FluoTime 300 EasyTau. A fluorescence spectrometer for beginners and experts

Transcription

1 FluoTime 300 EasyTau A fluorescence spectrometer for beginners and experts PicoQuant GmbH Rudower Chaussee 29 (IGZ) Berlin Germany Phone: +49-(0) Fax: +49-(0) info@picoquant.com

2 Foreword System Software EasyTau Page 8 11 System Components Page Steady-State Spectroscopy Dear Researcher, PicoQuant has a long and successful history in instrumentation for time-resolved spectroscopy and microscopy. Our picosecond diode lasers and photon counting electronics Vision Page 4 5 Page Specifications Page 21 can be found in more than a thousand working systems around the world. Their compactness and ease-of-use enabled scientists to perform once complex measurements on a daily basis. Nonetheless, we considered that time- Time-Resolved Spectroscopy Page resolved fluorescence measurements could be made even more easier, and therefore, developed a fully automated fluorescence lifetime and steady-state spectrometer the FluoTime 300. The system is in constant development and is always adapted to match the requirements of today s System Layout Page 6 7 PicoQuant for Scientists Page 22 research and to make the acquisition of fluorescence spectra easier than before. Measurement Examples If you are interested in this outstanding instrument, please Page contact us we are always happy to discuss your individual requirements in detail. Your needs will drive our development. Phone: +49-(0) info@picoquant.com 2 3

3 Vision Vision A fluorescence spectrometer for beginners and experts Ultra high sensitivity Single photon counting for ultimate sensitivity The FluoTime 300 is an advanced research-grade fluorescence lifetime spectrometer with steady-state option. Its intuitive system software EasyTau, along with the special application targeted software wizards, allows researchers to focus on their samples, without the need to worry about the best instrument settings for their experiments. Fully automated system All adjustable parts controllable from system software Turn-key excitation sources Picosecond pulsed diode lasers and LEDs, Xenon lamps, or other excitation sources Fluorescence spectroscopy has been established as one of the designed by scientists for scientists and will surely set a new fundamental spectroscopic laboratory methods. It is used for routine quality control as well as sophisticated measurements in research labs. An ideal instrument therefore combines the need of both worlds and makes the method usable for highly trained specialists as well as for the occasional user. This ambition was the fundamental design principle of the FluoTime 300. It was standard for fluorescence spectrometers. Wide lifetime range Fluorescence lifetimes < 10 ps and up to several hundred ms resolvable Wide spectral range Fluorescence detection between UV and NIR with nm resolution Large sample chamber Accommodates various sample holders for liquids and solids Comprehensive system software Intuitive user-interface with special application targeted wizards 4 5

4 System Layout System Layout Fully automated system with high quality components Standard detector Single photon sensitive detectors Single photon sensitive Photomultiplier Tubes (PMTs) or Microchannel Plate Photomultiplier Tubes (MCP-PMTs) are The FluoTime 300 is designed for maximum light throughput and highest sensitivity. This is achieved by single Emission monochromator available for the FluoTime 300. Various detector types cover the full wavelength span from 200 nm to 1700 nm and permit fluorescence lifetime measurements as short as 10 ps with photon counting data acquisition methods and careful appropriate system configurations. design and selection of all optical components. The heart of the FluoTime 300 is a large multifunctional sample Various excitation sources chamber with fully automated optical elements to control The FluoTime 300 can be equipped with various excitation intensity and polarization of the excitation beam and the fluorescence signal. Optional detector sources. Lifetime measurements are best performed with plug and use picosecond pulsed diode lasers or LEDs. For steadystate measurements different 300 W Xenon arc lamps provide Fully automated optical components continuous excitation light between 200 nm and 900 nm. The Fully automated polarizers and signal attenuators are installed range of excitation sources is completed by a sub-microsecond in all optical beam paths. The attenuators permit adjustment of pulsed Xenon flashlamp for phosphorescence measure- signal intensity over a wide range and thus adaptation of the setup to the corresponding sample requirements. The Glan- Signal attenuation Polarizer Filter wheel Reference detector ments. Other external excitation sources, such as Titanium:Sapphire lasers, can also be used for both Thompson polarizers can be freely rotated in 0.1 steps for lifetime and steady-state measurements. anisotropy studies. They feature a 15 mm clear aperture and transmit throughout the wavelength range from 200 nm to 2000 nm. Sub-microsecond flash lamp Steady-state Xenon arc lamp Multifunctional sample holders A single cuvette holder is the standard sample holder for the FluoTime 300. It can be supplied with a liquid cooling option or even along with a cryostat. Multiple sample measurements are possible by a four-position sample changer with full temperature control. Magnetic stirrers with variable speed are available for all holders. Solid samples can also be studied using a dedicated front face sample holder. Efficient monochromators The FluoTime 300 uses Czerny-Turner monochromators that can be equipped with different high quality diffraction gratings in order to achieve a high spectral resolution down to 0.2 nm over a broad spectral wavelength range from 200 nm up to 1700 nm. Motorized slits permit an easy adjustment of each monochromator s spectral bandpass. Excitation monochromator Laser input Sample holder Polarizer Polarizer Filter wheel 6 7

