2. Materials and methods
2.1 Algal culture
2.2 Excitation sources
2.3 Fluorometer system
2.4 Data Analysis
4. Decoupling of the PBPs under cold stress
5. Excited state populations according to a model of rate equations
8. About the author
Time- and wavelength-resolved fluorescence spectroscopy is an appropriate tool for quantitative and non-invasive investigations of living cells. Short measurement times with low excitation light intensities are necessary to observe variations of the fluorescence due to changes in the metabolism of the sensitive biological organisms.
With new techniques the fluorescence dynamics can be monitored simultaneously in a broad spectrum during a very short measurement time. That provides information about the spectral differences of the fluorescence dynamics which can vary in correlation with the metabolic changes.
The interaction of the photosynthetic subunits and especially the mechanisms regulating the energy transfer are presently interesting and open fields in photosynthesis research.
The phototrophic cyanobacterium Acaryochloris marina contains membrane extrinsic PBP antenna complexes and mainly Chl d containing membrane intrinsic core antenna complexes which absorb light and transfer excitation energy to the reaction center.
The results of our studies suggest a fast excitation energy transfer kinetics of 20-30 ps along the PBP antenna of A.marina followed by a transfer with a time constant of about 60 ps to Chl d.
Very often cells or cell fragments are kept at low temperature to decelerate ageing processes. Living cells of A. marina which were stored at 0°C for some time showed a reduced excitation energy transfer from the PBP to the Chl d antenna, which partially recovered when the sample had been kept at 25 °C for a short time.
The reduction of the excitation energy transfer might be caused by a mechanism that decouples the PBP antenna under cold stress conditions avoiding photo damage of the reaction center of PS II.
Keywords: Acaryochloris marina, fluorescence dynamics, spectroscopy, phycobiliprotein, chlorophyll d, fluorescence lifetime, excitation energy transfer, phycocyanin, allophycocyanin, cell metabolism, photosynthesis research, biotechnology, biomedicine, life sciences Veranderungen im Metabolismus lebender Zellen des Chl d- haltigen Cyanobakteriums Acaryochloris marina untersucht mittels zeit- und wellenlangenkorrelierten Einzelphotonenzahlens
Zeit- und wellenlangenaufgeloste Fluoreszenzspektroskopie ist ein geeignetes Verfahren zur quantitativen und nichtinvasiven Untersuchung lebender Zellen. Kurze Messzeiten bei niedrigen Anregungsintensitaten sind notwendig, um Veranderungen der Fluoreszenz, verursacht durch metabolische Anderungen in empfindlichen biologischen Organismen, zu beobachten.
Mit moderner Technik kann die Fluoreszenzdynamik simultan in einem breiten Spektrum innerhalb einer kurzen Messzeit abgebildet werden. Dies liefert Informationen uber spektrale Unterschiede der zeitabhangigen Fluoreszenz, welche in Korrelation zu metabolischen Veranderungen variieren kann.
Die Wechselwirkung der photosynthetischen Untereinheiten und insbesondere die Regulationsmechanismen des Energietransfers sind gegenwartig interessante und offene Fragen der Photosyntheseforschung.
Das phototrophe Cyanobakterium Acaryochloris marina enthalt membranextrinsische PBP Antennenkomplexe und uberwiegend Chl d haltige membranintrinsische Core- Antennenkomplexe, welche Licht absorbieren und die Anregungsenergie zum Reaktionszentrum weiterleiten.
Die Ergebnisse unserer Untersuchungen weisen auf einen schnellen Energietransfer entlang der PBP Antenne mit einer Kinetik von 20-30 ps hin, auf welchen ein Transfer zum Chl d mit einer Zeitkonstanten von etwa 60 ps folgt.
Sehr oft werden Zellen oder Zellfragmente gekuhlt, um Alterungsprozesse zu verlangsamen. Lebende Zellen von A. marina, die einige Zeit bei 0°C gelagert wurden, zeigten einen reduzierten Anregungsenergietransfer von der PBP Antenne zum Chl d, welcher sich innerhalb kurzer Zeit teilweise regenerierte, nachdem die Probe auf 25 °C erwarmt worden war.
Dieser Reduktion des Anregungsenergietransfers konnte ein Mechanismus zu Grunde liegen, welcher die PBP Antenne unter Kaltestress entkoppelt, um eine lichtinduzierte Zerstorung des Reaktionszentrums im PS II zu verhindern.
Acaryochloris marina which was discovered in 1996 has a unique composition of the light harvesting system. The chlorophyll (Chl) antenna of Photosystem II (PS II) contains mainly Chl d instead of the usually dominant Chl a and the Phycobiliprotein (PBP) antenna has a simpler rod shaped structure than in typical cynobacteria . The energy transfer processes are still not fully explained and further spectroscopic studies are necessary to answer the open questions.
