Fluorescence polarization and pigment orientation in photosynthetic bacteria

Fluorescence polarization of photosynthetic bacteria with various types of chromatophores suggests an orientation of bacteriochlorophylls, but not of carotenoids. Comparison of the in vitro fluorescence polarization spectra of bacteriochlorophyll at high and low concentration and at 77 °K with those in vivo at various temperatures and in the presence of carbowax indicates that dichroism of shape effects the in vivo spectra. Both orientation and shape effects are highest for bacteria containing lamellar-type chromatophores, and lowest in those containing vesicle-type ones.

The polarization values of the bacteria studied are similar for the various red bands, indicating a nearly parallel orientation of the adjacent bacteriochlorophylls.     

Scanning orientation and polarization effects for XRQA radiochromic film

Gafchromic XRQA radiochromic film, is an effective tool for quality assurance and dose assessment in kilovoltage radiotherapy and diagnostic applications. Like other Gafchromic film products, XRQA film exhibits a variation in dose to reflected optical density response with angle of rotation when analysed with a light source that is partially or fully polarised such as a desktop scanner. Although warnings are not given on manufacturers specifications, this can affect dosimetry accuracy and we recommend that it is essential to scan all XRQA films in the same orientation. The effect is not as pronounced as EBT Gafchromic film. The magnitude of this variation has been measured and shown to be up to 16 ± 2% (1SD) using a fully linear polarised light source was seen with a 90° angle rotation. This would be the maximum variation seen on a desktop scanner with a fully polarised light source. For our standard desktop scanner (Epson v700) a mean variation of 2 ± 1% from 0 cGy to 20 cGy applied dose was measured as compared to 8 ± 2% for EBT Gafchromic. We recommend that to decrease uncertainty in dose measurement, accurate alignment of the calibration films to experimental films be performed on a regular basis. This is especially important if your desktop scanner has a high degree of polarization of its light source.     

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.     

The role of skylight polarization in the orientation of a day-migrating bird species

To assess the role of skylight polarization in the orientation system of a day-migrating bird, Yellow-faced Honeyeaters (Lichenostomus chrysops, Meliphagidae) were tested in funnel cages for their directional preferences. In control tests in the natural local geomagnetic field under the clear natural sky, they preferred their normal migratory course. Manipulations of the e-vector by depolarizing the skylight or rotating the axis of polarization failed to affect the orientation as long as the natural geomagnetic field was present. When deprived of magnetic information, the birds continued in their normal migratory direction as long as they had access to information from the natural sky, or when either the sun or polarized light was available. However, when sun was hidden by clouds, depolarizers caused disorientation. — These findings indicate that polarized skylight can be used for orientation when no other known cues are available. However in the hierarchy of cues of this species, the polarization pattern clearly ranks lower than information from the geomagnetic field.     

Wavelength-dependent collagen fragmentation during mid-IR laser ablation

Mid-infrared free-electron lasers have proven adept in surgical applications. When tuned to wavelengths between 6 and 7 microm, such lasers remove defined volumes of soft tissue with very little collateral damage. Previous attempts to explain the wavelength-dependence of collateral damage have invoked a wavelength-dependent loss of protein structural integrity. However, the molecular nature of this structural failure has been heretofore ill-defined. In this report, we evaluate several candidates for the relevant transition by analyzing the nonvolatile debris ejected during ablation. Porcine corneas were ablated with a free-electron laser tuned to 2.77 or 6.45 microm-wavelengths with matched absorption coefficients for hydrated corneas that respectively target either tissue water or protein. The debris ejected during these ablations was characterized via gel electrophoresis, as well as Fourier transform infrared spectroscopy, micro-Raman and 13C-NMR spectroscopy. We find that high-fluence (240 J/cm2) ablation at 6.45 microm, but not at 2.77 microm, leads to protein fragmentation accompanied by the accumulation of nitrile and alkyne species. The candidate transition most consistent with these observations is scission of the collagen protein backbone at N-alkylamide bonds. Identifying this transition is a key step toward understanding the observed wavelength-dependence of collateral damage in mid-infrared laser ablation.     

Wavelength-dependent photocycle activity of xanthorhodopsin in the visible region

Xanthorhodopsin (xR) is a dual-chromophore proton-pump photosynthetic protein comprising one retinal Schiff base and one light-harvesting antenna salinixanthin (SX). The excitation wavelength-dependent transient population of the intermediate M demonstrates that the excitation of the retinal at 570 nm leads to the highest photocycle activity and the excitations of SX at 460 and 430 nm reduce the activity to ca. 37% relatively, suggesting an energy transfer pathway from the S₂ state of the SX to the S₁ state of the retinal and a quick internal vibrational relaxation in the S₂ state of SX prior to the energy transfer from SX to retinal.     

Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy

A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.     

Wavelength-dependent spectral changes accompany CN-hemin binding to human apohemoglobin

The interaction of apohemoglobin with two heme derivatives, CN-protohemin and CN-deuterohemin, was monitored at multiple Soret wavelengths (417–423 and 406–412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10°C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both CN-protohemin and CN-deuterohemin derivatives with the a chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of - and -chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral a propionyl 45(CE3); 58(E7); 61(E10) as well as the b vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the dimer interface.     

