Fluorolog and Fluorocube for Picosecond Molecular Dynamics. Lifetime Systems from HORIBA Jobin Yvon. Frequency Domain or Time Domain? Why Lifetimes?

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2 Fluorolog and for Picosecond Molecular Dynamics Time is always on your side with a lifetime system from HORIBA Jobin Yvon. Drawing on the expertise of Spex, SLM, and IBH, we ve put together solutions that fit any research need. Choose the ultimate sensitivity and precision of Time-Correlated Single-Photon Counting (TCSPC), or the speed and versatility of frequency-domain detection. Either way, you get TRUE picosecond performance. Lifetime Systems from HORIBA Jobin Yvon Why Lifetimes? Because of the unique sensitivity, selectivity, and non-destructive nature of fluorescence spectroscopy, the technique has become ever more popular, especially in biochemistry, pharmaceuticals, and materials science. It is an essential tool for DNAsequencing, cell-identification, in-vitro and in-vivo studies of biological events, clinical chemistry, and even in the frontiers of nanocrystals. Steady-state measurement of fluorescence produces an averaged picture of a substance: its absorption and resultant emission of light in the UV, visible, and IR region of the spectrum. By introducing timediscrimination, much more information is revealed. The motion, size, environment, intermolecular distances, and many other molecular parameters can be deduced from the behavior of a material s fluorescence as a function of time. For example, you can record similar fluorescence spectra from two molecular species. These two spectra, however, can be the product of very distinct, and different, mechanisms. The differences between the lifetimes recorded for each spectrum, however, could reveal that the molecule with the shorter lifetime is subject to collisional quenching by interacting with other molecules in the surrounding medium. On the other hand, the species with the longer lifetime could be trapped inside a protein, leaving it immune to external collisions. Even when spectra overlap, it may be possible to separate temporally contributions by lifetime, recording independent emission spectra at different lifetimes. This removes the interference between the two spectra. You might conclude that the difference between recording lifetime data and steady-state data is similar to the difference between a motion picture and a snapshot. Frequency Domain or Time Domain? There are two main, complementary techniques for obtaining lifetime, or dynamic, data from fluorescence samples: Frequency domain Time domain Intensity Source Sample Phase difference, Modulation, M = B/A b/a Degrees Figure 1. Frequency-domain plot of the excitation light (blue) and the fluorescent emitted response (red). Parameters extracted from comparing the excitation and emission are the modulation and the phase difference. The modulation and phase-difference are closely related to the lifetime of the fluorescence. In the frequency domain, the excitation source has its intensity modulated at a high frequency. As a result, the sample s emission is also modulated, but out of phase with the excitation. Both the phase-difference and the modulation of the emission are directly linked to the lifetime. b a B A 2

3 The Advantages of Time Domain Log(intensity) Log(intensity) Sample fluorescence Pulsed source Time Figure 2. Time-domain plot of excitation pulse (black) and the fluorescent emitted response (red). The fluorescence-intensity s decay-curve can be fitted to determine the fluorescence lifetimes of the sample. Reconvolution techniques correct for the finite duration of the excitation pulse. Time domain, when performed as TCSPC, offers the ultimate in precision and sensitivity that is inherent in photon-counting techniques. Other time-domain techniques, such as boxcar or strobe methods, lack this sensitivity, because they don t take advantage of the digital character of photons: either the photons are there, or they re not. With proper discriminators, you won t confuse photons with electronic noise. Our time-domain systems can be fitted with flash-lamps, for continuous wavelength, pulsed excitation, or our state-of-the art NanoLED sources that give an advantage in speed of acquisition, as well as being offered at wavelengths that span the UV to the near- IR. And don t forget that the time domain is easier to use on solid samples, as well as samples that require special environments, like being heated or chilled. And time-domain data is easier to acquire in the infrared. In the time domain, the sample is excited by a pulsed source with a pulse-width ideally less than the sample s lifetime. By collecting many emission photons, and timing the arrival of those photons relative to the excitation pulses, a decay trace is accumulated. From the decay trace, the lifetimes of the components can be calculated. The Advantages of Frequency Domain Frequency-domain measurements frequently can be faster and more convenient, especially with typical liquid samples in cuvettes emitting at a higher quantum efficiency. Frequency-domain measurements also produce two observables phase and modulation from which lifetimes can be calculated. Not only does this over-determine the lifetime, it also supplies additional precision at either longer or shorter lifetimes. What is more, frequency domain can be performed with a source that has continuous-wavelength output, allowing excitation anywhere in the UV visible near-ir range, to optimize emission intensity, while making it easy to switch to steady-state measurements. 3

4 Why Fluorolog? The Spex trademark of high-performance instrumentation is carried on by HORIBA Jobin Yvon. Fluorolog spectrofluorometers offer full automation, making experiments easy and reducing the chance of operator error. There are cheaper instruments without the same range that can do lifetime measurements, but not with the accuracy of the, and not with such convenience and simplicity of measurements. You would need a far larger investment in instrumentation to reach the same specifications with other techniques. Why Build Your Own System With Our Components? When your work demands a unique approach to lifetimes, or your budget is limited, you can begin with the simplest of lifetime systems, our FluoroCube, which uses filters instead of monochromators, and a single-wavelength pulsed source. From there, add monochromators when you need them, pulsed sources, and any of a host of accessories. Or start from scratch and use our photon-counting electronics, or our NanoLED pulsed sources, our picosecond detection module, etc. With proven IBH components, you become the champion of your sample and individual research. That s exactly what we did when we created the Fluorolog -TCSPC. Analyze single and multi-component fluorescence decays Figure 3 is an example of a simple lifetime-analysis in the frequency domain. The data points are fit to a single-exponential model. Two parameters are measured: the modulation and the phase-shift of the fluorescence from the sample. A least-squares simultaneous fit reveals a fluorescence lifetime of 4.03 ns. 4 Figure 3. Fluorescein in NaOH(aq), an example of single-exponential decay (τ = 4.03 ns) in the frequency domain. Note that two parameters (phase and modulation) are measured.

5 Why? Like the Fluorolog, the offers compact modular construction, so you can create exactly the instrument for your needs, without sacrificing precious bench space. Just about any configuration and detector are available. The is created by the pioneers of the coaxial nanosecond flash-lamp and pulsed UV LEDs, so we can offer the widest range of excitation sources of any manufacturer. External Ti:sapphire lasers are even compatible with the! Time-resolved spectroscopic capability is standard on the. A high-sensitivity optical system, with superior UV response, and a large aperture into the sample chamber, all highlight the. We are the only manufacturer that close-couples the detector, pre-amplifier, and discriminator to minimize data-corruption from correlated noise. You can start with a simple. Then, as your applications and budgets progress, add monochromators, other sources, and accessories. Attention to detail sets the and all our TCSPC systems apart from the rest. Why Fluorolog -TCSPC? The Fluorolog -TCSPC System combines the best of the Fluorolog and. We take the world s most sensitive steady-state spectrofluorometers, and combine them with the ultimate lifetime sensitivity of TCSPC. You can easily switch back and forth between dynamic and steady-state measurements. Choose a system as simple as a pulsed NanoLED diode, or as sweeping as a flash-lamp for excitation either way you get the best of both worlds. Many samples contain several species, or their decay mechanism involves several different fluorescent lifetimes. A multi-exponential lifetime analysis in the time domain is shown in Figure 4. Here is a graph showing the fluorescence decay-curve for HSA protein. A mathematical fit of the decay reveals three separate lifetimes, at 0.8 ns, 3.6 ns, and 7.2 ns. A fit postulating only two lifetimes is shown as inadequate. Figure 4. HSA protein exhibits tri-exponential decay, captured in the time domain. Testing various models to the data is easy with both the and the. Here a dual-lifetime model doesn t work, while three lifetimes gives excellent results. 5

6 Resolve picosecond-lifetimes Another superior characteristic of HORIBA Jobin Yvon s lifetime systems is their ability to measure ultra-short lifetimes in the picosecond range. Figure 5 shows frequency-domain data from acridine orange dissolved in benzene, revealing a lifetime of 57 ps. The fit is confirmed by the small χ 2 and low residuals. Figure 5. The s ability to resolve picosecond fluorescence lifetimes is demonstrated with acridine in benzene. The software fits a lifetime of 57 ps. Watch the time-evolution of spectra Molecules are dynamic systems, so their spectra are continuously evolving. HORIBA Jobin Yvon lifetime systems easily define the evolution of spectra with time. Figure 7 shows a time-resolved emission spectrum over the course of 160 ns, in a 3-D view, of a pyrene-excimer complex. Different emission characteristics appear when excited by different wavelengths. Individual decay-curves and spectra can be extracted for further analysis. Deconvolve multiple spectra A lifetime-resolved acquisition, or phase-resolved spectrum, can separate overlapping spectra with different lifetimes. Here, in Figure 6, the spectra of anthracene and 9-cyanoanthracene are deconvoluted within a methanol mixture using this technique in the frequency domain. Figure 7. The evolution of fluorescence spectra over a course of time can be examined with HORIBA Jobin Yvon instruments using a time-resolved emission spectrum. Here the evolution of a pyrene-excimer complex is plotted over the course of 160 ns and various emission wavelengths, using the. From the full plot, individual curves (white) can be studied, as shown below and above right. Figure 6. Using a lifetimeresolved acquisition, the has deconvoluted the overlapping spectra of anthracene (blue) and 9-cyanoanthracene (green) in methanol, using their different fluorescence lifetimes (τ anthracene = 4 ns; τ 9-cyanoanthracene = 12 ns). 6

7 Determine phosphorescence lifetimes Phosphorescence occurs over a much longer timespan than fluorescence. Using optional phosphorimeter accessories, both the and are able to examine luminescence lifetimes extending out to the millisecond or longer ranges. Figure 9 shows the phosphorescence from europium chloride, a luminescent compound with a lifetime of 112 µs, many orders Record time-resolved polarization spectra Figure 9. A phosphorescence decay of EuCl 3, recovering a lifetime of 112 µs, taken with the. Both the and can record this type of scan. of magnitude longer than fluorescence data. With optional automated polarizers, our lifetime spectrofluorometers run time-resolved emission experiments to determine: Complex rotational behavior of molecules Molecular size and shape Protein structure and dynamics Physical properties of polymers, liquid crystals, and membranes Figure 8, an anisotropy-decay acquisition, shows Figure 8. A time-resolved emission anisotropy what happens to experiment shows that perylene behaves like an molecules of the anisotropic rotor when it is dissolved in oil. aromatic hydrocarbon perylene when dissolved in oil: the two rotational axes of perylene are resolved, each with its own rotational correlation time, and perylene is shown to behave like an anisotropic rotor. Measure infrared luminescence Near-infrared emissions are widely studied in the fields of materials-science and nanotechnology. With optional near-ir detectors, HORIBA Jobin Yvon instruments can detect and analyze emissions from carbon nanotubes and other near-ir emitters. Figure 10 shows the time-resolved fluorescence of the Q-switching laser dye IR-140 that emits around 900 to 1000 nm, when excited in the visible range. Using our curvefitting software, a single-exponential fit gives a fluorescence lifetime of 1.26 ns. Because the near- IR detector is insensitive to the 408-nm NanoLED used to excite the laser dye, the inset shows the lamp profile of a NanoLED that emits at 1300 nm, a comparable model to the excitation source. Figure 10. Time-resolved near-ir emission spectrum of IR-140 laser dye in acetone. The red line shows the fit resulting in a fluorescence lifetime of 1.26 ns, when excited with a 408-nm NanoLED. The emission is > 950 nm, because the detector is sensitive to the nm range. No excitation prompt is shown because the 408 nm NanoLED is invisible to the detector. Instead, a comparable 1300-nm NanoLED s prompt is shown in the inset. Local support Who else can give you the service and applications support you need, in order to achieve the total potential from your instrument? HORIBA Jobin Yvon has full applications laboratories manned by fluorescence experts in the USA, Europe, and Asia. HORIBA Jobin Yvon s affiliates and sister companies are in the UK, Germany, France, Italy, China, Korea, and Japan. Add to this a worldwide network of representatives, and you can rest assured that you will have the support you expect only from HORIBA Jobin Yvon. 7

