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Ultrastructure of plant chloroplasts was studied by a single-molecule spectroscopy setup at a temperature of 77 K exploring spatial location of photosystems. Two chloroplast thylakoid membrane regions were visualized by fluorescence microscopy and detected at different wavelengths. The size of these regions and the spatial resolution of the microscope allowed us to measure their chlorophyll fluorescence emission spectra of these membrane domains. While the grana regions are characterized by a predominant presence of Photosystem II pigment–protein complexes emitting at 685 nm, Photosystem I complexes are localized in stroma regions and emit at 730 nm.

Low-temperature fluorescence and fluorescence excitation spectra of H2-tetra-4-tert-butyl phthalocyanine Langmuir-Blodgett films consisting of well-ordered phthalocyanines in domains and off-domain phthalocyanine molecules in random-oriented positions were measured. Persistent spectral hole burning was applied to study the role of fast excited energy transfer from domains to off-domain molecules.

Net photosynthetic rate (PN), productivity and the first phases of the fluorescence induction curve were investigated in leaves of two potato cultivars exposed to water stress. Water stress applied to potato plants at the beginning of their development (planting-bud formation) increased productivity but decreased PN and variable fluorescence (Fv) of leaves. The short-term influence of water stress on the same plants also diminished the Fv.

We observe a blue shift of the porous silicon photoluminescence peak with decreasing temperature in the range 305-180 K. Further, we study two-photon absorption (TPA) in porous silicon at 300 K through the luminescence excitation spectrum. The TPA shows an exponential tail with onset at 2hv ≈ 2.4 eV. This shape can be ascribed to potential fluctuations.

High spectral selectivity of hole burning in fluorescence spectra was used to study excited energy transfer in the CP 43 chlorophyll-protein complex of core antenna of photosystem II of Synechococcus . The observed energy transfer time is 7.6 ± 1.1 ps. The phenomenon of laser-induced hole filling was studied and explained in terms of energy transfer within the CP 43 complex.

The hole burning method was applied to the study of the photophysical properties of H 2 -tetra-4-tert-butyl phthalocyanine (H 2 -TTBPc) Langmuir-Blodgett film. The fluorescence spectra of this film together with the hole profile and the holewidth temperature dependence are presented. The influence of the fast excited state energy transfer on the presented results is discussed.

Low temperature hole burning excitation fluorescence spectra of a set of uroporphyrin I esters in amorphous (chloroform) matrix are presented. The hole burning spectra were excited within the region of the O-O S 1 → S 0 transition and measured in the temperature range 1.5 – 4.3 K. From the fluence the values of homogeneous linewidths were determined. The temperature dependence of the holewidth δ HB has been studied. The temperature dependence of δ HB follows the power law T α and coefficients α for uroporphyrin I esters are within the range 1.32 – 1.36, which is in good agreement with other results obtained for systems in amorphous matrices. The fluorescence lifetime values of Γ hom and zerotemperature extrapolated values are compared.

Results of persistent spectral hole burning in fluorescence spectra of photosystem II core antenna complexes CP 43 and CP 47 are presented. The holewidths (extrapolated to zero burning fluence) yield the excited state population decay times T 1 which are influenced by fast energy transfer within the antenna complexes. The obtained T 1 values range between 6.6 and 10 ps. The holewidths are independent on temperature within the studied temperature interval 1.5-4.2 K. The observed phenomenon of laser-induced hole filling in the spectra of CP 43 is explained in terms of energy transfer within the CP 43 pigment-protein complex.

Results of persistent hole burning fluorescence spectroscopy of green sulphur photosynthetic bacterium Chlorobium limicola are presented. Homogeneous spectral linewidths were determined from holewidths burned into inhomogeneously broadened NIR fluorescence spectra of chlorosomal bacteriochlorophyll-c at 1.8-4.2 K. The determined excited state relaxation time is strongly influenced by ultrafast energy transfer.