During the past 20 years there has been a remarkable growth in the use of fluorescence techniques in biological sciences. Quantification of toxic substances or health promoting components in fruits and vegetables, environmental monitoring, clinical chemistry and DNA sequencing are just a few areas of application .
With fluorescence spectrometry the wavelength- and also the time-dependency of the emitted light can be used to identify and quantify fluorescent substances.
In photosynthesis research information on excitation energy transfer among antenna pigments, charge separation within the reaction centers, forming of channels for non-photochemical quenching and the pigment-pigment or pigment-protein coupling can be gathered from analyses of the time decay and wavelength dependence of the emitted fluorescence ,.
In living cells changes in the fluorescence emission can be detected that correspond to the cell metabolism. The influence of stress factors like cold, heat, high light intensities or the starvation of essential components alters the conformation of the coupled pigment-protein complexes. In order to take full advantage of the information content of the fluorescence emission, it is necessary to monitor the time- and the wavelength dependency of fluorescence light with sufficient resolution because the broad fluorescence emission bands are the sum of the fluorescence of hundreds of pigments in different protein environments. Therefore the information about the electronic structure of a single pigment bound to a protein cannot be resolved. Mathematical data analysis and the theory of optical spectra which today delivers tools to calculate the absorption of pigment-protein complexes are helpful to extract information of specific molecules in a pool of fluorescing pigments .
When observing fast metabolic changes the signal has to be collected simultaneously in the time and wavelength domain, because the fluorescence decay at different wavelengths can only be compared if the time resolved fluorescence is measured at different wavelengths at the same time. Therefore fast acquisition of time resolved data in a broad spectral range is required which can be accomplished by new spectrometer systems.
In this study we use time- and wavelength-resolved single photon counting to investigate the excited states dynamics in living cells of the prokaryotic cyanobacterium Acaryochloris marina.
A.marina is a very special prokaryotic cyanobacterium. It is still the only known oxygenic photosynthetic organism containing Chl d as the dominant antenna pigment. Additionally Phycobiliproteins (PBPs) and small amounts of Chl a are present ,. Chl d has a formyl group on ring 1 of the porphyrin headgroup, in place of the vinyl group in Chl a, shifting the Qy absorption maximum to 696 nm in methanol, ~30 nm more to the red as compared to Chl a (665 nm in methanol). A.marina is therefore able to exploit the near infrared light that penetrates to the shady environment where it lives . In whole cells of A.marina, the main red absorption band is observed at 714-718 nm . The room temperature steady state fluorescence of A. marina exhibits a broad Chl d band at 724 nm . The red shift in absorbance of Chl d relative to Chl a is equal to an electronic energy gap of ~100 mV. Therefore it has been questioned how Chl d is able to split water in PS II ,,.
Absorption- and fluorescence-spectroscopic studies with time and wavelength resolution have clarified the pathway of excitation energy transfer on the molecular level . Also the mechanism of energy transfer and water splitting process has been analyzed in detail . Chl d was shown to be the primary donor of the reaction center (RC) of PS I in A. marina . The nature of the primary donor of PS II in A. marina is still in discussion. Recent studies suggest a start of the primary charge separation from the accessory Chl pigment which is Chl d (Chl d1). This Chl d molecule is stabilized by the so-called special pair which consists of Chl a. Then the charge is quickly localized at Chl a. Therefore both Chlorophyll types, Chl a and Chl d are essential for the photochemistry in PS II of A.marina .
Fig. 1 shows a scheme of the cell and the PS II antenna system of A.marina. The prokaryotic cells of A.marina are containing staples of the thylakoid membrane where the photosynthesis takes place. The membrane extrinsic antenna is represented by a PBP rod which is associated to the PS II core antenna containing Chl d .
Abbildung in dieser Leseprobe nicht enthalten
right side the Light harvesting antenna complexes and Reaction center of PS II are shown according to Marquardt et al. 
The PBPs of A.marina have been reported to form aggregates of a simpler structure than those in typical cyanobacteria (fig. 1) . They consist of four hexameric units, which resemble the peripheral rods of the typical cyanobacterial phycobilisomes (PBS) ,. Three of the hexamers were suggested to contain only phycocyanin (PC) and one to be a hetero-hexamer containing PC and allophycocyanin (APC). The excitation energy seems to be funneled directly from the APC-containing hetero-hexamer to Chl d of PS II without the involvement of an APC core as in typical cyanobacteria . Isolated PBP aggregates of A. marina exhibit a fluorescence maximum at 665 nm (from APC) with a shoulder at about 655 nm (from PC) at room temperature . This emission is also found in living cells of A.marina and the fluorescence decays with a time constant of 70 ps which is indicating the fast energy transfer from PBP to Chl d . For reviews on fluorescence of A. marina and comparison with other systems, see Mimuro  and Itoh .
