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LaserStrobe™
Fluorescence Lifetime Spectrofluorometer

 
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Fluorescence Lifetimes

 

 

Buyer's Guide

Looking for a compact bench-top time-resolved system instead?

EasyLife Fluorometer

If you are looking for a less expensive, simpler or easier to use luminescence system, we invite you to consider the compact, bench-top luminescence instruments offered by our sister company Optical Building Blocks Corporation (OBB). OBB provides simple, affordable, filter based systems for time-resolved luminescence research. For more information visit www.obbcorp.com/systems.

Each of PTI's diverse LaserStrobe™ systems is designed with particular user needs in mind. The LaserStrobe™ spectrofluorometer is designed for the user who needs the most powerful L-format system for the time-resolved fluorescence spectroscopy using a short time pulsed nitrogen laser.

LaserStrobe

Equipped with a computer-controlled scanning emission monochromator for instant wavelength selection and direct time-resolved spectra acquisition, the LaserStrobe™ provides unsurpassed wavelength coverage from 240 nm (frequency-doubler option required) to 990 nm. Very intense pulses make it ideal for weak samples, while the low repetition rate prevents photodecomposition. The LaserStrobe™ is suitable for virtually all fluorescence lifetime applications.

Recommended applications include:

  • Lifetimes of very weak or diluted fluorophores
  • Complex, heterogeneous kinetics
  • Lifetime and time-resolved spectra of tyrosine and tryptophan fluorescence in proteins (frequency-doubler option required)
  • Micellar studies
  • Polymers
  • Membranes
  • Porphyrins and chlorophylls
  • Excited state electron and proton transfer
  • Analytical and environmental assays
  • Phosphors, LEDs and solid state devices
  • Fluorescence resonance energy transfer (FRET)
  • Time-resolved fluorescence anisotropy

Data is collected and analyzed by proprietary Windows-based advanced fluorescence software with exclusive NTDA (non-linear timescale data acquisition) capability.

 

General Information

The LaserStrobe ™ is an L-format cuvette-based lifetime spectrofluorometer capable of making fast and simple fluorescence lifetime measurements from the ultraviolet to the visible, from 100 picoseconds to tens of microseconds, with single or multiple exponential decays. The illumination source is a pulsed nitrogen dye laser. The excitation wavelength selection is by means of various dyes used in the dye laser (in addition to the nitrogen line at 337.1 nm). Emission wavelength selection is by means of a ¼ meter type monochromator. The QuadraCentric™ sample compartment comes standard with a 10 x 10 mm cuvette holder. The compartment is roomy and will accept various options such as polarizers, solid or powdered sample holders, and other accessories.

The modular architecture of the system allows for many additional options and accessories at any time as your budget allows or to meet your changing needs.

The system comes complete with electronics, acquisition and analytical software, hardware interface and computer, as well as installation.

The Evolution of Fluorescence Lifetime Instruments

PTI's fluorescence lifetime instrument development team was established in 1975 at the dawn of modern fluorescence lifetime instrumentation. We would like to believe we have made a significant contribution towards its evolution.

In 1975, an instrument using the time correlated single-photon counting (TCSPC) technique was introduced by PTI's development team. Although there have been many new developments in TCSPC over the years, no one has yet been able to make it affordable—a major drawback that has hampered the growth of fluorescence lifetime measurements.

While TCSPC produces extremely accurate results, it also requires a considerable length of time to make measurements—hours, in most cases. This characteristic severely restricted its uses in the life science area where unstable samples are intolerant measurement times.

Shortly after the introduction of TCSPC, the Phase, or phase modulation, technique became available as an alternative. Phase was cheaper and could measure faster, but because it only operated at a few frequencies, it was of limited use in the accurate measurement of fluorescence lifetimes.

The development of multi-frequency Phase in the 1980's improved the usefulness of Phase dramatically by allowing it to measure more complex lifetimes with improved accuracy. Unfortunately, this innovation raised the cost of Phase beyond the already expensive TCSPC instruments, and also increased the complexity of operation. Although still faster than TCSPC, measurement times increased.

Until, recently, TCSPC was the technique of choice if you wanted accuracy - assuming that your sample was stable over the length of the measurement time required. If you wanted rapid data acquisition, Phase would be the most appropriate choice. Both methods, though, are expensive and complicated to use.

PTI recognized that, if fluorescence lifetime measurements were to become more widely used, a technique would have to be commercialized that had the accuracy of TCSPC, the speed of Phase, but would be inexpensive and easy to operate.

The strobe technique was first described in the literature in 1960. Unfortunately, the electronics and computers of that time were not sophisticated enough for the theory to be put into practical use.

By 1987, advancements in electronics allowed PTI to introduce the patented LS-100 Strobe-based system for the measurement of lifetimes. The LS-100 offered both speed and accuracy, and it was easy to operate. But while less costly than TCSPC or Phase systems, it was still expensive.

With the introduction of the patented StrobeMaster and LaserStrobe™ fluorescence lifetime systems, PTI has achieved its goal.

A Strobe system that has accuracy of TCSPC and is faster than Phase— yet it is easy to operate and costs about the same as a steady state fluorometer!

LaserStrobe™ System

The patented LaserStrobe™ is based on the Strobe technique for measuring fluorescence lifetimes.  In addition to the benefits offered by the Strobe-Master, the LaserStrobe’s nitrogen/dye laser source provides a clean 500 picosecond laser pulse for measuring lifetimes below one nanosecond with greater precision. Excitation wavelengths extend to 990 nanometers, and the optional frequency doubler allows for powerful UV output down to 235 nanometers.  The only commercially available fluorescence lifetime system that can surpass the StrobeMaster in measurement speed is the LaserStrobe™ and is still very affordable.

The LaserStrobe™ base system includes PTI’s nitrogen/dye laser excitation system, all Strobe electronics, a spacious sample compartment with single 10 mm cuvette holder, two filter holders and a lid-activated emission port shutter, detector housing with power supply and photomultiplier tube, FeliX™ software package, reference manual, installation, training, and a one-year warranty.

PTI Lifetime Techniques

PTI lifetime instruments employ two basic techniques: our patented, award winning Stroboscopic Technique for time-resolved fluorescence and our Gated Voltage-Controlled Integrator (VCI) for phosphorescence. Both techniques utilize pulsed light sources.

The Stroboscopic Technique
The Stroboscopic Technique is the simplest, fastest and the most direct way of measuring fluorescence lifetimes. It employs either our high-performance thyratron-gated nanosecond flash lamp or our powerful pulsed nitrogen/dye laser system, which features extraordinary wavelength coverage. In fact, the laser source, with an optional frequency doubler, can cover the excitation wavelength range from 235 nm to over 900 nm in an almost continuous fashion. The key feature of the stroboscopic technique is a pulsed photomultiplier as part of the detection system. A very short electrical pulse, whose timing is synchronized by a high precision crystal clock with the optical excitation pulse, activates the photomultiplier. The timing of the clock is under software control. By repetitive pulsing and by varying the timing of the gating pulse, the decay curve is recorded by measuring the photocurrent on the photomultiplier. The pulsed photomultiplier is extremely sensitive and achieves excellent temporal resolution. The timing is very precise, with steps as small as 5 ps. As a direct consequence of software control over the timing clock, the stroboscopic technique has a unique ability to acquire data with a nonlinear time base and at random time intervals. For example, with the use of our Logarithmic Timebase Acquisition protocol it is possible to measure lifetimes differing by four orders of magnitude in one single experiment! The Random Acquisition mode, on the other hand, eliminates bias when measuring samples that are inherently unstable. The measurement of time-resolved spectra is as easy as with the steady state system: the required time delay after the flash is entered in the software menu and the emission monochromator scans the desired wavelength range.

The Gated VCI Technique
The technique for measuring phosphorescence is also very simple. Since the time domain is microseconds and longer, the triggering of the light source, (either our versatile Xenon flash lamp or the pulsed nitrogen/dye laser) and the timing of the readout gate of the detector signal are under software control. This way, a decay curve is easily recorded by moving the detector gate over the time window of interest or a time-resolved spectrum is measured by fixing the gate position and scanning the monochromator. By varying the gate position and the gate width, fluorescence and phosphorescence spectra can be easily separated due to their lifetime difference.

Complementary Fluorescence Techniques

There are two main types of luminescence: Fluorescence and Phosphorescence. Both represent emission of light from photo-excited molecules or atoms. The rules that govern these emissions are described by quantum mechanics. The main distinction between the two processes is that fluorescence is an allowed transition, i.e. it happens between electronic states of the same multiplicity (e.g. singlet-tosinglet), while phosphorescence is a forbidden transition between states of different multiplicity, i.e. triplet-to-singlet. This determines the time scale for these transitions: fluorescence is a much faster process and the emission of a photon happens typically in some hundreds of picoseconds to hundreds of nanoseconds after the excitation, while phosphorescence emission, since it is forbidden, takes much longer, typically on the microsecond-to-second time scale. The difference in the lifetimes makes these two techniques complementary, e.g. some studied phenomena that happen on a faster time scale will affect fluorescence of the probe molecule, while slower events will be imprinted on phosphorescence. PTI open architecture design permits combining these two measurements in a single system thus providing an unsurpassed capability of measuring lifetimes spanning 10 orders of magnitude with the same instrument.

Applications using time-resolved luminescence have been growing very rapidly during the last decade. They encompass very diverse disciplines, ranging from such traditional areas as photochemistry, photophysics and photobiology to medicine, numerous applications in industry and even agriculture. The lifetime not only reflects intrinsic properties of the excited molecule, but is also affected strongly by properties of the environment and by various interactions with surrounding molecules. For example, conformational changes in proteins, nucleic acids or other macromolecules can be monitored and detected by measuring the emission decay of the probe molecule. Distances between two chromophore groups of a macromolecule can be determined by studying the fluorescence resonance energy transfer (FRET), which changes the lifetime of the donor molecule. Information about polarity, viscosity of the environment, ion transport, and local electric field will be "imprinted" in the lifetime of the excited molecule. Emission decays often show more than one lifetime and in some cases are described by even more complex kinetic behavior. PTI systems include a complete software package, which contains analysis modules for virtually any time-resolved application. Another important technique is time-resolved emission anisotropy, which is an emission decay measurement with polarized light. The temporal behavior of anisotropy depends on rotational freedom of the emitting molecule and thus can be used to determine microviscosity of a membrane, the size of a protein, to follow folding and unfolding of a protein or to monitor curing processes of polymers.

If you are looking for a less expensive, simpler or easier to use luminescence system, we invite you to consider the compact bench-top luminescence instruments offered by our sister company Optical Building Blocks Corporation (OBB). OBB provides simple, affordable, integrated bench-top systems for steady state and time-resolved luminescence research. For more information visit www.obbcorp.com

 
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