Theory of fluorescence polarization of oriented pigment molecules in spherical arrays.

Theoretical results are presented which are appropriate for the analysis of the static polarized fluorescence experiment with oriented pigment molecules in spherical arrays (vesicles). Though the global orientation mediated over the whole sphere is isotropic, the fluorescent molecules may have preferred local orientation with respect to the local plane. As in a former paper, concerning fluroescence polarization in planar arrays, three basic (local) orientation distributions of the electronic transition moments are investigated, which may be expected to describe a wide class of real cases with sufficient accuracy. Analytic expressions for the degree of polarization are derived. One important result is that the degree of polarization may be extremely dependent on the local orientation of transition moments. Hence the usual method of determination of microviscosities from experiments with vesicles with the use of the theory of fluorescence polarization for macromolecules in solutions should be regarded with great caution.     

Theory of fluorescence polarization of oriented pigment molecules in spherical arrays

Summary Theoretical results are presented which are appropriate for the analysis of the static polarized fluorescence experiment with oriented pigment molecules in spherical arrays (vesicles). Though the global orientation mediated over the whole sphere is isotropic, the fluorescent molecules may have preferred local orientation with respect to the local plane. As in a former paper, concerning fluorescence polarization in planar arrays, three basic (local) orientation distributions of the electronic transition moments are investigated, which may be expected to describe a wide class of real cases with sufficient accuracy. Analytic expressions for the degree of polarization are derived. One important result is that the degree of polarization may be extremely dependent on the local orientation of transition moments. Hence the usual method of determination of microviscosities from experiments with vesicles with the use of the theory of fluorescence polarization for macromolecules in solutions should be regarded with great caution.I wish to thank prof. P. LÄuger and Dr. G. Pohl for interesting discussions. This work has been financially supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 138).     

Dynamical theory of fluorescence polarization in a planar array of oriented pigment molecules.

A theory is developed appropriate for the analysis of fluorescence polarization experiments with pigment molecules in a planar array (plane membrane). Especially rotatory and oscillatory dynamics of the pigment molecules are considered. Three model calculations are performed, which describe the following different situations: (a) Rotational diffusion of molecules around the normal to the plane membrane. (b) Oscillatory diffusion of molecules with respect to this normal. (c) As a two-dimensional example the independent superposition of both types of motion. Central point of these model calculations is the determination of an intensity of emission function, from which in practical application the measured fluorescence intensities may uniquely be calculated.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluoresence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400–680 nm) and emission wavelengths (675–750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, P_IN and P_AN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization P_IN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Q_y (≈ 660−680 nm) absorption band of chlorophyll in vivo. With 670–680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts, P_IN is shown to be +5±1%. The excitation wavelength dependence studies of P_IN indicate that the carotenoid and chlorophyll Q_y transition moments tend to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

Computer aided fluorescence imaging of photosynthetic systems

A fluorescence video imaging system utilizing relatively inexpensive commercial components is described. The instrument utilizes a black and white CCD video camera detector, a commercial video imaging board and a IBM-AT compatible computer. The color output of the imaging board greatly aids in the users ability to visually discriminate areas of interest in the video field. Software development that enables the user to capture kinetic traces in real time from the video images is also described. The system is used to monitor fluorescence from photosynthetic systems. The usefulness of the system in screening for photosynthetic mutants is also demonstrated. The cost of the system can be kept below $12,000.Abbreviations CCD charge-coupled device - DCMU diuron, 3-[3,4-Dichlorophenyl]1,1-dimethylurea - AGC automatic gain control - LUT look-up table - AOI area of interest - CPU central processing unit - RAM random access memory - ADC analog-to-digital converter - FVIPS fluorescence video image processing software - I/O input/output - F₀ dark-level fluorescence - OIDPSMT characteristic transient components, where O is dark level, I is intermediary peak, D is dip, P is peak of fast transient, S is quasi-steady state level, M is second maximum, T is terminal level     

Epi-illumination optical design for fluorescence polarization measurements in flow systems.