8 Specifications A Fluorolog - or a is a complete, stand-alone spectrofluorometer with both lifetime and steady-state modes. All HORIBA Jobin Yvon spectrofluorometers operate on user-friendly Windows XP-compatible software, offering instrument control, data-acquisition, and data-processing, with non-linear least-squares lifetime modeling. Major components Frequency synthesizers 35 W RF amplifiers Pockels-cell modulator compartment with laser-input port High-intensity CW source T-format sample compartment Reference and signal detectors Excitation and emission monochromators (single-or double-grating) SpectrAcq system controller Data-acquisition and modeling software Pulsed source Optional excitation and emission monochromator Optional x-y-z sample positioner Large sample compartment Optical interlock system Picosecond photon-counting detector Driving and timing electronics Data-acquisition and analysis software Lifetime range 10 picoseconds to 10 microseconds (for a 1 phase-shift at 250 MHz, and 0.1 modulation at 1 MHz) TCSPC <100 ps to 400 µs MCS <10 µs to minutes (subject to accessories) Frequency range () 0.1 to 310 MHz Acquisition modes Frequency domain for fluorescence dynamics Photon-counting for steady-state measurements TCSPC for fast fluorescence decays Multi-channel scaling for phosphorescence measurements Source 450-W CW xenon lamp in an air-cooled housing, with integrated power supply. Laser port standard for additional source. Choice of interchangeable flashlamps, NanoLED laser diodes, NanoLED pulsed LED sources, SpectraLEDs (for measuring longer lifetimes), or laser-input optics for external laser systems. Spectrometers All-reflective f/3.6 Czerny-Turner, with kinematic, interchangeable gratings from UV to IR. Range is nm (up to 4000 nm with optional gratings), minimum step size is nm, accuracy is ±0.5 nm (with a 1200 grooves/mm grating); resolution is 0.2 nm; speed is 80 nm/s. f/3 low time-dispersion, Seya-Namioka configuration, with concave holographic sinusoidal gratings. Monochromator has vertical dispersion to match cuvette geometry. Spectrosil-B lenses. Range is nm (to 1600 nm with optional gratings), bandwidth externally adjustable from 1 32 nm. Computer-optimized focusing with motorized lenses. Sample holder Automated, motorized, 4-position, thermostatted cuvette holder with magnetic stirrer. Temperature range = 10 C to +80 C (with optional waterbath). Cuvette holder with optional x-y-z sample positioner and facility for recirculating liquid for temperature control. Front-face holder optional. Temperature range = 70 C to +95 C with cold-finger accessory. signal-to-noise ratio 4000:1, in steady-state mode, with the Raman band of water at 397 nm, at 5-nm bandpass, excitation at 350 nm, integration time of 1 s. (First standard deviation in signal-to-background-noise at 450 nm). Detectors R928P photomultiplier tube is standard for nm response. Other optional detectors to 1100 nm. TBX-04 photomultiplier tube module, response nm. Dark counts < 80 cps. Timing jitter 250 ps FWHM. Optional cooled extended-red detectors are available to 850 nm. Optional R928P photomultiplier tube with nm response. Accessories Microwell-plate reader, phosphorimeter, temperature baths, detectors, gratings, microscope interfaces, automated polarizers, solid-sample holder, stopped-flow accessory, and more! Multiple excitation sources, front-face sample holder, cooling baths, polarizers, extended-red detectors, gratings, and more! TCSPC on Fluorolog Excitation sources Steady-state Broadband 450-W xenon arc lamp from UV to near-ir. TCSPC Fixed-wavelength Plug-and-play interchangeable NanoLED pulsed laser-diodes and Available LEDs. Wavelengths from UV to near-ir for laserdiodes and LEDs. Standard optical pulse durations are < 200 ps (< 100 ps typical) for laser-diodes, < 1.5 ns for LEDs. High repetition rate. 5000F coaxial nanosecond flashlamp with broadband output from nm. Thyratron-gated spark and pulse generator (10 Hz 100 khz). Minimum optical pulse-width is 0.6 ns. Spectrometers Czerny-Turner, with classically-ruled gratings and all reflective optics. Optional double-grating spectrometers available. For standard 1200-grooves/mm, Resolution is 0.2 nm Accuracy ± 0.5 nm Speed 80 nm/s Range nm Detectors Steady-state and TCSPC TBX-04 photomultiplier tube module, response nm. Dark counts < 80 cps. Timing jitter typically 200 ps FWHM, 250 ps maximum. Optional cooled extended-red detectors are available to 850 nm. Inquire for other wavelength ranges. Sensitivity Steady-state Signal-to-noise ratio = 4000:1 for a Raman scan of double-distilled ICP-grade water at 397 nm with 5-nm bandpass, 1 s integration time, and background noise first standard deviation at 450 nm. (with Triple Illuminator, 450 W CW Xe lamp, and R928P photomultiplier tube) Lifetimes measurable TCSPC <100 ps to 400 µs MCS (subject to accessories) Contact us for more details. We ll show you how to achieve the right instrument for your samples, for ONLY HORIBA Jobin Yvon offers both Time and Frequency Domain Systems. Because <10 µs to minutes We re serious about fluorescence. (All HORIBA Jobin Yvon companies were formerly known as Jobin Yvon) USA: HORIBA Jobin Yvon Inc., 3880 Park Avenue, Edison, NJ , Toll-Free: jobinyvon Tel: , Fax: , info@jobinyvon.com, France: HORIBA Jobin Yvon S.A.S., 16-18, rue du Canal, Longjumeau Cedex, Tel: +33 (0) , Fax: +33 (0) , Japan: HORIBA Ltd., JY Optical Sales Dept, Higashi-Kanda, Daiji Building, Higashi-Kanda Chiyoda-ku, Tokyo , Tel: +81 (0) , Germany: +49 (0) Italy: UK: +44 (0) China: +86 (0)