The persistent spectral hole burning technique was applied to the fluorescence spectra of free base tetra-4- tert -butyl phthalocyanine and chlor-aluminium tetra-4- tert -butyl phthalocyanine multilayer Langmuir-Blodgett films. Resonant zero phonon holes were burnt into the O-O fluorescence band near 700 nm at 4.2 K. A set of red shifted off-resonant holes is explained as due to localized molecular vibrational states. For both free base and central metal phthalocyanine Langmuir-Blodgett films the structure of the hole burned spectra indicates a photophysical hole burning mechanism.

The low-temperature fluorescence and fluorescence excitation hole-burning (HB) spectra at 4.2 K of uroporphyrin in amorphous (chloroform) matrix are presented. Persistent photochemical holes were burnt using both pulse and cw ring dye lasers within the region of OO transition. The HB mechanism was attributed to the tautomerisation of two inner protons of the porphyrin ring. The study of the hole width dependence on burning time and power has provided the zero extrapolated value of the homogeneous linewidth at 4.2 K (480±40) MHz. Moreover, HB kinetics were measured and HB quantum yield determined.

The persistent spectral hole burning technique has been applied to free base and central metal tetraphenylporphyrins with different substituents. The upper limits of the singlet-singlet homogeneous widths determined from the observed persistent holes have been correlated with the fluorescence lifetimes at low temperatures. Hole burning kinetics and mechanisms of both free base and central metal pigments are discussed.

This paper reports transient and persistent hole-burning of photosynthetically active as well as chemically reduced and heat inactivated photosystem II particles isolated from cyanobacteria. Transient spectra of active and non-active particles are significantly different. For both, the possible origin of the bottle-neck state is discussed. Persistent holes were ascribed to the antenna complex of photosystem II. From their width the energy transfer rate was estimated to be 4.8 ps at 4.2 K.

Photosystem II core antenna of blue-green algae Synechococcus isolated in gel was studied by optical hole burning spectroscopy at 4·2 K. Persistent holes were burned into fluorescence spectra throughout the region 680–696 nm. The hole width extrapolated to zero burning fluence yielded a value 1·0±0·2 cm−1. A theoretical interpretation of the hole profile in fluorescence is presented. The dependence of saturated hole depth on burning wavelength is related to inhomogeneous site distribution function.

Persistent and transient fluorescence hole-burning spectra of both higher plants and blue-green algae were measured to study the role of structural and functional properties of photosynthetic materials prepared by means of different biochemical methods: intact cells, thylakoide membranes, photosystem II particles and isolated antenna pigments. The width of persistent zero-phonon hole was interpreted due to fast excited energy transfer in antennae. The unstructured transient holes observed on reaction centres of photosystem II particles are connected with a charge-separated bottle-neck.

The hole burning technique was applied in fluorescence excitation spectra of free base tetra-4-tert-butyl phthalocyanine multilayer Langmuir-Blodgett films. Persistent holes were burnt into the spectral region near 615 nm (attributed to the absorption of domains of parallel molecules) and detected near 700 nm at the OO fluorescence band (attributed to the emission of off-domain molecules in the Langmuir-Blodgett films). The fast excited energy transfer running from the domains to the off-domain molecules results in the decrease of the excited state lifetime and corresponding broadening of the measured holewidth δ HB = 0.25 cm −1 at 4.2 K. The obtained δ HB temperature dependence yielding δ HB (T) = 0.20 + 0.0085T (1.15±0.1) cm −1 is discussed in the frame of the Foerster excited energy transfer model.

Persistent spectral hole-burning fluorescence spectra of photosystem II core antenna at 4.2 were measured. The obtained hole widths were interpreted in terms of fast excited energy transfer within pigment-protein complexes. The energy transfer efficiency, the inhomogeneous distribution function and hole-burning mechanism are discussed with respect to the role of the surrounding protein environment (polymer, glass). The observed light induced hole filling is also interpreted as a result of efficient energy transfer.

