LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program

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1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: Helyszín / Place: OKTÓBER 10. / OCTOBER 10, 2018 SZBK NÖVÉNYBIOLÓGIA, 113. LAB. / LAB # 113, PLANT BIOLOGY, BRC SZEGED, TEMESVÁRI KRT. 62. Gyakorlati foglalkozás címe / Title of the practical demonstration: TIME-RESOLVED OPTICAL SPECTROSCOPY Gyakorlatvezető / Demonstrator: PETAR LAMBREV Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

2 Theoretical background Time resolved fluorescence Time-resolved fluorescence spectroscopy measures the decay of fluorescence intensity over time after a short excitation pulse. Since fluorescence is emitted from the excited fluorophore, the fluorescence intensity directly reflects the concentration of excited states, which typically follows an exponential decay law: where is the fluorescence lifetime, one of the most important characteristics of a fluorophore. It represents the average time the fluorophore remains in the excited state and so depends on all possible excited-state deactivation pathways, hence all interactions of the excited fluorophore with its environment: where are deactivation rate constants. The lifetime also determines the fluorescence quantum yield: where is the radiative decay rate constant and is the radiative, or natural lifetime of the fluorophore. The steady-state fluorescence measured under continuous excitation light is simply the integrated time-resolved fluorescence decay and is proportional to the lifetime: Even in the ideal case of identical fluorophores decaying with a single lifetime, a timeresolved fluorescence measurement has an advantage over the steady-state fluorescence in that it is an absolute measurement of the fluorescence lifetime, independent from fluorophore concentration and instrumental factors, whereas steady-state fluorescence is always a relative measurement. The main advantages of time-resolved fluorescence however are in that it reveals molecular information about the fluorophore in terms of its excited state dynamics. Time-resolved fluorescence can reveal fluorophore conformational states, solvent interactions, quenching mechanisms, excitation energy transfer kinetics, and much more. page 2

3 Principles of time correlated single photon counting (TCSPC) TCSPC is one of several possible technical approaches to conduct time-resolved fluorescence measurements in the time domain. Compared to other techniques, TCSPC has the advantages of requiring low excitation power, which is critical for sensitive biological materials, very high signal-to-noise ratio, or high dynamic range, over a very large time range, usually from several picoseconds up to microseconds. Consequently, one can use iterative fitting analysis to extract several fluorescence lifetimes present in the sample, from a single measurement. As with all time-domain measurements the sample under investigation is excited by short excitation pulses, typically from a mode-locked laser or laser diode. In TCSPC excitation pulses of low power and high repetition rate are used, so that fluorophores are excited no more than once during a single pulse and after each excitation event there is a low probability that a single emitted photon of fluorescence will be detected. The detector is then operated in photon-counting mode the analog amplitude of the detector signal is discarded and only the detection event is registered. When a photon is detected, the time elapsed between the excitation pulse and the detection event is recorded. After the detection of many photons, a histogram of the distribution of measured times is accumulated (Fig. 1), which has the shape of the original waveform, or fluorescence decay function. Original Waveform (Distribution of photon probability) Detector Signal: Period 1 Period 2 Period 3 Period 4 Period 5 Period 6 Period 7 Period 8 Period 9 Period 10 Time Period N Result after many Photons Fig. 1. Principle of TCSPC measurements. From [1]. page 3