5 System Software EasyTau System Software EasyTau Acquiring fluorescence spectra has never been easier Acquiring fluorescence anisotropy spectra and especially time-resolved fluorescence measurements are very often considered to be a task for experts. With the operation and analysis software EasyTau of the FluoTime 300, this prejudice will surely be overcome. The software features a number of specific application wizards that guide the inexperienced user through all necessary steps to acquire high quality fluorescence data. An alternative customized measurement mode, with full control over all system parameters and a scripting language, makes the software also attractive to experts in the field. System status at a glance The system status of the FluoTime 300 is permanently monitored and displayed. The user is always informed about all the important parameters, such as current excitation and detection wavelength, polarizer position, signal intensity, temporal resolution and sample temperature. Step 1: The user is asked to enter information about the sample and to define relevant measurement parameters, such as solvent, excitation, and detection wavelength. WIZARDS INCLUDED Excitation / Emission spectra Excitation / Emission anisotropy Intensity and lifetime kinetics Fluorescence decays Time-resolved anisotropy Time-resolved emission spectra Intelligent data storage The EasyTau software is based on unique and well structured data storage architecture. All measurement data files and all related analysis results are stored in a clearly arranged workspace. Measurement data is sorted according to the studied sample, which permits a very quick assessment of sample properties. Measuring time-resolved anisotropy Complete logging of parameters The complete system and acquisition settings for each measurement are logged and stored with each data file, and can thus be recapped at any time later. As a feature for multiuser facilities, the EasyTau software even permits to store the current user along with the measurement data. Step 2: The wizard automatically optimizes the spectrometer for best performance by varying signal intensity, temporal resolution, laser repetition rate, etc. Powerful application wizards A special feature of the EasyTau software is the application wizards concept that allows even inexperienced users to acquire high quality fluorescence data. The wizards include a considerable amount of knowledge and experience acquired in more than 15 years of fluorescence spectroscopy at PicoQuant. In essence, every wizard takes over the adjustment and optimization of the spectrometer according to the detected sample properties. It sets suited measurement conditions and even detects conflicts, such as the possible detection of Raman scattering. Step 3: After successful optimization, the fluorescence decays at the parallel and perpendicular polarizer orientation (as well as at magic angle, if selected by the user) are measured. Step 4: A second optimization run is performed in order to adjust the system for record- ing the Instrument Response Function (IRF). After the measurement, decays and IRF are stored in the hierarchical workspace for further analysis. 8 9

6 System Software EasyTau System Software EasyTau Full instrument control The EasyTau software is also attractive to experts in the field of fluorescence spectroscopy, who prefer to have full instrument control. The customized measurement mode provides full control to the user, allowing the selection and adjustment of every part of the instrument including, but not limited to, excitation and detection wavelength, polarizer position, attenuator settings, and shutter state. The customized measurement mode supports time-resolved as well as steady-state measurements. In steadystate mode it is even possible to measure intensity as a function of various parameters, including polarizer angle, time, and collection lens position as examples. All measurement results are stored in the hierarchical workspace for further analysis. The FluoTime 300 makes timeresolved measurements accessible to all laboratories. Joseph R. Lakowicz, Center for Fluorescence Spectroscopy, University of Maryland Complex data arithmetic The EasyTau software provides direct processing and analysis of the experimental data using a powerful mathematical function processor. Basic operations, such as subtractions or multiplications, and even advanced operations, such as smoothing, derivatives, integrations or trigonometric functions can be applied on either experimental data or results from previous calculations. Data can be normalized and displayed in linear or logarithmic plots with varying axis scaling. Plots can be freely zoomed, and all measurement or analysis results can be