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LaserStrobe™
Fluorescence Lifetime Spectrofluorometer

 
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Fluorescence Lifetimes

 

 

Applications

LaserStrobe™ Application Areas

Membrane Fluidity

One of the classical biological applications of time-resolved fluorescence spectroscopy. Typically, an elongated hydrophobic (i.e. water insoluble) molecule is used as a probe (e.g. DPH) and the anisotropy decay is measured. Due to topology of the membrane, the probe rotations are limited to the space within a cone. The rotational correlation time and the cone angle are obtained from the anisotropy data.

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Protein Structure and Dynamics

A distance between two protein sites labeled with an energy donor and acceptor can be measured via FRET (fluorescence resonance energy transfer). Energy transfer efficiency is obtained from a decrease of the donor lifetime. Segmental and global motion of proteins can be studied by anisotropy decays of intrinsic (tryptophan, tyrosine) or artificial fluorescence probes. Localization of certain groups in proteins (e.g. accessibility to water) can be studied by the effect of external quenchers on the lifetime of the probe. Protein folding/unfolding can be monitored by changes in the probe lifetime and/or rotational correlation time.

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Properties of Excited States

A very basic research area, where a fluorescing molecule is a research object on its own rather than a means of studying something else. Fluorescence lifetimes are measured in order to gain insight about the nature of electronic transition, determine radiative and nonradiative rate constants, follow excited state relaxation processes, intrinsic changes in molecular geometry, electron and energy transfer, interactions with solvent, etc.

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Surfactants (micelles)

Micelles are molecular aggregates that are formed when soaps and detergents are dissolved in water. Fluorescence decays are used mainly to determine micelle aggregation number, critical micelle concentration, polydispersity (i.e. distribution of micelle sizes) and diffusion rates in micelles.

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Polymers

Time resolved fluorescence is used to study intermolecular chain dynamics, end-to-end distances, secondary structure, viscosity, and association of polymers. Usual techniques are: FRET, anisotropy decays, excimer or exciplex formation and lifetimes.

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Time-Resolved Luminescence Applications

Biological Surfactants Polymers

  • Oligonucleotide conformation via FRET
  • End-to-end distance distribution in oligonucleotides
  • Intercalation into nucleic acids
  • Time-resolved fluorescence and phosphorescence immunoassays
  • Membrane (lipid, phospholipid) phase transitions
  • Membrane polarity and fluidity
  • Ion transport across membrane
  • Membrane heterogeneity
  • Conformational changes in proteins (enzymes)
  • Protein folding/unfolding
  • Protein/membrane interaction with drugs
  • Time-resolved fluorescence of porphyrin/chlorophyll in photosynthesis
  • Protein aggregation, size and shape
  • Fluorescence lifetime microscopy in single cells
  • Time-resolved autofluorescence of tissue (cancer research)
  • Sensitizers for photodynamic therapy
  • Studies of vision pigments

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Surfactants

  • Determination of micellar aggregation
  • Numbers determination of critical micelle concentration (CMC)
  • Microviscosity and of micelles
  • Solubilization of organics in micelles
  • Quenching and diffusion in micelles

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Polymers

  • Latex film formation
  • Polymer-surfactant interactions
  • Polymer and copolymer association
  • Properties of cellulose
  • Microviscosity of polymers
  • Curing of polymers

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Lifetime-Based Luminescence Sensing

  • Oxygen sensing
  • Glucose sensing
  • Chloride and heavy atom sensing
  • Temperature sensing

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Molecular Photophysics and Photochemistry

  • Lifetimes of excited states
  • Excimer and exciplex formation kinetics
  • Triplet excimers
  • Excited state electron transfer
  • Excited state proton transfer
  • Resonance energy transfer
  • Solvation dynamics
  • Molecular isomerization
  • Sensitized phosphorescence
  • Room temperature phosphorescence