2. Materials and Methods
2.1 Algal culture
A. marina was grown as described in  in artificial sea water at 301 ± 2 K (28° ± 2 °C) under an illumination intensity of 5 Wm-2 and continuous aeration. Before the measurements, the cells were spun down and gently re-suspended in a smaller amount of the growth medium to increase the cell density to about 5 pM of Chl d. This was done in order to obtain sufficiently high count rates during the fluorescence measurements while using low excitation intensities (1-5 Wm-2) under continuous stirring necessary to avoid closing of the PS II reaction centers.
2.2 Excitation sources
For time resolved measurements a picosecond diode laser module was used for excitation at 632 nm (BHL-600, FWHM 60 ps, repetition frequency 20 MHz, Becker & Hickl GmbH, Berlin). The measurements were performed in a 3 x 10 mm cuvette shielded from room light. Fluorescence was detected at a right angle to the excitation beam. In order to suppress the scattered excitation light a long-pass emission filter was inserted between the cuvette and the detector (640ALP at an angle of 0° for 632 nm excitation (Omega Inc, cut-off wavelength 640 nm)).
In a modified setup to analyze the spectrum of the PBP emission, a LED was used for excitation at 600 nm (FWHM 800 ps, repetition frequency 8 MHz, Picoquant, Berlin). The long-pass emission filter (640ALP) was used at an angle of 30° (resulting in a cut-off wavelength 620 nm) giving additional information of the emission spectrum of PBPs in the range from 620 nm to 640 nm.
2.3 Fluorometer system
Fig. 2 shows two different types of single photon counting fluorometer systems with time- and wavelength resolution which are used for the fluorescence spectroscopic studies.
In Fig. 2 a) the system based on the technique of a delay-line detector is shown. During the measurement time- and space- correlation is used to determine simultaneously time- and wavelength information about the collected photons.
The double correlated single photon counting is achieved by using a microchannel plate photomultiplier (MCP-PMT) with delay-line anode (Europhoton GmbH, Berlin). In combination with a 120 mm crossed Czerny-Turner polychromator (MultiSpec, LOT) equipped with a 600 grooves/mm grating as a dispersive element, the space coordinate can be used for wavelength resolution .
Abbildung in dieser Leseprobe nicht enthalten
Fig. 2 a) Double correlated time- and wavelength resolved fluorometer system as described in . The time difference between the reference pulse and the first incoming photon is measured by a time- amplitude converter (TAC2) and also the time spread in the delay-line detector, which depends on the frequency of the photons (TAC1)
As a function of the wavelength the fluorescence photons are deflected onto the photocathode of the MCP-PMT in the focal plane of the polychromator. A photoelectron is emitted at the inner side of the cathode and amplified by two microchannel plates producing a spatially limited electron cloud, which finally impinges on the delay-line anode. The electric charge moves to the opposite ends of the delay-line and arrives at both ends at different times depending on the position where the electron cloud hit the anode. The measured time difference between these two output pulses is then used to calculate the space-coordinate of the photon which allows the determination of its wavelength. Therefore this detector setup allows simultaneous monitoring of the time and wavelength dependence of fluorescence light with picosecond time resolution.
The two outputs of the delay-line anode are amplified by two 1GHz preamplifiers (Ortec 9306) and further processed by constant fraction discriminators (CFDs) (Tennelec TC 454). The Output of one of the CFDs provides the start signal for two time-to-amplitude converters
(TACs, Ortec 457 for space domain and Tennelec TC 864 for time domain). The stop signal for the space-domain TAC is provided by the output of the second CFD. A small fraction of the excitation laser light is reflected onto a fast photodiode that provides the stop signal for the time-domain TAC. The outputs of the two TACs are processed by a personal computer. The resulting data is stored in a two-dimensional channel matrix of size 256 x 1024, with 256 channels in the space domain corresponding to spectral resolution (wavelength coordinate) and 1024 channels in the time domain corresponding to temporal resolution (time coordinate of the fluorescence decay). The wavelength coordinate is provided directly by the output of the space-domain TAC. A correction for different photon wavelengths is applied to the output values of the time domain TAC in order to obtain the correct value of the time coordinate.
The technique of single photon counting operates with high signal to noise ratio which was yN~js > 100 in all measurements performed in this study. With the setup presented in Fig. 2a) this S/N ratio can be achieved in measurement times of < 10 min. for up to 30 wavelength sections of 5-10 nm spectral widths. The instrumental response function (IRF) of this system has 150 ps full width at half maximum (FWHM), limiting the time resolution to about 30 ps.