An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90 degrees, multiangle light scattering, and high precision cell-sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.     

On the quenching of the fluorescence yield in photosynthetic systems

A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria.     

Mechanism of photoinactivation in photosynthetic systems IV. Light-induced changes in the fluorescence transient

1. 1. Light-induced changes in the fluorescence transient (685nm) of spinach chloroplast fragments at room temperature wereinvestigated in an attempt to correlate these changes with photoinactivationin photosystem II.
2. 2. Parallel decreases in the steady-statelevel of fluorescenceand in the variable fraction, observedunder aerobic light-treatment,were not related processes butseparate reactions as indicatedby an anaerobic-interruptionexperiment where the decrease inthe steady-state level occurredonly after the disappearanceof induction.
3. 3. Anaerobic light-treatmentcaused an increase in the initiallevel of fluorescence parallelto photoinactivation in photosystemII, and a more rapid partialdecrease in the teady-state levelof fluorescence.
4. 4. Thesteady-state level of fluorescence showed pronouncedpH dependency,and had an optimum at about pH 6.5, while theinitial levelwas practically independent of environmental pHwithin a neutralrange. Aerobic or anaerobic light-treatmentcompletely eliminatedpH dependency.
5. 5. Effects of electron acceptors, dichlorophenyl-dimethylurea,dithionite, and of electron donors for photosystem II on thefluorescence transient of photoinactivatedchloroplast fragmentswere investigated. Based on the data presented here, it seemsreasonable to assume that photoinactivation in photosystem IIis closely related to the state of reaction centers in the photosystem.

(Received July 8, 1970; )     

Direct fluorescence polarization immunoassay.

Direct fluorescence polarization immunoassay of serum cortisol was established. Non-specific binding of fluorescent-labelled cortisol to serum proteins was successively eliminated by sodium dodecyl sulfate. The minimal amount of cortisol detected was 0.1 ng/tube and serum concentration of 1.0 microgram/dl to 100 microgram/dl of cortisol could be measured. This fluorescence polarization immunoassay satisfied the standard criteria of accuracy and precision. The values correlated well with those obtained by radioimmunoassay.     

Small angle x-ray scattering studies of magnetically oriented lipid bilayers.

Magnetically oriented lipid/detergent bilayers are potentially useful for studies of membrane-associated molecules and complexes using x-ray scattering and nuclear magnetic resonance (NMR). To establish whether the system is a reasonable model of a phospholipid bilayer, we have studied the system using x-ray solution scattering to determine the bilayer thickness, interparticle spacing, and orientational parameters for magnetically oriented lipid bilayers. The magnetically orientable samples contain the phospholipid L-alpha-dilauroylphosphatidylcholine (DLPC) and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in a 3:1 molar ratio in 70% water (w/v) and are similar to magnetically orientable samples used as NMR media for structural studies of membrane-associated molecules. A bilayer thickness of 30 A was determined for the DLPC/CHAPSO particles, which is the same as the bilayer thickness of pure DLPC vesicles, suggesting that the CHAPSO is not greatly perturbing the lipid bilayer. These data, as well as NMR data on molecules incorporated in the oriented lipid particles, are consistent with the sample consisting of reasonably homogeneous and well dispersed lipid particles. Finally, the orientational energy of the sample suggests that the size of the cooperatively orienting unit in the samples is 2 x 10(7) phospholipid molecules.     

Angle resolved fluorescence depolarization experiments on oriented lipid membrane systems

It is demonstrated that angle resolved steady state fluorescence depolarization experiments on oriented lipid membrane systems can be an useful alternative to time-resolved fluorescence depolarization experiments on vesicles. It is shown that some basic assumptions underlying time-resolved experiments are not always valid. The usefulness of the measurement of an additional order parameter, less than P4 greater than is demonstrated.     

Theory of proton hyperfine interactions in bacterial photosynthetic systems. Bacteriopheophytin cation and anion.