Chlorobium limicola is a green sulfur photosynthetic bacteria. Its light harvesting system consists of huge extra-membrane ellipsoidal structures (100-150 nm long and 30-40 nm in diameter) called chlorosomes. Each chlorosome contains several thousands of bacteriochlorophyll-c (BChl-c) molecules (organized in long rod-like pigment-protein complexes) together with a significant amount of carotenoids. The whole chlorosome is enveloped with a BChl-a containing layer and attached to the cytoplasmic membrane through a water soluble BChl-a polypeptide link complex. Light energy absorbed in BChl-c is transfered via BChl-a in the envelope and via BChl-a in the link polypeptide complex to the BChl-a containing core antenna which surrounds the reaction center and is located within the cytoplasmic membrane. In the last few years, the dynamics of the excited energy transport (EET) in the whole light harvesting system of Chlorobium limicola and related species have been extensively studied. Fast EET from BChl-c to BChl-a was studied by means of ps absorption [1] and ps fluorescence decay [2]. Both methods yield similar values of the shortest decay components which characterize the EET: 21 ps and 26 ps, respectively. Detailed hole burning study of the BChl-a containing link antenna of Prosthecochloris aestuarii revealed excitonic structure of the antenna subunits and provided the decay times (on the order of 100 fs) of upper excitonic levels [3].

Uroporhyrin, like other natural porphyrins such as coproporphyrin or protoporphyrin, is an important intermediate occurring in the metabolic pathway to protoheme. These porphyrins are important indicators of pathogenic states in an organism. Luminescence spectra of these molecules consist of (≈ 100 cm–1) broad bands due to an inhomogeneous broadening. Fluorescence line narrowing and hole burning spectroscopies enable to remove an inhomogeneous broadening and high resolution spectra can be obtained (≈ 5 cm–1 and ≈ 0.01 cm–1, respectively). Low temperature optical spectra of coproporphyrin and protoporphyrin in various matrices have been measured [1-4]. The aim of this contribution is to present hole burning and fluorescence line narrowing spectra of uroporphyrin.

Langmuir—Blodget (LB) films are thin or ultrathin molecular layer systems. They are good basis for the creation of molecular encembles with predetermined architecture including superlattices and quasi two-dimensional systems. This opens new possibilities for a detailed investigation of various intermolecular coupling. In this paper we present preliminary fluorescence hole burning data of metal-free tetra-4-terc-butyl phthalocyanine (H2TTBPc) in LB film.

Phthalocyanine Lanngmuir Blodgett (LB) films represent ultra thin monomolecular layers with molecules in tight contact. Most of the phthalocyanine molecules in LB films are ordered in domains where they occupy well defined (parallel) positions. There is also a minority of non-ordered molecules in off-domain positions in LB films. This fact is well documented in X-ray diffraction and transmission electron diffraction [1]. Moreover, significant inhomogeneous broadening of absorption spectra of LB film has been observed (see fig. 1), in comparison with absorption spectra of isolated molecules. On the other hand the observed fluorescence is very weak, nevertheless its spectral profile well corresponds to that of isolated molecules. This phenomenon can be well explained on the basis of strong aggregation and/or fast excited energy transfer (EET) [2]. Persistent hole burning (PHB) (in fluorescence excitation spectra) has recently been applied on LB film of related porphyrin - poly-heptyl-cyanoacrylate matrix [3]. The PHB study of tetraaazoporphyrin in LB films suggested this EET interpretation. The role of EET in free base phthalocyanines was also discussed in [4] together with the analysis of off-resonant holes in fluorescence spectra. The aim of this paper is to determine the excited state lifetimes T1 strongly influenced by fast EET from PHB measurements (in fluorescence excitation spectra).

The application of hole burning spectroscopy (HB) in the study of photosynthetic systems offers an independent method for determining excited state lifetimes of particular chromophores. The rate constants of excitation energy transfer (EET) in photosynthetic antennae can be directly determined by time resolved fluorescence spectroscopy. For most bacterial and higher plants antennae is of the order of 10-12 s–1 [1]. Efficient EET in pigment-protein complexes causes significant shortening (three orders of magnitude) of the excited state lifetimes T1 in comparison with isolated pigments.