Simple multipulse excitation for enhanced detection of long-lived fluorophores

Joseph Kimball, Dmytro Shumilov, Rafal Fudala, Sangram Limbaji Raut, Ignacy Gryczynski, Julian Borejdo, Irina Akopova, Ryszard Grygorczyk, Zygmunt Gryczynski

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review


Typically the signal-to-background ratio is the limiting aspect of fluorescence-based detecting and imaging. The background signal can be composed of a variety of sources-excitation scattering, contaminants, and autofluorescence from cellular constituents. Most of these sources have a short-lived lifetime (ps to ns range). In order to increase the signal-to-background ratio, fluorophores with high brightness or in large concentrations are typically used along with time-gated detection. This unfortunately sacrifices the probe's signal unless it has a very long lifetime. Herein we are presenting a simple method to enhance the detection of widely available and well-known mid-range lifetime (∼20 ns) fluorophores' signal against short-lived backgrounds. This requires a repetition rate of ∼300 MHz to pump a 20 ns probe sufficiently. Typical laser sources today are not equipped with repetition rates above 80 MHz. However, this multipulse method allows these rates to be attainable for nearly any pulsed laser source. Multiple pulses of excitation are separated by a variable temporal length, which is short compared to the lifetime of the long-lived fluorophore, to increase the excited state population of a long-lived fluorophore, while the short-lived background decays almost completely between pulses. This is accomplished by simply redirecting the pulsed excitation beam through glass and then a delay length any number of times and lengths as desired to control the number of pulses and separation times.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VII
ISBN (Print)9780819498632
StatePublished - 1 Jan 2014
EventSingle Molecule Spectroscopy and Superresolution Imaging VII - San Francisco, CA, United States
Duration: 1 Feb 20142 Feb 2014


OtherSingle Molecule Spectroscopy and Superresolution Imaging VII
Country/TerritoryUnited States
CitySan Francisco, CA


  • Long lived probe
  • Multiple pulse excitation


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