4 Unlike any analog recording technique, the time resolution of the single-photon counting measurement is not limited by the response time of the detector, i.e. the time between the arrival of a photon at the sensor and the appearance of an electric signal at its output, but only by the transit time spread, i.e. the variance of the time response, which is much narrower than the time response. The time resolution is characterised by the instrument response function (IRF). The IRF width of instruments equipped with microchannel plate (MCP) detectors is down to 30 ps. With a proper IRF deconvolution technique one can readily resolve lifetimes shorter than 10 ps. A block scheme of a standard TCSPC instrument is shown in Fig. 2. Reference pulses from light source threshold CFD zero cross start Range Gain Memory Histogram Preamplifier threshold stop TAC AMP ADC Address (time) Detector CFD zero cross Singlephoton pulses Fig. 2. Scheme of a TCSPC instrument. From [1]. Offset = control elements data +1 Adder The electronic block of the instrument has two input channels one from the excitation laser source, and one from the detector of fluorescence. Both electrical signals are converted into digital on/off pulses by constant-fraction discriminators (CFD), eliminating their analog amplitude. Using CFDs gives accurate timing of the pulses irrespective of their original amplitude. The CFD pulses are used as the start and stop signals for a time-to-amplitude converter (TAC). The TAC operates as a stopwatch, generating signal amplitude that is proportional to the time elapsed between the start and the stop signals. The amplified TAC signal is digitized by an analog-to-digital converter (ADC) and stored as a time signal in the appropriate histogram bin in the data memory. A modern variation of the TCSPC instrumentation uses so-called time-to-digital converter (TDC) that combines the functions of the TAC and ADC, circumventing the limited number of time channels imposed by the ADC (typically 4096 for a 12-bit ADC) and thus allowing to extend the time span of the measurement. page 4

5 The PicoQuant FluoTime 200 spectrometer Description The FluoTime 200 spectrometer from PicoQuant (Germany) is a high performance fluorescence lifetime system. It contains the complete optics and electronics for recording fluorescence decays by TCSPC (as well as multichannel scaling). The system is optimized for high temporal resolution and can be used with femtosecond or picosecond lasers. With the FluoTime 200, decay times down to a few picoseconds can be resolved. The system allows operation at laser repetition rates as high as 100 MHz and count rates up to several million counts/sec. The setup used in this practical demonstration is equipped with a high time resolution MCP detector (Hamamatsu R-3809U) and a computer-controlled monochromator for measuring time-resolved spectra. The instrument s modules are illustrated in Fig. 3. A Fianium WhiteLase Micro supercontinuum laser (1) provides 6 ps white-light excitation pulses at a repetition rate of 20 MHz. The laser beam passes through an optical filter assembly (2) which holds a band-pass filter to select excitation wavelength and attenuating neutral density filters. The beam is focused onto the sample cell holder in the measuring chamber of the FT200 spectrometer (3). The reference signal from the laser is amplified by the PAM 102 amplifier (4), passed through a nanosecond delay box (5), and connected to Channel 0 of the PicoHarp 300 electronic block (6). The detector signal from the spectrometer is connected to Channel 1. The PicoHarp 300 is a stand-alone TCSPC system that contains all the necessary electronics to collect histograms with time bins with 4 ps minimal width. The collected data is transferred to the computer via USB interface. Fig. 3. Diagram of the FluoTime 200 spectrometer setup page 5

6 FluoTime 200 Operating instructions Startup 1. Switch on the mains power 2. Make sure that all modules are powered: a. PicoHarp 300 b. FluoTime 200 c. WhiteLase Micro d. PAM 102 e. PC 3. Make sure the sample holder is empty 4. Start the PC and run the PicoHarp software 5. Open the monochromator dialog box (press the button on the toolbar) 6. Initialize the monochromator. The operating motor is audible. 7. Start the laser turn the key clockwise Caution! Do not remove the fiber guide from its holder! Do not look directly into the laser beam! 8. Make sure the laser repetition rate (1,96e+007) is displayed on the Input0/cps box of the main program window. 9. Turn on the detector power supply, first press the POWER switch, then the OUTPUT button. page 6

7 10. Gradually increase the voltage using the CV knob to 3,000 V. Above 2,900 V observe the appearance of detector signal in the Input1/cps box on the PC screen. Caution! High count rates can saturate and damage the MCP detector. Do not allow the detector count rate to rise above 100k cps (1e+005 at Input1) at any time! Should the count rate rise above that level, immediately open the lid of the measuring compartment to engage the detector shutter! Shutdown 1. Remove any samples from the sample holder. 2. Turn off the laser, turning the key counterclockwise. 3. Decrease the MCP power supply voltage to 0 using the CV knob. 4. Press the output button, then the power switch. 5. Shut down the computer. 6. Switch off the mains power. Measuring IRF 1. Becker, W., Advanced time correlated single photon counting techniques. Vol : Springer. page 7