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Inorganic Luminescence

  • Time-resolved luminescence of doped crystals
  • Photoluminescence of phosphors
  • Characterization of electroluminescent phosphors
  • Development of laser diodes and LEDs

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Environmental

  • Binding of organic solutes to humic acids (soil research)
  • Detection and identification of aromatics in environment
  • Surfactant-mediated oil recovery
  • Studies on detoxification of environment polluted by hydrophobic organic compounds Characterization (finger-printing) and detection of crude oils

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Petroleum Research

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Others

  • Surface photochemistry
  • Studies of zeolites
  • Supra-molecular systems
  • Molecular switches
  • Photographic materials
  • Langmuir-Blodgett films
  • Agriculture (sensitizers for optimum wavelengths for crops)
  • Development and properties of laser dyes
  • Inclusion complexes with cyclodextrins (pharmaceutical)
  • Time-resolved fluorescence chromatography

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General Applications

Time-Resolved Spectra of Pgp

PTI--LaserStrobe

Pgp (P-glycoprotein, courtesy of Prof. Frances Sharon, Univ. of Guelph). The Pgp protein is important in cancer research, as it is responsible for multi-drug resistance of the cell. The Time-Resolved Spectra (TRES) were reconstructed from decay curves measured at different emission wavelengths and the time delay between two adjacent curves is 0.5 ns (spectra shift from left to right). The TRES show temporal evolution of the fluorescence spectrum coming from multiple tryptophans of Pgp during the excited state lifetime. This spectral evolution may be caused by intrinsic heterogeneity of Trp moieties and/or excited state relaxation processes.

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Human Serum Albumin Decay

Human Serum Albumin (HSA) decay and analysis courtesy of Dr. John Brennan, McMaster University, Hamilton, Ontario. A 3-exponential model was used to fit the data resulting in the following parameters: a1 = 0.18, t1= 0.45 ns; a2 = 0.25, t2 = 3.10 ns; a3 = 0.15, t3 = 6.51ns.

PTI

PTI

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Fluorescence Decay of BSA

PTI

Fluorescence decays of Bovine Serum Albumin (BSA) illustrating an effect of protein unfolding (denaturation) on the protein lifetimes. The first decay is that of BSA in buffer (native, folded structure) and the second one is with a micellar detergent SDS added, which causes the BSA to unfold. The decays were acquired with an arithmetic timescale.

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Luminescence Decay of Chelated Europium Crystal

PTI

We are the only company to offer logarithmic time scaling due to our unique technique. This illustration show how you can acquire and analyze vastly different lifetimes on complex decay kinetics with several different lifetimes as well as rise time. This is done with a small number of data points in a single decay acquisition. (Sample courtesy of Dr. Mary Berry, Univ. Of South Dakota)

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Felix Software Application Templates

Fluorescence Decay: Laser

Int Time
This is the time window in microseconds within which the signal is integrated for each laser pulse. The window should be long enough so that the emission signal is fully contained within it. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

PTI

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Phosphorescence Decay: Xenon Flash Lamp

Int Time
This is the width of the integration window for each lamp pulse. Since, in this case, the observation window is defined by the integration time, it is normal to choose the integration time to be comparable to the channel spacing. Choosing an integration time of 1000 µs when the channel spacing is only 1 µs loses time resolution while choosing an integration time of 1 µs when the channel spacing is 100 µs loses sensitivity.

Shots
Enter the number of lamp pulses to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time. For a XenonFlash, 20 shots is an acceptable number.

Frequency
The lamp frequency can be set up to 100 Hz. For very long-lived samples, the phosphorescence from one pulse may not have completely decayed before the next pulse arrives. At least ten sample lifetimes should be allowed between each lamp pulse. Thus a lamp frequency of 100 Hz may be used for samples whose lifetimes are shorter than 1000 µs.

