TimeMaster™ 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.
Back to top.
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.
Back to top.
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.
Back to top.
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.
Back to top.
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.
Back to top.
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
Back to top.
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
Back to top.
Polymers
- Latex film formation
- Polymer-surfactant interactions
- Polymer and copolymer association
- Properties of cellulose
- Microviscosity of polymers
- Curing of polymers
Back to top.
Lifetime-Based Luminescence Sensing
- Oxygen sensing
- Glucose sensing
- Chloride and heavy atom sensing
- Temperature sensing
Back to top.
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
Back to top.
Inorganic Luminescence
- Time-resolved luminescence of doped crystals
- Photoluminescence of phosphors
- Characterization of electroluminescent phosphors
- Development of laser diodes and LEDs
Back to top.
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
Back to top.
Petroleum Research
- Characterization (finger-printing) and detection of crude oils
Back to top.
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
Back to top.
General Applications
Time-Resolved Spectra of Pgp
Click on image to enlarge.
Back to top. |
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 0.5ns (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. |
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.
Back to top. |
Click on image to enlarge. |
Fluorescence Decay of BSA
Click on image to enlarge. |
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.
Back to top. |
Luminescence Decay of Chelated Europium Crystal
 Click on image to enlarge.
Back to top. |
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 rinse time. This is done with a small nu8mber of data points in a single decay acquisition. (Sample courtesy of Dr. Mary Berry, Univ. Of South Dakota) |
Time-Dependent Fluorescence Anisotropy
Time-dependent fluorescence anisotropy is the correct definition of the study of polarization effects when measuring fluorescence decays; however, in common usage, it is also referred to as polarization or anisotropy measurements (the terms are roughly equivalent in meaning). This is somewhat confusing since there are also polarization or anisotropy measurements that can be performed using steady state measurement of fluorescence. In order to understand what the customer is trying to do - and hence to be able to meet his/her need - it is very important to determine precisely what the customer means.
In the following material, the subject will relate to polarization measurements that are made simultaneously with fluorescence lifetime measurements.
Polarization studies generally deal with measurement of molecular rotation. Fluorescent molecules exhibit a change in polarization of fluorescence during molecular rotation. The measurement is accomplished by using polarized light, which is accomplished by placing a polarizing filter between the light source and the sample. A second polarizing filter is placed between the sample and the detector.
Polarized fluorescence lifetime measurements are taken with the two filters in parallel (both excitation and emission polarizers have the same alignment) and crossed (excitation and emission polarizers have opposite alignments). This is generally accomplished by rotating the emission polarizer (either manually or under computer control) 90° between the two measurements.
With an L-format system, two polarizing filters are used. With a T-format system, three filters are used, and the two measurements (parallel and crossed) can be made simultaneously. Sheet polarizers are normally used where the spectral range is above 300 nm. Sheet polraizers are inexpensive, but do not work in the UV below 300 nm. Quartz Glan Thompson polarizers work below 300 nm. These polarizers are an order of magnitude greater in price than sheet polarizers.
By definition, polarizing filters pass less than 50% of the original light. Glan-Thompson polarizers, because of their more limited acceptance angle, pass considerably less than 50%. Therefore, the sensitivity of the instrument is degraded by polarization by at least a factor of two. With their superb sensitivity, PTI systems have an advantage for TCSPC and Strobe. Because steady state sources are used for the Phase technique, Phase instruments are comparable to PTI.
Back to top.
Time Resolved Spectra
Time-resolved spectroscopy (TRES) is a process whereby the excitation spectrum of a sample is measured as a function of wavelength at varying delay times following excitation with a narrow pulse of light. There are two different methods used to determine time-resolved spectra:
- Fluorescence decay data are collected within a pre selected time window and time delay as the wavelength is scanned, or
- Fluorescence decay curves are collected at various consecutive wavelengths and a computer is used now to assemble the parts of the decay curves that have the same time, and thus form a three-dimensional representation of the time-resolved spectrum.
(Insert Diagrams)
PTI's Strobe system uses either method. To perform time-resolved spectroscopy, specialized software is required, as well as a scanning monochromator on the emission channel.
To date, time-resolved spectra has not been done with the Phase technique.
Back to top.
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.
Back to top. |
 |
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.
Back to top. |
 |
Phosphorescence Decay: Laser
 |
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.
Back to top. |
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.
Back to top. |
 |
Fluorescence Time Resolved Spectra: Nanosecond Flash Lamp
 |
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.
Back to top. |
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.
Back to top. |
 |
Fluorescence 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 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.
Back to top. |
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.
Back to top. |
 |
|
Applications
Specifications
