Photoswitchable cyan fluorescent protein for protein tracking

In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780-840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms.     

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.     

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.     

In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.     

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.     

Visualization of the post-Golgi trafficking of multiphoton photoactivated transferrin receptors

Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn(+)EEA1(+) endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway.     

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.     

Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one-photon and two-photon excitation.

We measured the emission spectra, intensity decays and anisotropy decays of the single tryptophan residue of human serum albumin (HSA) resulting from one-photon (295-298 nm) and two-photon (590-596) excitation. The emission spectra and intensity decays were independent of the mode of excitation. The anisotropy decays were superficially similar for one- and two-photon excitation. However, upon consideration of the different orientation photoselection for one- and two-photon excitation, the anisotropy data reveal different angles between the absorption and emission oscillators for one-photon and two-photon excitation. This result suggests different relative one-photon and two-photon cross-sections for the 1La and 1Lb transitions of the indole residue. This first report of the time-resolved anisotropy decay of a protein resulting from two-photon excitation suggests that such measurement will yield insights into the complex photophysical properties of tryptophan residues in proteins.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state ³P700, created via radical pair formation and recombination. ³P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at _f > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10^-4 cm^-1 and E=38.5×10^-4 cm^-1, were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the ³P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < _f < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < _f < 700 nm (inner core antennae, F690), and _f > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of ³P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.Abbreviations Chl chlorophyll - CP1 core chlorophyll protein of Photosystem I - EPR electron paramagnetic resonance - F690, F720 chlorophyll forms having fluorescence maximum at 690–695 and 720 nm, respectively - F(A)(O)DMR fluorescence (absorption) (optical) detected magnetic resonance - FF fluorescence fading - ISC intramolecular intersystem crossing - _f fluorescence emission wave-length - LHC I light harvesting chlorophyll a/b protein of Photosystem I - P700 primary donor of Photosystem I - PS I Photosystem I - RC reaction centre - RP radical pair - SDS sodium dodecyl sulphate - ZFS zero field splitting     

Spectral resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 through direct excitation

We have measured fluorescence spectra from Photosystem I (PS I) on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 at room temperature as a function of excitation wavelength. Our data show a gradual enhancement of long-wavelength fluorescence at 710 nm as the excitation wavelength is increased from 695 to 720 nm. This verifies the presence of low-energy chlorophylls (LE Chls), antenna Chls with energy levels below that of the primary electron donor, P700. The change in fluorescence with excitation wavelength is attributed to the finite time it takes for equilibration of excitations between the bulk and LE Chls. The spectra were deconvoluted into the sum of two basis spectra, one an estimate for fluorescence from the majority or bulk Chls and the other, the LE Chls. The bulk Chl spectrum has a major peak at 688 nm and a lower amplitude vibrational band around 745 nm and is assumed independent of excitation wavelength. The LE Chl spectrum has a major peak at 710 nm, with shoulders at 725 and 760 nm. The relative amplitude of emission at the vibrational side bands increases slightly as the excitation wavelength increases. The ratio of the fluorescence yields from LE Chls to that from bulk Chls ranges from 0.3 to 1.3 for excitation wavelengths of 695 to 720 nm, respectively. These values are consistent with a model where the LE Chls are structurally close to P700 allowing for direct transfer of excitations from both the bulk and LE Chls to P700.     

Dual-photon excitation of albumin

Fluorescence emission after two-photon excitation at 580 nm is observed in albumin by means of Nd:YAG laser at room temperature. The two-photon excitation spectral range 550-590 nm was obtained. The experimental results show that albumin fluorescence originates from tryptophan residues.     

Purification of the yellow fluorescent protein from Vibrio fischeri and identity of the flavin chromophore

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.     

The two-photon induced fluorescence of the tumor localizing photosensitizer hematoporphyrin derivative via 1064 nm photons from a 20 ns Q-switched Nd-YAG laser

We demonstrate the direct 1064 nm two-photon excitation of hematoporphyrin derivative (HPD), a complex mixture of photosensitizing porphyrins which is selectively retained in tumor tissue and used in cancer photochemotherapy. Although 1064 nm is outside of the one-photon HPD absorption spectrum, two-photon induced fluorescence from HPD was observed following excitation by the 20 ns output of an amplified, Q-switched Nd-YAG laser at peak power levels of 0.1 to 3 GW/cm2. Evidence for the successful two-photon excitation to vibrational levels of the S1 state consists of the observation of the known HPD fluorescence spectrum exhibiting peaks at approximately 615 and 675 nm, with the observed two-photon induced fluorescence intensity exhibiting a quadratic dependence on the excitation laser intensity as required for a direct two-photon process. More generally, these results suggest the possibility for the achievement of photosensitized oxidations utilizing photons of lower energy than that required for single photon excitation, offering the potential for both greater selectivity and a reduction in competing photochemical processes.     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.