The electronic structures of the cation and anion of bacteriopheophytin alpha monomer are investigated by the self-consistent charge extended Hückel procedure including both pi and sigma electrons in the molecule. The calculated electron distributions are tested by comparison of the predicted hyperfine fields at proton sites with experimental data in both the ions and most important, by their ability to explain the observed trend in the hyperfine fields in going from the cation bacteriopheophytin+ alpha to the anion, a trend that is similar in many respects to the corresponding observed trend for the bacteriochlorophyll alpha cation and anion. Good agreement is obtained with experiment both for the absolute values of the observed proton hyperfine fields in both bacteriopheophytin a cation and anion as well as the ratio of the corresponding fields for the two systems. In particular, our calculated electron distributions in the two molecules lead, for the cation, to substantially different proton hyperfine fields for the two methyl groups attached to rings I and III, while for the anion, the corresponding fields are much closer to each other, a trend in good agreement with recent data. Also explained are the features of larger methine hyperfine constants in the anion as compared to the cation and the reverse trend for the protons in rings II and IV. Other features of the calculated electron distributions in the cation and anion are discussed and compared with each other. Possible additional measurements in the two systems that could provide further tests of the theoretically obtained electron distribution will be pointed out.     

Multiple excitations in photosynthetic systems.

The yield of fluorescence in Chlorella from a 7 ns pulse of light is found to decrease gradually as a function of the number of hits in the photosynthetic units. The fivefold decrease in yield is spread over some three orders of magnitude of pulse energy and strongly suggests another random process in addition to that of photon absorption. Evidence supports the view that this random process is not in the time but in the spatial domain. The model used to fit the data is that of a unit with multiple traps for the singlet excitation. An excitation is captured by an open trap or destroyed by a filled trap with equal probability. These studies give evidence for the connectivity of the photosynthetic energy transfer apparatus on the short time scale. The short fluorescence lifetimes following picosecond pulse excitation of photosynthetic systems reported by several laboratories may be explained by the effect of multiple excitations.     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

Theory of proton hyperfine interactions in bacterial photosynthetic systems.: Bacteriopheophytin cation and anion

The electronic structures of the cation and anion of bacteriopheophytin a monomer are investigated by the self-consistent charge extended Hückel procedure including both π and σ electrons in the molecule. The calculated electron distributions are tested by comparison of the predicted hyperfine fields at proton sites with experimental data in both the ions and most important, by their ability to explain the observed trend in the hyperfine fields in going from the cation bacteriopheophytin⁺a to the anion, a trend that is similar in many respects to the corresponding observed trend for the bacteriochlorophyll a cation and anion. Good agreement is obtained with experiment both for the absolute values of the observed proton hyperfine fields in both bacteriopheophytin a cation and anion as well as the ratio of the corresponding fields for the two systems. In particular, our calculated electron distributions in the two molecules lead, for the cation, to substantially different proton hyperfine fields for the two methyl groups attached to rings I and III, while for the anion, the corresponding fields are much closer to each other, a trend in good agreement with recent data. Also explained are the features of larger methine hyperfine constants in the anion as compared to the cation and the reverse trend for the protons in rings II and IV. Other features of the calculated electron distributions in the cation and anion are discussed and compared with each other. Possible additional measurements in the two systems that could provide further tests of the theoretically obtained electron distribution will be pointed out.     

High-order fluorescence and exciton interaction in photosynthetic bacteria.

We have observed fluorescence at visible wavelengths from chromatophores of photosynthetic bacteria excited with infrared radiation which we attribute to bacteriochlorophyll of the antenna system. The fluorescence is prompt (no delay greater than 5 ns). Its spectrum shows peaks at 445, 530 (broad) and 600 nm when excited with either 694 or 868 nm. Quantum yield is of the order of 10(-9). The dependence on intensity indicates generation by mainly third-order processes which could involve triplet state in combination with excited singlets. Second-order single-singlet fusion could also contribute. The high-order fluorescence can also be explained as arising from absorption of a second photon by singlet excited states.     

Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.