Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue

Ryan M. Rich, Ignacy Gryczynski, Rafal Fudala, Julian Borejdo, Dorota L. Stankowska, Raghu R. Krishnamoorthy, Sangram Raut, Badri P. Maliwal, Dmytro Shumilov, Hung Doan, Zygmunt Gryczynski

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Applications of fluorescence based imaging techniques for detection in cellular and tissue environments are severely limited by autofluorescence of endogenous components of cells, tissue, and the fixatives used in sample processing. To achieve sufficient signal-to-background ratio, a high concentration of the probe needs to be used which is not always feasible. Since typically autofluorescence is in the nanosecond range, long-lived fluorescence probes in combination with time-gated detection can be used for suppression of unwanted autofluorescence. Unfortunately, this requires the sacrifice of the large portion the probe signal in order to sufficiently filter the background.We report a simple and practical approach to achieve a many-fold increase in the intensity of a long-lived probe without increasing the background fluorescence. Using controllable, well separated bursts of closely spaced laser excitation pulses, we are able to highly increase the fluorescence signal of a long-lived marker over the endogenous fluorescent background and scattering, thereby greatly increasing detection sensitivity. Using a commercially available confocal microscopy system equipped with a laser diode and time correlated single photon counting (TCSPC) detection, we are able to enhance the signal of a long-lived Ruthenium (Ru)-based probe by nearly an order of magnitude. We used 80. MHz bursts of pulses (12.5. ns pulse separation) repeated with a 320. kHz repetition rate as needed to adequately image a dye with a 380. ns lifetime. Just using 10 pulses in the burst increases the Ru signal almost 10-fold without any increase in the background signal.

Original languageEnglish
Pages (from-to)292-298
Number of pages7
Issue number2
StatePublished - 15 Mar 2014


  • Confocal microscopy
  • Fluorescence imaging
  • High contrast imagine
  • Long-lived probe
  • Multi-pulse excitation
  • Time-resolved fluorescence


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