exported as presentation-ready images or as multicolumn ASCII data for further analysis. The experimental data files are not modified in any analysis step ensuring data authenticity for further analysis procedures. Scripted data acquisition Besides full instrument control, the customized measurement mode includes a very unique feature a scripting language that allows development of user-defined measurement protocols. The scripting language can access and control every single component of the FluoTime 300. It supports timeresolved as well as steady-state measurements. Through the use of control structures, loops, and mathematical functions, the scripting language allows the experienced user to develop even the most complex measurement procedures. Measurement protocols that have to follow predefined procedures will greatly benefit from the scripted data acquisition. This is often the case in routine applications such as quality control. Multiexponential decay analysis For analysis of time-resolved measurement results, a direct data link to the established FluoFit software is provided. FluoFit is a powerful global data analysis software for fluorescence decay and anisotropy measurements. Tail fitting as well as a numerical reconvolution algorithm to account for the Instrument Response Function (IRF) can be applied. The association of the experimental data curves to the decay and IRF or the different polarization orientations for anisotropy measurements is automatically performed. Exponential decay models up to 4th order or, alternatively, to different lifetime distribution models can be fitted to the decay data. Initial values for the fitted parameters are automatically estimated. Reduced chi-square, weighted residuals, and autocorrelation trace are shown for assessment of the goodness of fit. Advanced error analysis using support plane or bootstrap analysis assigns realistic confidence intervals to the fitted parameters

7 Steady-State Spectroscopy Steady-State Spectroscopy Steady-state measurements are classical tasks performed with a fluorescence spectrometer. They refer to a measurement condition in which the fluorescence is not temporally resolved, but integrated over time. Typical examples are excitation and emission spectra or steady-state anisotropy. Ultimate sensitivity Spectral correction with a mouse click The FluoTime 300 is based on single photon counting data Spectral properties of the excitation source, the monochromator, acquisition, which is the most suited method for measuring and the detector affect the recorded excitation and emission weak fluorescence with the highest dynamic range. The signalto-noise ratio achievable with this method is therefore higher these influences have to be taken into spectra. For quantitative measurements, all than for any other detection method. This allows measurements account. With the FluoTime 300, excitation spectra are easily corrected of fluorescence spectra of extremely diluted dye solutions even down to femtomolar concentration range. The sensitivity using the automatically recorded is also demonstrated by the water Raman spectrum, which signal from a built-in reference is traditionally used to compare the performance of different detector, which monitors spectrometers. With the FluoTime 300, a signal-to-noise of a fraction of the > 6500:1 can be routinely attained using a standard blue excitation sensitive detector. light. For correction of emission spectra, calibration files are delivered with each spectrometer. The correction routines are all included in the EasyTau system software and can be applied by a simple mouse click. Numerous applications Steady-state fluorescence spectroscopy can be used in many research areas, such as: analytical chemistry identify species by their excitation or emission spectra and determine their concentration photochemistry characterize photochromes or follow reaction kinetics industrial quality control monitor the quality of polymers, analyze proteins or DNA environmental science monitor traces of organic or inorganic substances in solvents or solid samples food science study growth or aging phenomena of fruits or plants Examples Examples Water Raman spectrum: For this measurement an emission spectrum of HPLC grade water in a 1 cm path length quartz cuvette was recorded from 360 nm to 450 nm with a 5 nm detection bandwidth, 1 nm step size, and 1 s integration time per wavelength. The excitation source was a 300 W coaxial Xenon arc lamp with the excitation monochromator set to 350 nm with a 5 nm bandwidth. The signal was detected with a standard blue sensitive photomultiplier. The resulting signalto-noise ratio is > 6500:1 based on the measured peak counts at 397 nm and the background signal at 450 nm. Sensitivity in the red spectral range: Maintaining high sensitivity in the spectral range between 600 nm and 900 nm requires special detectors and balanced performance of the other spectrometer components. The result shows a benchmark test using a popular red fluorescent dye, ATTO 655-COOH dissolved in aqeous buffer (PBST). The sample was excited at 640 nm using a vertically polarized cw laser. The fluorescence was detected at magic angle conditions at 1 s accumulation per wavelength step using a standard red sensitive cooled PMT. The signal intensity (inset) at the peak wavelength shows excellent linear dependence on sample concentration down to 10 pm. At this concentration the emission intensity is equal to the Raman scattering intensity of the water. By subtracting the corresponding blank spectrum (blue) from the measured intensity (green) it is still possible to recover the correct ATTO 655 emission spectrum (red). As a comparison, the emission curve recorded at 100 pm concentration is shown (black dashed), scaled for comparison. This sensitivity limit can be pushed further down to 1 pm by removing the emission polarizer, increasing the excitation power or increasing the accumulation time

