The fluorescence-based methods of single-molecule optical detection have opened up unprecedented possibilities for imaging, monitoring, and sensing at a single-molecule level. However, single-molecule detection methods are very slow, making them practically inapplicable. In this paper, we show how to overcome this key limitation using the expanded laser spot, laser excitation in a nonfluorescent spectral window of biomolecules, and more binding fluorescent molecules on a biomolecule that increases the detection volume and the number of collected photons. We demonstrate advantages of the developed approach unreachable by any other technique using detection of single cardiac troponin-T molecules: (i) 1000-fold faster than by known approaches, (ii) real-time imaging of single troponin-T molecules dissolved in human blood serum, (iii) measurement of troponin-T concentration with a clinically important sensitivity of about 1 pg/mL. The developed approach can be used for ultrafast, ultrasensitive detection, monitoring, and real-time imaging of other biomolecules as well as of larger objects including pathogenic viruses and bacteria.
Keywords: cardiomarkers; real-time bio-imaging; single biomolecule detection; single-molecule counting techniques in sensorics; troponin-T.