Simple multipulse excitation for enhanced detection of long-lived fluorophores

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

doi:10.1117/12.2045432

4 March 2014 Simple multipulse excitation for enhanced detection of long-lived fluorophores

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

Author Affiliations +

Proceedings Volume 8950, Single Molecule Spectroscopy and Superresolution Imaging VII; 895005 (2014) https://doi.org/10.1117/12.2045432
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

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.

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Joseph Kimball, Dmytro Shumilov, Rafal Fudala, Sangram Raut, Ignacy Gryczynski, Julian Borejdo, Irina Akopova, Ryszard Grygorczyk, and Zygmunt Gryczynski "Simple multipulse excitation for enhanced detection of long-lived fluorophores", Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 895005 (4 March 2014); https://doi.org/10.1117/12.2045432

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KEYWORDS

Molecules

Signal detection

Glasses

Neon

Laser sources

Quantum efficiency

Scattering

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