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Phosphorescence Decay: Laser

PTI

Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time it is normal to choose the integration time to be comparable to the channel spacing. Choosing an integration time of 100 µs when the channel spacing is only 1 µs loses time resolution while choosing an integration time of 1 µs when the channel spacing is only 100 µs loses sensitivity.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Fluorescence Time Resolved Spectra: Laser

Int Time
This is the time window in microseconds within which the signal is integrated for each pulse. The window should be long enough so that the emission signal is fully contained within. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ration at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

PTI

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Fluorescence Time Resolved Spectra: Nanosecond Flash Lamp

PTI

For this system the frequency of lamp pulses is set in the hardware configuration to be 18 to 20 kHz. The electronics convert this to an essentially DC signal from the detector.

Integration
Enter the time is seconds over which the signal will be averaged for each point of each scan. Extra integration time will improve the signal to noise ratio at the expense of additional acquisition time.

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Phosphorescence Steady State Emission Scan: Laser (Gated Emission Scan)

Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time, increasing the integration time will increase the signal at the expense of lifetime resolution while decreasing the integration time will increase the lifetime resolution at the expense of signal strength. In particular, when the instrument is being used to separate fluorescence spectra from phosphorescence spectra, care must be used in selecting the integration time. Since fluorescence is essentially over in the first 5 to 10 µs after the excitation pulse, the delay should be set to the excitation peak and the integration time to 5 to 10 µs. Longer integration times will contaminate the fluorescence with phosphorescence. When collecting phosphorescence, the delay should be set 5 to 10 µs after the excitation pulse and the integration time chosen to be larger to maximize sensitivity.

Shots
Enter the number of laser shots to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ration at the expense of additional acquisition time. For statistical reasons, it is generally preferable to average over several scans than over more shots on a single scan. Thus averaging three scans with five shots each scan is better than one scan with fifteen shots.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire decay data. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Fluorescence Timebased: Laser

PTI

Points/sec
The value in this box is completely defined by the choice of shots and frequency and cannot be chosen directly.

Int Time
This is the time window in microseconds within which the signal is integrated for each laser pulse. The window should be long enough so that the emission signal is fully contained within. Set this parameter to 50 µs.

Shots
Enter the number of laser shots to be collected and averaged for each point for each scan. Extra shots will improve the signal to noise ratio at the expense of time resolution. When using a timebased experiment to adjust the instrument hardware this value is set rather low so that the effects of adjustments can be seen quickly.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies shorten the time required to acquire data and can improve time resolution. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Ten pulses per second is a reasonable choice for most experiments.

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Phosphorescence Timebased: Laser

Points/sec
The value in this box is completely defined by the choice of shots and frequency and cannot be chosen directly.

Int Time
This is the width of the integration window for each laser pulse. Since, in this case, the observation window is defined by the integration time, increasing the integration time will increase the signal at the expense of lifetime resolution while decreasing the integration time will increase the lifetime resolution at the expense of signal strength. In particular, when the instrument is being used to separate fluorescence spectra from phosphorescence spectra, care must be used in selecting the integration time. Since fluorescence is essentially over in the first 5 to 10 µs after the excitation pulse, the delay should be set to the excitation peak and the integration time to 5 to 10 µs. Longer integration times will contaminate the fluorescence with phosphorescence. When collecting phosphorescence, the delay should be set 5 to 10 µs after the excitation pulse and the integration time chosen to be larger to maximize sensitivity.

Shots
Enter the number of laser pulses to be collected and averaged at each point for each scan. Extra shots will improve the signal to noise ration at the expense of time resolution. When using a timebased experiment to adjust the instrument hardware this value is set rather low so that the effects of adjustments can be seen quickly.

Frequency
This determines the frequency of laser firing and may be set at up to 20 Hz. Higher frequencies can improve time resolution. However, the consumption of nitrogen gas increases substantially at higher frequencies and the energy per pulse drops. Smaller frequencies may be useful when very long timebases are run, otherwise extremely large amounts of data will be collected.

PTI

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