmol.