Generating multiple-pulse bursts for enhanced fluorescence detection

Dmytro Shumilov, Ryan M. Rich, Ignacy Gryczynski, Sangram Limbaji Raut, Karol Gryczynski, Joe Kimball, Hung Doan, Thomas J. Soørensen, Bo W. Laursen, Julian Borejdo, Zygmunt Gryczynski

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)

Abstract

The signal-to-background ratio is the limiting factor for fluorescence based detection, sensing, and imaging. A typical background signal will include direct scattering of excitation and Raman scattering of the sample as well as autofluorescence from the sample and additives. To improve the signal-to-background ratio, fluorophores of high brightness and/or high concentration of the fluorophores need to be used. Most of the background is instantaeous and short-lived (picosecond to nanosecond time scale), and using long-lived fluorescence probes combined with time-gated detection allows for significant suppression of unwanted background. Unfortunately, this approach requires substantial sacrifice of the probe signal in order to sufficiently filter the background unless the fluorescence lifetime of the probe is very long. However, long lived probes like ruthenium bipyridyl have relatively low brightness compared to probes that have shorter, 10-30 ns fluorescence lifetimes. We recently presented an approach based on bursts of multiple pulses that allowed for high probe signal amplification using long-lived ruthenium based probe (Ru) and an 80 MHz repetition-rate laser excitation. Unfortunately, Ru represents an extreme case for probe lifetime, and a probe with a shorter lifetime of 20 ns will require excitation from a pulsed source with much higher repetition rate to significantly enhance its signal. Such high repetition rates are not possible to generate with most of today's available electronics. In this report we present new approaches to optimize and generate bursts of pulses with high repetition rate within the burst and no need for new or improved electronics. The high repetition rates originate from a low-repetition source and are highly tunable. We demonstrate that a burst of 2-10 pulses spaced 3 ns apart (corresponding to a 'burst repetition rate' of 330 MHz) allows for high signal enhancement of the 20 ns probe over the sub-nanosecond/nanosecond background. Such an approach can be applied for any sensing format, allowing much higher sensitivity for detection. Since the energy of a single pulse is spread over a few pulses in the burst, the fluorophore's photostability also improves.

Original languageEnglish
Article number024009
JournalMethods and Applications in Fluorescence
Volume2
Issue number2
DOIs
StatePublished - 1 Jun 2014

Fingerprint

bursts
Fluorescence
fluorescence
repetition
probes
pulses
Fluorophores
life (durability)
Ruthenium
ruthenium
Luminance
brightness
Electronic equipment
excitation
2,2'-Dipyridyl
Laser excitation
scattering
electronics
format
Amplification

Keywords

  • Fluorescence based imaging
  • Fluorescence detection
  • Fluorescence microscopy
  • Long lived fluorophores
  • Multi-pulse

Cite this

Shumilov, Dmytro ; Rich, Ryan M. ; Gryczynski, Ignacy ; Raut, Sangram Limbaji ; Gryczynski, Karol ; Kimball, Joe ; Doan, Hung ; Soørensen, Thomas J. ; Laursen, Bo W. ; Borejdo, Julian ; Gryczynski, Zygmunt. / Generating multiple-pulse bursts for enhanced fluorescence detection. In: Methods and Applications in Fluorescence. 2014 ; Vol. 2, No. 2.
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Generating multiple-pulse bursts for enhanced fluorescence detection. / Shumilov, Dmytro; Rich, Ryan M.; Gryczynski, Ignacy; Raut, Sangram Limbaji; Gryczynski, Karol; Kimball, Joe; Doan, Hung; Soørensen, Thomas J.; Laursen, Bo W.; Borejdo, Julian; Gryczynski, Zygmunt.

In: Methods and Applications in Fluorescence, Vol. 2, No. 2, 024009, 01.06.2014.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Generating multiple-pulse bursts for enhanced fluorescence detection

AU - Shumilov, Dmytro

AU - Rich, Ryan M.

AU - Gryczynski, Ignacy

AU - Raut, Sangram Limbaji

AU - Gryczynski, Karol

AU - Kimball, Joe

AU - Doan, Hung

AU - Soørensen, Thomas J.