8 Time-Resolved Spectroscopy Time-Resolved Spectroscopy In time-resolved measurements the detected fluorescence photons are registered with respect to the last excitation pulse. This allows the study of phenomena ranging from the picosecond to the millisecond time scale. Typical examples are measurements of the fluorescence and phosphorescence lifetime or time-resolved anisotropy. Measurement results within seconds Dynamics from ps to ms The time-resolved data acquisition of the FluoTime 300 uses the With TCSPC, a fast Microchannel Plate Photomultiplier Tube method of Time-Correlated Single Photon Counting (TCSPC), (MCP-PMT), and excitation sources with short pulse widths and which is the most sensitive and precise method to measure high repetition rates such as picosecond pulsed diode lasers, fluorescence lifetimes. The raw measurement result directly lifetimes down to a few picoseconds can be resolved. represents the temporal change in fluorescence intensity and By changing the repetition rate of the excitation sources, which is thus very intuitive. By plotting the data in logarithmic scale, is easily accomplished with picosecond pulsed diode lasers, it is easy to check whether decays exhibit only a single lifetime TCSPC can also be used to measure lifetimes up to several or multiple lifetimes even polarization effects can be instantly hundred nanoseconds. Even phosphorescence studies with observed. With TCSPC, fluorescence decays can be measured lifetimes up to a few milliseconds can be performed with the in seconds or even fractions of a second. FluoTime 300 along with a dedicated Multichannel Scaling (MCS) board and excitation sources such as pulsed Xenon flash lamps. A probe for the molecular environment The fluorescence lifetime is characteristic for each fluorophore, and can thus be used to characterize a sample. It is, however, also influenced by the chemical composition of its environment. Additional processes like Förster Resonance Energy Transfer (FRET), quenching, charge transfer, solvation dynamics, or molecular rotation also have an effect on the decay kinetics. Lifetime changes can therefore be used to gain information about the local chemical environment, to follow reaction mechanisms, or as a tool for quality control in industrial applications. Acquiring fluorescence spectra has never been easier. Zygmunt Gryczynski, Texas Christian University Numerous applications Time-resolved fluorescence spectroscopy can be used in many research areas, such as: analytical chemistry identify or separate species by their fluorescence lifetime, monitor changes in the environment biochemistry study protein folding or signaling pathways photobiology detect singlet oxygen for photodynamic therapy biophysics study membrane rigidity or enzyme/substrate interactions industrial quality control monitor the quality of wafers, semiconductors, or solar cells Examples Principle of TCSPC Counts Start-Stop-Time 1 Start-Stop-Time 2 Fit of an exponential decay function to the histogram envelope yields the fluorescence lifetime Start-Stop-Time Time-Correlated Single Photon Counting (TCSPC) is based on the precise measurement of the time difference between the moment of excitation and the arrival of the first fluorescence photon at the detector. The measurement of the time difference is repeated many times to account for the statistical nature of fluorescence emission and all measured time differences are sorted into a histogram. This histogram of photon arrival times can then be analyzed to extract the fluorescence lifetime and signal amplitude. Lifetimes shorter than instrument response Fluorescence decays usually follow an exponential dependence on time. Extracting the fluorescence lifetime is therefore easily possible by fitting a suited exponential decay function to the experimental data, taking the characteristics of the instrument into account. These characteristics, which include e.g., the finite pulse width of the excitation source, are commonly described by the Instrument Response Function (IRF), which can be precisely measured with the FluoTime 300. Elaborate data analysis corrects for the finite width of the IRF, and thus, allows resolving fluorescence lifetimes much shorter than the IRF itself. A state-of-the-art fluorescence spectrometer with outstanding fluorescence lifetime capabilities. Niko Hildebrandt, Université Paris-Sud Fluorescence lifetime analysis of a 1 µm Fluorescein solution in ph 10 phosphate buffer. The plot shows the acquired TCSPC histogram (blue) along with the additionally measured Instrument Response Function, IRF (red). During the analysis process, the finite width of the IRF is taken into account by a numerical reconvolution process. The weighted residual trace (bottom panel) clearly shows an excellent agreement between fit and experimental histogram, which would not be possible without treatment of the IRF. The recovered lifetime is 3.99 ± 0.01 ns. The measurement was completed in less than 10 seconds