Fig. 2b) shows a time- and wavelength resolved multi anode detector system with 16 output (anode) elements (PML-16 , Becker&Hickl, Germany). Compared to the sensitive delay-line anode system the multi anode system yields a dramatically increased detection rate up to 1.000.000 counts/sec with the mentioned 20 MHz diode laser. The core of the PML-16 is a Hamamatsu R5900 16 channel multi-anode photomultiplier tube with 16 separate output (anode) elements and a common cathode and dynode system as described in . The wavelength resolution of the multi anode system is limited by the number of anodes in comparison to the whole detected spectral bandwidth that is determined by the grating. Using the polychromator with a 600 grooves/mm grating the spectral bandwidth of the PML-16 is about 6 nm /channel.
New techniques using several photomultiplier tubes allow even higher count rates because pile up can be neglected if detection systems with detector arrays are used .
Measurements with higher time resolution were achieved employing a monochromator system (McPherson Instrument) with a MCP-PM-tube (Hamamatsu). The IRF of this system is 90 ps FWHM allowing a time resolution which is shorter than 20 ps. These measurements were performed in a time window with 4096 channels and 5 ns in total. Typical calibration values were 1.22 ps per channel in the time domain.
A Peltier cooling/heating system (Peltron GmbH, Germany) allows the choice of any temperature in the range of 260 K to 330 K in every setup. The regulation of the sample temperature is necessary to avoid warming up of the sensitive cells during the measurement. Furthermore the choice of different temperatures was necessary to investigate the influence of cold stress. For further details see also .
2.4 Data analysis
For a correct data analysis the knowledge of the response of the system to the laser pulse without fluorescence is necessary. The temporal width of this signal is caused by the laser pulse duration and additional electronic broadening processes. The deconvolution of the instrumental response function (IRF) with the fluorescence signal helps to better the time resolution down to 20 % of the laser pulse FWHM.
After every experiment the IRF was measured by detecting the attenuated excitation light scattered from a cuvette filled with distilled water using the same conditions as in measuring fluorescent samples. The fluorescence decay was analyzed employing Levenberg-Marquardt algorithm for the minimization of the reduced (ft2) . The algorithm was implemented using Matlab© software (The MathWorks Inc.). The decay was fitted to a multi-exponential decay model
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with up to four components (n=4). The quality of the fit was judged by the value of xr2 and by the degree of randomness of residuals (difference between the experimental data points and the fit). As an alternative to the Matlab implementation the software of Globals Unlimited (University of Illinois, Urbana, USA) providing equivalent analysis options was used (also see Gilmore ).
Fig. 3 a) shows a fluorescence measurement of whole cells of A.marina collected after excitation with 632 nm at room temperature. The number of the registered photons at each wavelength and each time channel was stored in a 2-dim. data matrix. In this matrix the lines contain 1024 entries which represent the 1024 time channels. The columns contain 256 entries representing the wavelength channels. In Fig. 3a) the data matrix is shown as a color intensity plot (CIP). A CIP is a plot of the fluorescence intensity (pictured by the color or greyscale) in dependency of wavelength (y-axis) and time (x-axis). The fluorescence decay curves of any wavelength section can be plotted from this matrix (Fig. 3 b).
Fig. 3 c) shows the fluorescence spectrum at different times. Plots of the spectra at different time intervals are called time resolved spectra. Time resolved spectra help to identify the fluorescence of certain fluorophores at later times when the fluorescence emission of other pigments or scattered light already decayed.
In fig. 3 c) the main emission is observed at 645 - 660 nm (PBP emission) immediately after excitation (0 ps) while one will find strongest fluorescence at 725 nm (Chl d emission) after 1 ns. For better visibility the spectrum after 1 ns is multiplied with a factor 5.
The fluorescence decay curves of different wavelength sections are fitted according to the multiexponential model (eq. 1).
The multiexponential fits of several decay curves are often fitted together (global fit) with common values of lifetimes Tj (linked parameters) and wavelength-dependent pre-exponential factors aj(X) (non-linked parameters). The result of such analysis is usually plotted as graph of aj(X) for all wavelength independent lifetimes Tj representing so-called decay associated spectra (DAS) showing the energetic position of individual decay components (fig. 3d).
For further details on the data analysis see also .
- Arbeit zitieren
- Schmitt (Autor)Theiss (Autor)Wache (Autor)Fuesers (Autor)Andree (Autor)Eichler (Autor)Eckler (Autor), 2007, Fluorescence spectrocopy of excitation energy transfer processes in Acaryochloris marina, München, GRIN Verlag, https://www.grin.com/document/159775