Label‐free multi‐parametric imaging of single cells: dual picosecond optoacoustic microscopy

Advances in microscopy with new visualization possibilities often bring dramatic progress to our understanding of the intriguing cellular machinery. Picosecond optoacoustic micro‐spectroscopy is an optical technique based on ultrafast pump‐probe generation and detection of hypersound on time durations of picoseconds and length scales of nanometers. It is experiencing a renaissance as a versatile imaging tool for cell biology research after a plethora of applications in solid‐state physics. In this emerging context, this work reports on a dual‐probe architecture to carry out real‐time parallel detection of the hypersound propagation inside a cell that is cultured on a metallic substrate, and of the hypersound reflection at the metal/cell adhesion interface. Using this optoacoustic modality, several biophysical properties of the cell can be measured in a noncontact and label‐free manner. Its abilities are demonstrated with the multiple imaging of a mitotic macrophage‐like cell in a single run experiment.      

Alzheimer's disease amyloid-β peptide analogue alters the ps-dynamics of phospholipid membranes

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-β (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-β (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290 K and 320 K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-β (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-β (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-β (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-β (25-35) peptide induced membrane alteration even at only 3 mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.     

Electron and nuclear dynamics in many-electron atoms, molecules and chlorophyll-protein complexes: A review

It has been shown [V.A. Shuvalov, Quantum dynamics of electrons in many-electron atoms of biologically important compounds, Biochemistry (Mosc.) 68 (2003) 1333-1354; V.A. Shuvalov, Quantum dynamics of electrons in atoms of biologically important molecules, Uspekhi biologicheskoi khimii, (Pushchino) 44 (2004) 79-108] that the orbit angular momentum L of each electron in many-electron atoms is L = mVr = n? and similar to L for one-electron atom suggested by N. Bohr. It has been found that for an atom with N electrons the total electron energy equation E = −(Z^eff)²e⁴m/(2n²?²N) is more appropriate for energy calculation than standard quantum mechanical expressions. It means that the value of L of each electron is independent of the presence of other electrons in an atom and correlates well to the properties of virtual photons emitted by the nucleus and creating a trap for electrons. The energies for elements of the 1st up to the 5th rows and their ions (total amount 240) of Mendeleev' Periodical table were calculated consistent with the experimental data (deviations in average were 5 × 10^− 3). The obtained equations can be used for electron dynamics calculations in molecules. For H₂ and H₂⁺ the interference of electron-photon orbits between the atoms determines the distances between the nuclei which are in agreement with the experimental values. The formation of resonance electron-photon orbit in molecules with the conjugated bonds, including chlorophyll-like molecules, appears to form a resonance trap for an electron with E values close to experimental data. Two mechanisms were suggested for non-barrier primary charge separation in reaction centers (RCs) of photosynthetic bacteria and green plants by using the idea of electron-photon orbit interference between the two molecules. Both mechanisms are connected to formation of the exciplexes of chlorophyll-like molecules. The first one includes some nuclear motion before exciplex formation, the second one is related to the optical transition to a charge transfer state.     

Interactions between PTB RRMs Induce Slow Motions and Increase RNA Binding Affinity

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. ¹⁵N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.     

Spectral characteristics of fluorophores formed via interaction between all-trans-retinal with rhodopsin and lipids in photoreceptor membrane of retina rod outer segments

It is shown that all-trans-retinal under model conditions of its excessive accumulation in photoreceptor membranes interacts with amino groups of rhodopsin and lipids, forming at least three distinct fluorophores with fluorescence quantum yield 20–40 times higher than that of free all-trans-retinal. These retinal derivatives are likely precursors of photo- and cytotoxic fluorophores of lipofuscin and in particular of A2E. Spectral characteristics of fluorophores have been described. Picosecond time-resolved laser fluorescence spectroscopy was used to study kinetics of fluorescence decay of both free and bound all-trans-retinal; fluorophores were determined and their lifetimes have been measured. Based on calculations it is shown that the decay kinetics of all-trans-retinal derivatives consists of three components with lifetimes equal to 48, 208, and 900 ps; kinetics of free all-trans-retinal is monoexponential with lifetime of 31 ps. The chemical nature of fluorophores with the lifetimes obtained is discussed.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

The rate of formation of P700+—A0 - in photosystem I particles from spinach as measured by picosecond transient absorption spectroscopy

Photosystem I particles containing 30–40 chlorophyll a molecules per primary electron donor P700 were subjected to 1.5 ps low density laser flashes at 610 nm resulting in excitation of the antenna chlorophyll a molecules followed by energy transfer to P700 and subsequent oxidation of P700. Absorbance changes were monitored as a function of time with 1.5 ps time resolution. P700 bleaching (decrease in absorbance) occurred within the time resolution of the experiment. This is attributed to the formation of ¹P700.^* This observation was confirmed by monitoring the rise of a broad absorption band near 810 nm due to chlorophyll a excited singlet state formation. The appearance of the initial bleach at 700 nm was followed by a strong bleaching at 690 nm. The time constant for the appearance of the 690 nm bleach is 13.7±0.8 ps. In the near-infrared region of the spectrum, the 810 nm band (which formed upon the excitation of the photosystem I particles) diminished to about 60% of its original intensity with the same 13.7 ps time constant as the formation of the 690 nm band. The spectral changes are interpreted as due to the formation of the charge separated state P700⁺—A₀ ^-, where A₀ is the primary electron acceptor chlorophyll a molecule.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves

Higher plants must dissipate absorbed light energy that exceeds the photosynthetic capacity to avoid molecular damage to the pigments and proteins that comprise the photosynthetic apparatus. Described in this minireview is a current view of the biochemical, biophysical and bioenergetic aspects of the primary photoprotective mechanism responsible for dissipating excess excitation energy as heat from photosystem II (PSII). The photoprotective heat dissipation is measured as nonphotochemical quenching (NPQ) of the PSII chlorophyll a (Chl a) fluorescence. The NPQ mechanism is controlled by the trans-thylakoid membrane pH gradient (ΔpH) and the special xanthophyll cycle pigments. In the NPQ mechanism, the de-epoxidized endgroup moieties and the trans-thylakoid membrane orientations of antheraxanthin (A) and zeaxanthin (Z) strongly affect their interactions with protonated chlorophyll binding proteins (CPs) of the PSII inner antenna. The CP protonation sites and steps are influenced by proton domains sequestered within the proteo-lipid core of the thylakoid membrane. Xanthophyll cycle enrichment around the CPs may explain why changes in the peripheral PSII antenna size do not necessarily affect either the concentration of the xanthophyll cycle pigments on a per PSII unit basis or the NPQ mechanism. Recent time-resolved PSII Chi a fluorescence studies suggest the NPQ mechanism switches PSII units to an increased rate constant of heat dissipation in a series of steps that include xanthophyll de-epoxidation, CP-protonation and binding of the xanthophylls to the protonated CPs; the concerted process can be described with a simple two-step, pH-activation model. The xanthophyll cycle-dependent NPQ mechanism is profoundly influenced by temperatures suboptimal for photosynthesis via their effects on the trans-thylakoid membrane energy coupling system. Further, low temperature effects can be grouped into either short term (minutes to hours) or long term (days to seasonal) series of changes in the content and composition of the PSII pigment-proteins. This minireview concludes by briefly highlighting primary areas of future research interest regarding the NPQ mechanism.