9 Measurement Examples Measurement Examples Lifetime shorter than instrument response The excellent stability of the laser, the good temporal resolution of the compact PMT detector, and the low jitter of the timing electronics make it possible to routinely resolve decay times on the order of tens of picoseconds, even without a MCP-PMT detector. The figure shows the fluorescence decay of a 100 nm solution of Erythrosin B in distilled water. The sample was excited with 531 nm laser light (laser pulse width 72 ps, FWHM) and the fluorescence was detected at 550 nm ± 3 nm through a polarizer set at magic angle conditions. The IRF was determined to be 200 ps (FWHM). Data analysis using numerical reconvolution resulted in a single liftetime of 89 ps ± 4 ps (χ 2 =1.08), which is Spectral correction Spectral properties of the excitation source, the monochromator, and the detector affect the recorded excitation and emission spectra. With the FluoTime 300, these influences can be corrected using the built-in reference detector and the calibration files delivered with each FluoTime 300 system. The effect of spectral correction is shown for a 10 nm Fluorescein (free acid) solution in ph=9 buffer. Excitation spectrum recorded at 550 nm detection wavelength. Emission spectrum recorded with 485 nm excitation. A spectral bandpass of 2 nm was set on both monochromators. The effect of spectral correction is clearly visible and demonstrates the necessity of this procedure for obtaining quantitative data. in excellent agreement with the published literature value of 89 ps ± 3 ps. Dynamic anisotropy Motorized, large aperture, UV-transparent Glan-Thompson fluorescence lifetime proved to describe the system very well, Decay Associated Spectra (DAS) The outstanding sensitivity of the FluoTime 300, together with the high performance pulsed light sources, TCSPC technology, and analysis software from PicoQuant, allows the user to routinely perform studies that previously required special-purpose set-ups and custom software development. Examples are Time-Resolved Emission Spectra (TRES) and Decay Associated Spectra (DAS) of Tryptophan. A 17 µm solution of Tryptophan in a ph 7.4 phosphate buffered saline τ τ τ was excited with a pulsed LED at 290 nm and the fluorescence was detected from 310 nm to 460 nm. An IRF and 31 decay curves were automatically collected within less than 12 minutes. Global analysis reveals that all 31 decay curves can be well described by three global lifetimes: 360 ps, 2.5 ns and 7.4 ns. By plotting the wavelength dependent relative amplitudes of these three components, one gets the DAS of Tryptophan, which reveals a relatively slow spectral relaxation. The 360 ps decay component depopulating the blue edge of the emission spectrum contributes to the long wavelength side as a rising (negative amplitude) component. polarizers, programmable sample temperature control, and EasyTau scripting are the key components of the complex measurement sequence that leads to the results presented below. Sample: Coumarin 6 in ethylene glycol excited at 440 nm, emission detected at 510 nm with 3 nm bandpass. The system automatically set the sample temperature, and at each temperature step, four measurements were automatically performed: an IRF determination, and VV, VH and VM polarized decay measurements. A quick analysis of VV and VH decays clearly shows temperature dependent behavior of the emission anisotropy. For detailed quantitative results, global reconvolution anisotropy analysis of the information-rich data set was per- as expected for a small, highly polar particle in polar solvent. The result reveals the slightly temperature dependent single exponential fluorescence lifetime of Coumarin 6. The obtained rotational correlation times could even be fitted to a theoretical curve taking the temperature dependence of viscosity into account. Even a precise calculation of the steady state anisotropy values was possible, which where found to be in perfect agreement with the anisotropy values estimated by the Perrin equation. formed with the FluoFit software. A model of a single, spherical rotating particle with a single exponential 16 17

10 System Components System Components Compact units with ultimate performance The FluoTime 300 is characterized by exceptional sensitivity in combination with unprecedented ease-of-use. Many of these features are a result from the unique fusion of highly sophisticated state-of-the-art technologies. The underlying key technologies are the proven picosecond pulsed diode lasers and the Time-Correlated Single Photon Counting (TCSPC) electronics developed by PicoQuant, complemented by high-end optomechanics, detectors and accessories. High quality optical components Efficient monochromators The FluoTime 300 includes various high quality optical elements The FluoTime 300 uses Czerny-Turner monochromators with such as lenses, polarizers, attenuators, and filter wheels. All focal lengths of either 150 mm or 300 mm. Both monochromators can be equipped with various high quality diffraction grat- components are motorized and their position is either automatically optimized by the EasyTau software wizards or manually ings