Wavelength-dependent conformational changes in collagen after mid-infrared laser ablation of cornea

We ablated porcine corneas with a free electron laser tuned to either 2.77 or 6.45 μm, two matched wavelengths that predominantly target water and protein, respectively. The ejected nonvolatile debris and the crater left behind were examined by circular dichroism, Raman spectroscopy, and scanning electron microscopy to characterize the postablation conformation of collagen proteins. We found near-complete unfolding of collagen secondary and tertiary structure at either ablating wavelength. On the other hand, we found excess fibril swelling and evidence for excess cis-hydroxyproline in the 6.45-μm debris. These results support the hypothesis that the favorable ablative properties of protein-targeting wavelengths rest on selective heating of tissue proteins.     

Morphology of the Daphnia nervous system: A comparative study on Daphnia pulex,Daphnia lumholtzi,and Daphnia longicephala

The freshwater crustacean Daphnia is well known for its phenotypic plasticity, in which environmental cues are perceived by the nervous system and transformed into phenotypic adaptations beneficial under current conditions. Critical knowledge regarding the distribution and localization of neuronal antigens or neurotransmitters and differentially expressed proteins is sparse. Here, we applied immunohistochemical and histological‐staining methods in combination with confocal laser scanning microscopy on whole mount preparations in Daphnia pulex, Daphnia longicephala, and Daphnialumholtzi. We document the nervous system, elements of the sensory system, and cell clusters with secretory characteristics in the Daphnia head. This is the first report on the nervous system of the species D.longicephala and D.lumholtzi. The methods established in this investigation will help to foster research on specific neuronal mechanisms in this rapidly advancing model system of life science research. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Choosing orientation: influence of cargo geometry and ActA polarization on actin comet tails

Networks of polymerizing actin filaments can propel intracellular pathogens and drive movement of artificial particles in reconstituted systems. While biochemical mechanisms activating actin network assembly have been well characterized, it remains unclear how particle geometry and large-scale force balance affect emergent properties of movement. We reconstituted actin-based motility using ellipsoidal beads resembling the geometry of Listeria monocytogenes. Beads coated uniformly with the L. monocytogenes ActA protein migrated equally well in either of two distinct orientations, with their long axes parallel or perpendicular to the direction of motion, while intermediate orientations were unstable. When beads were coated with a fluid lipid bilayer rendering ActA laterally mobile, beads predominantly migrated with their long axes parallel to the direction of motion, mimicking the orientation of motile L. monocytogenes. Generating an accurate biophysical model to account for our observations required the combination of elastic-propulsion and tethered-ratchet actin-polymerization theories. Our results indicate that the characteristic orientation of L. monocytogenes must be due to polarized ActA rather than intrinsic actin network forces. Furthermore, viscoelastic stresses, forces, and torques produced by individual actin filaments and lateral movement of molecular complexes must all be incorporated to correctly predict large-scale behavior in the actin-based movement of nonspherical particles.     

Sunset,skylight polarization and the migratory orientation of yellow-rumped warblers,Dendroica coronata

Celestial light cues visible at sunset appear to play an important role in the nocturnal orientation of several species of night-migrating birds. The pattern of skylight polarization, an especially prominent geographical reference at sunrise and sunset, influences the orientation behaviour of migratory birds. Yellow-rumped warblers were capable of seasonally appropriate cage orientation at dusk and were sensitive to manipulation of the axis of skylight polarization (E-vector). A series of experimental treatments was designed to examine the relationship between sunset position and skylight polarization. The window panels of hexagonal enclosures were fitted with a depolarizer and a polaroid filter to rotate the E-vector, and mirrors to reflect the position of sunset. The results indicate that this migrant minimizes sunset position as an orientation relative to skylight polarization and may depend upon the latter to orient at dusk. The possibility that yellow-rumped warblers calibrate their sun compass in relation to polarized light remains a question for future research.     

Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.     

Wavelength-dependent xanthophyll cycle activity in marine microalgae exposed to natural ultraviolet radiation

The wavelength dependency of xanthophyll cycling in two marine microalgae (Thalassiosira weissflogii and Dunaliella tertiolecta) was studied by establishing biological weighting functions (BWFs) during exposure to natural ultraviolet radiation. High-(HL) and low-(LL) light-acclimated cultures of both species were exposed outdoors for up to 60 min under a series of UVR (280–400 nm) cut-off filters, after which the de-epoxidation state of xanthophyll cycle pigments, radiocarbon assimilation and photochemical quantum yield were measured. Exposures were repeated 4–8 times during the daily cycle to create exposure–response curves for each wavelength condition. UVR affected the three target processes significantly in both species and biological weights increased with decreasing wavelength, particularly in the UVBR region (280–315 nm). Minor wavelength dependency was observed between 315 and 360 nm. After BWF normalization to 300 nm, the LL cultures showed highly similar responses when comparing the three target processes, while the BWFs for the HL cultures differed significantly. The observed enhanced xanthophyll cycling activity in the UVR region implied that xanthophylls had an active role in diminishing UVR stress. However, this enhancement seems to be an indirect effect of damage within the dark reactions of photosynthesis. Hence, another vital target process further downstream in the photosynthetic process, possibly involved in the dark reactions, seems to be responsible for the high similarity in BWFs.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.