Photon echo studies of photosynthetic light harvesting

The broad linewidths in absorption spectra of photosynthetic complexes obscure information related to their structure and function. Photon echo techniques represent a powerful class of time-resolved electronic spectroscopy that allow researchers to probe the interactions normally hidden under broad linewidths with sufficient time resolution to follow the fastest energy transfer events in light harvesting. Here, we outline the technical approach and applications of two types of photon echo experiments: the photon echo peak shift and two-dimensional (2D) Fourier transform photon echo spectroscopy. We review several extensions of these techniques to photosynthetic complexes. Photon echo peak shift spectroscopy can be used to determine the strength of coupling between a pigment and its surrounding environment including neighboring pigments and to quantify timescales of energy transfer. Two-dimensional spectroscopy yields a frequency-resolved map of absorption and emission processes, allowing coupling interactions and energy transfer pathways to be viewed directly. Furthermore, 2D spectroscopy reveals structural information such as the relative orientations of coupled transitions. Both classes of experiments can be used to probe the quantum mechanical nature of photosynthetic light-harvesting: peak shift experiments allow quantification of correlated energetic fluctuations between pigments, while 2D techniques measure quantum beating directly, both of which indicate the extent of quantum coherence over multiple pigment sites in the protein complex. The mechanistic and structural information obtained by these techniques reveals valuable insights into the design principles of photosynthetic light-harvesting complexes, and a multitude of variations on the methods outlined here

Two-color Three-pulse Photon Echo Studies on the Photosynthetic Bacterial Reaction Center

Photosynthesis begins with absorbing the sun light by the light harvesting complexes. The solar energy is then funneled into the reaction center (RC) via the energy transfer between the light harvesting complexes at ultrafast rates (~1/100fs ) with extremely high quantum efficiency (~100 %). Most of the complexes are composed of pigments and protein matrices that tightly bind them. The pigments are responsible for absorbing and transferring the energy. The roles of the protein environment of photosynthetic pigment-protein complexes have been suggested, but the detailed mechanisms are still not fully understood. In this dissertation, non-linear spectroscopic methods using ultrashort pulses (~ 40-fs FWHM), three-pulse photon echo studies are presented to investigate the roles of protein environment of the photosynthetic bacterial RC. The technique characterizes the protein dynamics around the pigments (a bacteriochlorophyll a, B and a bacteriopheophytin a, H) in the RC. In particular, two-color three-pulse electronic coherence photon echo technique is used to observe the quantum coherence between the excited states of coupled H and B, whose life time is sensitive to the protein dynamics. I found a long-lasting quantum coherence suggesting that the protein actively preserves the quantum coherence. A scenario in which the long-lasting coherence can accelerate the rate of energy trap is described with a simple Bloch model simulation. In addition, one- and two-color three-pulse photon echo peak shift (1C- and 2C3PEPS) techniques are used to measure the coupling strength between H and B in the wild type RC. The coupling strength is facilitated from the geometry between the pigments governed by the protein environment. The simulation based on the standard response function formalism is used to obtain the coupling strength. 2C3PEPS signal from H and B of the oxidized RC is reproduced to extract the coupling constant between them by quantum-master equation which efficiently incorporates pulse overlap effect and bath memory effect. The values will enable the molecular level of studies on the photosynthetic energy and electron transfer

Rapid and Simple Detection of Ochratoxin A using Fluorescence Resonance Energy Transfer on Lateral Flow Immunoassay (FRET-LFI)

The detection of mycotoxins is crucial because of their toxicity in plants, animals, and humans. It is very important to determine whether food products are contaminated with mycotoxins such as ochratoxin A (OTA), as mycotoxins can survive heat treatments and hydrolysis. In this study, we designed a fluorescence resonance energy transfer (FRET)-based system that exploits antibody-antigen binding to detect mycotoxins more rapidly and easily than other currently available methods. In addition, we were able to effectively counteract the matrix effect in the sample by using a nitrocellulose membrane that enabled fluorescence measurement in coffee samples. The developed FRET on lateral flow immunoassay (FRET-LFI) system was used to detect OTA at a limit of detection (LOD) of 0.64 ng∙mL−1, and the test can be completed in only 30 min. Moreover, OTA in coffee samples was successfully detected at a LOD of 0.88 ng∙mL−1, overcoming the matrix effect, owing to the chromatographic properties of the capillary force of the membrane. We believe that the developed system can be used as a powerful tool for the sensitive diagnosis of harmful substances such as mycotoxins and pesticides for environmental and food quality control monitoring

Stochastic Photon Emission from Nonblinking Upconversion Nanoparticles

Because of their well-known optical properties, upconversion nanoparticles (UCNPs) are regarded as some of the most promising nanomaterials for bioimaging, biosensors, and solar cells. The nonblinking nature of their upconversion emissions has been a particularly beneficial advantage for live-cell imaging. However, the origin of this unique property has never been seriously investigated. We report, for the first time, the observation of stochastic photon emission (SPEM) in core/shell UCNPs (NaYF₄:Yb³⁺,Er³⁺/NaYF₄) on the microsecond and nanosecond time scales, even under continuous irradiation at 980 nm. This SPEM was attributed to slow “upconversion cycles”. We consider that the conventionally reported, nonblinking nature of UCNP emissions can be attributed to the averaging of SPEMs from multiple Er³⁺ ions and the low temporal resolution of previous observation. The off-time distribution, which possesses kinetics information for the upconversion pathways, was well fitted to a single exponential indicating involvement of a single rate-determining step. The distinct behaviors of the green and red emissions confirm their different photophysical pathways

The preferred upconversion pathway for the red emission of lanthanide-doped upconverting nanoparticles, NaYF4:Yb3+,Er3+

Lanthanide-doped upconverting nanoparticles (UCNPs, NaYF4:Yb3+, Er3+) are well known for emitting visible photons upon absorption of two or more near-infrared (NIR) photons through energy transfer from the sensitizer (Yb3+) to the activator (Er3+). Of the visible emission bands (two green and one red band), it has been suggested that the red emission results from two competing upconversion pathways where the non-radiative relaxation occurs after the second energy transfer (pathway A, I-4(15/2) -> I-4(11/2) -> F-4 -> -> H-2(11/2) -> S-4(3/2) -> F-4(9/2) -> I-4(15/2)) or between the first and the second energy transfer (pathway B, I-4(15/2) -> I-4(11/2) -> I-4(13/2) -> F-4(9/2) -> I-4 ->). However, there has been no clear evidence or thorough analysis of the partitioning between the two pathways. We examined the spectra, power dependence, and time profiles of UCNP emission at either 980 nm or 488 nm excitation, to address which pathway is preferred. It turned out that the pathway B is predominant for the red emission over a wide range of excitation powers.