in order to achieve a high spectral resolution down to adjustable in the customized measurements mode. Even the 0.2 nm over a broad wavelength range. Wavelength tuning is entrance and exit slits of the monochromators are under complete computer control, leaving the placement of the sample nm. The spectral bandpass of the monochromators can easily completely computer controlled with a minimum step size of 0.1 to be the only necessary manual step for measuring a fluorescence be adjusted by changing the width of the entrance and exit slit. spectrum. Excitation subsystem The excitation subsystem consists of compact turn-key picosecond diode lasers (LDH Series) or LEDs (PLS Series), which cover the wavelength range from 255 nm to the infrared. The lasers emit pulses as short as 50 ps and even the LEDs reach pulse widths well below 1 ns. Special lasers additionally support continuous wave operation. All lasers and LEDs are variable in output power and repetition rate, and are thus ideal excitation sources for fluorescence lifetime measurements. A common driver unit of the PDL Series makes wavelength changes easy and simple. For excitation spectra, different 300 W Xenon arc lamps provide continuous excitation light between 200 nm and 900 nm. The range of excitation sources is completed by a pulsed Xenon flash lamp for phosphorescence measurements. Other external lasers such as Titanium:Sapphire lasers can also be used for both lifetime and steady-state measurements. TCSPC data acquisition An outstanding Time-Correlated Single Photon Counting (TCSPC) data acquisition unit, the PicoHarp 300, is one of the key components of the FluoTime 300. The PicoHarp 300 is a stand-alone unit that is connected to the system computer via a highspeed USB connection. It features a temporal resolution of 4 ps and can be used to measure fluorescence lifetimes up to several hundred nanoseconds as well as steady-state fluorescence. In case of phosphorescence measurements a compact multichannel scaler board is used, which allows measurement of lifetimes up to several hundred milliseconds. Cryogenic temperatures For applications that require cryogenic sample conditions, the FluoTime 300 can be equipped with liquid nitrogen or helium cryostats from Oxford Instruments (Optistat Series). These cryostats are inserted into the sample chamber and enable low temperature photoluminescence measurements from 2.3 K to 500 K. The heart of the FluoTime 300 is a large multifunctional sample chamber with fully automated optical elements and support for different sample holders including cryostats. Compact pulse diodes lasers with picosecond pulses or LEDs with sub-nanosecond pulses are typically used for excitation

11 System Components System Components Specifications Detectors The FluoTime 300 uses one or two single photon counting detectors of various types, attached to an exit slit of the emission monochromator. Each detector includes an electromechanical shutter, optional cooling, and an overload protection, which can be operated from the system software. The available detectors are either Photomultiplier Tubes (PMTs), Microchannel Plate Photomultiplier Tubes (MCP-PMTs), or Hybrid- PMT modules. PMTs of the PMA Series cover the spectral range from 200 nm to 900 nm. They are selected and configured for optimum detection efficiency and temporal resolution. The PMA Series PMTs are the best choice for resolving lifetimes down to 50 ps. Quantum efficiency as a function of wavelength of the available detector types. The efficiency of MCP-PMTs is similar to the efficiency of the PMTs. For applications that require very high temporal resolution, MCP-PMTs can be provided. Their very fast response time permits resolving lifetimes even down to 10 ps if operated along with an appropriate short pulsed laser system. All MCP-PMTs can be optionally equipped with a thermoelectric cooler to reduce dark counts. The Hybrid-PMT features a very high detection efficiency up to 45 % at 500 nm and can resolve lifetimes down to 30 ps owing to its faster response time. Infrared optimized PMTs cover the spectral range between 950 nm and 1700 nm. All NIR-PMTs are supplied with internal thermoelectric cooler that eliminates the need for liquid nitrogen and cooling water. The response time of these detectors is fast enough to resolve lifetimes down to 100 ps. Optical configuration L-Geometry Mode of operation Time-Correlated Single Photon Counting (TCSPC) Multichannel Scaling (MCS) Sensitivity Signal-to-noise ratio typically better than 6500:1, as measured from a water Raman spectrum, excitation wavelength 350 nm, spectral bandwidth 5 nm, integration time 1 s Lifetime range 50 ps to 10 µs with PMT detector and TCSPC electronics < 10 ps to to 10 µs with MCP-PMT detector, TCSPC electronics and suitable laser Up to several 100 ms with PMT or MCP-PMT and MCS electronics Excitation sources Picosecond pulsed diode lasers or LEDs with repetition rates up to 80 MHz, common driver unit Sub-microsecond pulsed Xenon flash lamp 300 W Xenon arc lamp External lasers such as Titanium:Sapphire lasers or pulsed DPSS lasers Monochromators Czerny-Turner type Focal length: 150 mm or 300 mm, single or dual exit slits Grating with 1200 g/mm, blazed at 500 nm (other gratings on request) Slit width adjustable between 10 µm and 4 mm (continuously adjustable, full computer control) Stray light rejection