AU - Laursen, Bo W.

AU - Borejdo, Julian

AU - Gryczynski, Zygmunt

PY - 2014/6/1

Y1 - 2014/6/1

N2 - The signal-to-background ratio is the limiting factor for fluorescence based detection, sensing, and imaging. A typical background signal will include direct scattering of excitation and Raman scattering of the sample as well as autofluorescence from the sample and additives. To improve the signal-to-background ratio, fluorophores of high brightness and/or high concentration of the fluorophores need to be used. Most of the background is instantaeous and short-lived (picosecond to nanosecond time scale), and using long-lived fluorescence probes combined with time-gated detection allows for significant suppression of unwanted background. Unfortunately, this approach requires substantial sacrifice of the probe signal in order to sufficiently filter the background unless the fluorescence lifetime of the probe is very long. However, long lived probes like ruthenium bipyridyl have relatively low brightness compared to probes that have shorter, 10-30 ns fluorescence lifetimes. We recently presented an approach based on bursts of multiple pulses that allowed for high probe signal amplification using long-lived ruthenium based probe (Ru) and an 80 MHz repetition-rate laser excitation. Unfortunately, Ru represents an extreme case for probe lifetime, and a probe with a shorter lifetime of 20 ns will require excitation from a pulsed source with much higher repetition rate to significantly enhance its signal. Such high repetition rates are not possible to generate with most of today's available electronics. In this report we present new approaches to optimize and generate bursts of pulses with high repetition rate within the burst and no need for new or improved electronics. The high repetition rates originate from a low-repetition source and are highly tunable. We demonstrate that a burst of 2-10 pulses spaced 3 ns apart (corresponding to a 'burst repetition rate' of 330 MHz) allows for high signal enhancement of the 20 ns probe over the sub-nanosecond/nanosecond background. Such an approach can be applied for any sensing format, allowing much higher sensitivity for detection. Since the energy of a single pulse is spread over a few pulses in the burst, the fluorophore's photostability also improves.

AB - The signal-to-background ratio is the limiting factor for fluorescence based detection, sensing, and imaging. A typical background signal will include direct scattering of excitation and Raman scattering of the sample as well as autofluorescence from the sample and additives. To improve the signal-to-background ratio, fluorophores of high brightness and/or high concentration of the fluorophores need to be used. Most of the background is instantaeous and short-lived (picosecond to nanosecond time scale), and using long-lived fluorescence probes combined with time-gated detection allows for significant suppression of unwanted background. Unfortunately, this approach requires substantial sacrifice of the probe signal in order to sufficiently filter the background unless the fluorescence lifetime of the probe is very long. However, long lived probes like ruthenium bipyridyl have relatively low brightness compared to probes that have shorter, 10-30 ns fluorescence lifetimes. We recently presented an approach based on bursts of multiple pulses that allowed for high probe signal amplification using long-lived ruthenium based probe (Ru) and an 80 MHz repetition-rate laser excitation. Unfortunately, Ru represents an extreme case for probe lifetime, and a probe with a shorter lifetime of 20 ns will require excitation from a pulsed source with much higher repetition rate to significantly enhance its signal. Such high repetition rates are not possible to generate with most of today's available electronics. In this report we present new approaches to optimize and generate bursts of pulses with high repetition rate within the burst and no need for new or improved electronics. The high repetition rates originate from a low-repetition source and are highly tunable. We demonstrate that a burst of 2-10 pulses spaced 3 ns apart (corresponding to a 'burst repetition rate' of 330 MHz) allows for high signal enhancement of the 20 ns probe over the sub-nanosecond/nanosecond background. Such an approach can be applied for any sensing format, allowing much higher sensitivity for detection. Since the energy of a single pulse is spread over a few pulses in the burst, the fluorophore's photostability also improves.

KW - Fluorescence based imaging

KW - Fluorescence detection

KW - Fluorescence microscopy

KW - Long lived fluorophores

KW - Multi-pulse

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U2 - 10.1088/2050-6120/2/2/024009

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