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 contributionResearchpeer-review

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.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VII
PublisherSPIE
Volume8950
ISBN (Print)9780819498632
DOIs
StatePublished - 1 Jan 2014
EventSingle Molecule Spectroscopy and Superresolution Imaging VII - San Francisco, CA, United States
Duration: 1 Feb 20142 Feb 2014

Other

OtherSingle Molecule Spectroscopy and Superresolution Imaging VII
CountryUnited States
CitySan Francisco, CA
Period1/02/142/02/14

Fingerprint

Fluorophores
life (durability)
excitation
Lasers
repetition
pulses
Pulsed lasers
Excited states
probes
Glass
Luminance
Laser pulses
Fluorescence
Pumps
contaminants
Scattering
Impurities
pulsed lasers
Imaging techniques
brightness

Keywords

  • Long lived probe
  • Multiple pulse excitation
  • TCSPC

Cite this

Kimball, J., Shumilov, D., Fudala, R., Raut, S. L., Gryczynski, I., Borejdo, J., ... Gryczynski, Z. (2014). Simple multipulse excitation for enhanced detection of long-lived fluorophores. In Single Molecule Spectroscopy and Superresolution Imaging VII (Vol. 8950). [895005] SPIE. https://doi.org/10.1117/12.2045432
Kimball, Joseph ; Shumilov, Dmytro ; Fudala, Rafal ; Raut, Sangram Limbaji ; Gryczynski, Ignacy ; Borejdo, Julian ; Akopova, Irina ; Grygorczyk, Ryszard ; Gryczynski, Zygmunt. / Simple multipulse excitation for enhanced detection of long-lived fluorophores. Single Molecule Spectroscopy and Superresolution Imaging VII. Vol. 8950 SPIE, 2014.
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Kimball, J, Shumilov, D, Fudala, R, Raut, SL, Gryczynski, I, Borejdo, J, Akopova, I, Grygorczyk, R & Gryczynski, Z 2014, Simple multipulse excitation for enhanced detection of long-lived fluorophores. in Single Molecule Spectroscopy and Superresolution Imaging VII. vol. 8950, 895005, SPIE, Single Molecule Spectroscopy and Superresolution Imaging VII, San Francisco, CA, United States, 1/02/14. https://doi.org/10.1117/12.2045432

Simple multipulse excitation for enhanced detection of long-lived fluorophores. / Kimball, Joseph; Shumilov, Dmytro; Fudala, Rafal; Raut, Sangram Limbaji; Gryczynski, Ignacy; Borejdo, Julian; Akopova, Irina; Grygorczyk, Ryszard; Gryczynski, Zygmunt.

Single Molecule Spectroscopy and Superresolution Imaging VII. Vol. 8950 SPIE, 2014. 895005.

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

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AU - Shumilov, Dmytro

AU - Fudala, Rafal

AU - Raut, Sangram Limbaji

AU - Gryczynski, Ignacy

AU - Borejdo, Julian

AU - Akopova, Irina

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N2 - 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.

AB - 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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KW - Multiple pulse excitation

KW - TCSPC

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U2 - 10.1117/12.2045432

DO - 10.1117/12.2045432

M3 - Conference contribution

SN - 9780819498632

VL - 8950

BT - Single Molecule Spectroscopy and Superresolution Imaging VII

PB - SPIE

ER -

Kimball J, Shumilov D, Fudala R, Raut SL, Gryczynski I, Borejdo J et al. Simple multipulse excitation for enhanced detection of long-lived fluorophores. In Single Molecule Spectroscopy and Superresolution Imaging VII. Vol. 8950. SPIE. 2014. 895005 https://doi.org/10.1117/12.2045432