typically 1:10 5 Detector Cooled or uncooled detectors Photomultiplier Tubes (PMTs) with different spectral ranges between 200 nm and 910 nm Micochannel Plate Photomultiplier Tubes (MCP-PMTs) with various spectral ranges between 185 nm and 910 nm Infrared sensitive Photomultiplier Tubes (PMTs) with different spectral ranges between 950 nm and 1700 nm Hybrid-PMT with spectral range between 300 nm and 750 nm Software Easy to use and comprehensive Windows based system and analysis software Data archiving in workspace, data export features, data arithmetic Application wizards for several typical measurement tasks Customized measurement mode with full instrument control Scripting language for user-defined data acquisition and measurement sequences Lifetime analysis based on numerical reconvolution procedure, up to 4th exponential decay functions, lifetime distributions, anisotropy, global analysis, rigorous error analysis Specifications are subject to changes

12 PicoQuant for Scientists PicoQuant for Scientists PicoQuant GmbH PicoQuant GmbH The annual workshop on Single Molecule Spectroscopy brings together the top researchers in the field. Application lab PicoQuant always welcomes scientists to visit our application microscopy and its applications. Both courses are intended for labs, to see the FluoTime 300 working, and to do test measurements with their own samples. We perform a quick and qualified of fluorescence spectroscopy and microscopy and their individuals seeking an in-depth introduction to the principles investigation of your scientific problems. We discuss your needs applications to the life sciences. They consist of lectures by and try to offer a solution tailored to your requirements. Of course, renowned scientists as well as comprehensive instrumentation all of our other products, including the single molecule sensitive and software hands-on training. MicroTime 200 microscope system, are also available for testing and evaluation. Workshop on Single Molecule Spectroscopy Since 1995, the scientists from PicoQuant organize the annual Courses on Time-Resolved Fluorescence workshop on Single Molecule Spectroscopy and Ultrasensitive To improve the understanding and usefulness of timeresolved fluorescence spectroscopy and microscopy, researchers in the field. With this event, we continue Analysis in the Life Sciences, which brings together the top PicoQuant established the European Short Course to encourage the exchange of knowledge and new on Principles and Applications of Time-resolved ideas between the experts in single molecule Fluorescence Spectroscopy as an annual event spectroscopy, interested scientists from other since In 2009 an additional event was fields, and potential users introduced focusing on time-resolved from life science industry. PicoQuant GmbH was founded in 1996 to develop robust, compact and easy to use time-resolved instrumentation and systems. Today, PicoQuant GmbH is known as a company leading in the field of single photon counting and time-resolved fluorescence instrumentation. Our instruments are used all over the world. They help to prepare papers in Nature and Science as well as carrying out routine quality control and production processes of global industrial players. Starting from traditional time-resolved fluorescence detection in bioanalytics, the range of applications is continuously increasing and includes semiconductor quality control, diffuse optical tomography, quantum information processing, optical detector testing and telecommunications. Due to our easy to use products, researchers can now focus on their problems in biology, medicine, environmental science, or chemistry without needing a large background in physics, electronics or optics. Our intention is to offer state-of-the-art technology, which has been co-developed and tested by renowned researchers, at a price affordable to scientific groups and cost sensitive industry. Following this philosophy, we are always looking for new challenges. PicoQuant especi-ally encourages OEM inquiries for its products, notably for applications where time-resolved techniques were considered too expensive and cumbersome in the past. PicoQuant s innovative and dynamic team of physicists, chemists, biologists, designers, and electronic and mechanical engineers work together to offer you a full range of modules for optical excitation, photon counting, as well as complete and automatic instrumentation for a wide range of electrooptical measurement tasks. The combination of more than 15 years R & D work, several thousand units sold, and cooperation with experts for special applications provides a stable basis for new outstanding developments always driven by our customer s needs and inspirations. We invite you to visit our website or contact our specialists directly to discuss your specific needs. And, of course, you are always welcome to visit our application labs during your travel to Germany. Our annual workshops and courses are another perfect opportunity to learn about new techniques and to discuss your needs. Published by: PicoQuant GmbH, Rudower Chaussee 29 (IGZ), Berlin April 2011 Concept: united communications GmbH, Berlin Editorial and production: PinkLemon Werbeagentur Tina Stundner, Berlin Print: dhs, Berlin Photos: PicoQuant 22 Corporate names, trademarks stated herein are the property of their respective companies.

