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

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

Abstract

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
JournalMethods
Volume66
Issue number2
DOIs
StatePublished - 15 Mar 2014

Fingerprint

Molecular imaging
Molecular Imaging
Optical Imaging
Ruthenium
Fluorescence
Tissue
Semiconductor Lasers
Fixatives
Cellular Structures
Photons
Confocal Microscopy
Lasers
Coloring Agents
Laser excitation
Confocal microscopy
Semiconductor lasers
Laser pulses
Scattering
Imaging techniques
Processing

Keywords

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

Cite this

@article{2e1a5fbcb36a4127a05c8deb1ca2a7c6,
title = "Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue",
abstract = "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.",
keywords = "Confocal microscopy, Fluorescence imaging, High contrast imagine, Long-lived probe, Multi-pulse excitation, TCSPC, Time-resolved fluorescence",
author = "Rich, {Ryan M.} and Ignacy Gryczynski and Rafal Fudala and Julian Borejdo and Stankowska, {Dorota Luiza} and Raghu Krishnamoorthy and Raut, {Sangram Limbaji} and Maliwal, {Badri P.} and Dmytro Shumilov and Hung Doan and Zygmunt Gryczynski",
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pages = "292--298",
journal = "Methods",
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Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue. / Rich, Ryan M.; Gryczynski, Ignacy; Fudala, Rafal; Borejdo, Julian; Stankowska, Dorota Luiza; Krishnamoorthy, Raghu; Raut, Sangram Limbaji; Maliwal, Badri P.; Shumilov, Dmytro; Doan, Hung; Gryczynski, Zygmunt.

In: Methods, Vol. 66, No. 2, 15.03.2014, p. 292-298.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue

AU - Rich, Ryan M.

AU - Gryczynski, Ignacy

AU - Fudala, Rafal

AU - Borejdo, Julian

AU - Stankowska, Dorota Luiza

AU - Krishnamoorthy, Raghu

AU - Raut, Sangram Limbaji

AU - Maliwal, Badri P.

AU - Shumilov, Dmytro

AU - Doan, Hung

AU - Gryczynski, Zygmunt

PY - 2014/3/15

Y1 - 2014/3/15

N2 - 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.

AB - 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.

KW - Confocal microscopy

KW - Fluorescence imaging

KW - High contrast imagine

KW - Long-lived probe

KW - Multi-pulse excitation

KW - TCSPC

KW - Time-resolved fluorescence

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U2 - 10.1016/j.ymeth.2013.08.026

DO - 10.1016/j.ymeth.2013.08.026

M3 - Article

VL - 66

SP - 292

EP - 298

JO - Methods

JF - Methods

SN - 1046-2023

IS - 2

ER -