13 Supplement Supplement New system add-ons Extended specifications Cryostat accessory The sample chamber is large enough to accommodate an Oxford Instruments OptistatDN series cryostat. This optional component allows low temperature photoluminescence measurements to be made. Precise control of the sample temperature is possible with various cryostat types from 2.3 to 500 K. A stand-alone digital cryostat controller is included. Rapid kinetic (stopped-flow) accessory Rapid kinetic accessories (SFA-20 series) from TgK Scientific Ltd. make possible to monitor fast reactions (on millisecond time scale) in solution, like enzyme kinetics, quenching, association/dissociation, etc. The accessory has an empirical deadtime < 8 ms. Microvolume version, pneumatic drive, anaerobic kit, variable ratio mixing, multimixing versions and advanced analysis software (Kinetic Studio) are available as options. Monochromators Type Czerny-Turner design Czerny-Turner design Focal length 150 mm 300 mm Aperture F/4.2 F/3.9 Stray light rejection Grating* 1200 g/mm, blazed at 500 nm 1200 g/mm, blazed at 500 nm Resolution 0.4 nm 0.1 nm (at nm, slit width 10 µm, 1200 g/mm grating) (at nm, slit width 10 µm, 1200 g/mm grating) Step size (min) 0.01 nm 0.01 nm Slit width adjustable between 10 µm - 4 mm 10 µm - 4 mm (continously adjustable, completely motorized) (continously adjustable, completely motorized) Wavelength accuracy 0.25 nm (1200 g/mm grating) 0.2 nm (1200 g/mm grating) Wavelength repeatability 0.1 nm 0.1 nm Excitation sources Light source Laser Diode Heads (LDH Series) pulsed LEDs (PLS Series) pulsed Xenon lamp cw Xenon lamp Wavelengths (nm) , 530, 595, Pulse width ps 400 ps - 1 ns < 1 µs --- Repetition rate up to 80 MHz up to 40 MHz up to 300 Hz --- Detectors Type* PMT (PMA-C Series) MCP-PMT NIR-PMT Spectral range (nm) , , Dark counts (cps, < 150 < 900 < 50 < 500 < at 20 C, typ. value) FluoTime 300 with integrated cryostat from Oxford Instruments FluoTime 300 with integrated stopped-flow accessory Data acquisition Type PicoHarp 300 NanoHarp 250 Number of time channels/curve up to up to Count depth 16 bit 18 bit Time resolution (bin width) 4 ps 4 ns or 32 ns Differential non-linearity < 0.1 % rms, < 5 % pp. < 0.1 % rms, < 1 % pp. Dead time < 95 ns 8 ns Discriminator level, zero cross adjust software adjustable software adjustable Collection time 1 ms - 10 h 1 ms - 10 h Operating environment Computer system Power requirements Windows XP/Vista/7 110 to 230 V, 50/60 Hz Dimensions (base unit) Without steady-state option 900 mm 550 mm 400 mm (w d h) With steady-state option 900 mm 1100 mm 400 mm (w d h) * Other types are available upon request. All Information given here is reliable to our best knowledge. However, no responsibility is assumed for possible inaccuracies or omissions. Specifications and external appearances are subject to change without notice. Trademarks or corporate names are used for explanation and identification, to the owner s benefit and without intent to infringe. Supplement last updated: January Supplement FluoTime 300 Supplement FluoTime 300