Reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing

Wavefront shaping can compensate the wavefront distortions in deep tissue focusing, leading to an improved penetration depth. However, when using the backscattered signals as the feedback, unexpected compensation bias may be introduced, resulting in focusing position deviations or even no focus in the illumination focal plane. Here we investigated the reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing by measuring the position deviations between the foci in the illumination focal plane and the epi‐detection plane. The experimental results show that when the penetration depth reaches 150 μm in mouse brain tissue (with scattering coefficient ~22.42 mm^?1) using a 488 nm laser and an objective lens with 0.75 numerical aperture, the center of the real focus will deviate out of one radius range of the Airy disk while the optimized focus in the epi‐detection plane maintained basically at the center. With the penetration depth increases, the peak to background ratio of the focus in the illumination focal plane decreases faster than that in the epi‐detection plane. The results indicate that when the penetration depth reaches 150 μm, feedback based on backscattered signals will make wavefront shaping lose its reliability, which may provide a guidance for applications of non‐invasive precise optogenetics or deep tissue optical stimulation using wavefront shaping methods. A, Intensity distribution in the epi‐detection plane and the illumination focal plane before and after correction, corresponding to brain sections with 250 and 300 μm thickness, respectively. Scale bar is 2 μm. B, Averaged focusing deviations in the epi‐detection plane (optimized) and the illumination focal plane (monitored) after compensation. The unit of the ordinate is one Airy disk diameter. Black dashed line represents one Airy disk radius. Bars represent the SE of each measurement set.      

Electrical recording and ultrastructure of stylet penetration by the greenhouse whitefly

Earlier studies have indicated that interior (physical and/or chemical) properties of a plant may be responsible for feeding-site selection by the greenhouse whitefly, Trialeurodes vaporariorum (Westw.). In order to study the process of feeding-site selection further, stylet-penetration activities and the pathway followed by the stylets in host-plant tissue were investigated using a DC electrical recording method and transmission electron microscopy (TEM). Penetrating whiteflies attached to a gold wire were included in an electrical circuit to record electrical penetration graphs (EPGs). Seven EPG patterns have been distinguished, five of which could be correlated with components of the stylet-penetration process: 1) one with penetration of the leaf surface, 2) one with intercellular penetration and salivary-sheath secretion, 3) one with sieve element penetration and ingestion, 4) one with short penetration of a cell, and 5) one with xylem penetration. The stylet pathway is almost completely intercellular before the phloem is reached and in contrast to aphids, brief symplast punctures are very rare. In general, it takes T. vaporariorum more than half an hour from the start of a penetration to reach a sieve element. Rejection of feeding sites occurs within a few minutes of penetration by adult whiteflies, a time span in which stylets are presumed to penetrate just beyond the epidermis. Properties of the apoplast close to the leaf surface seem therefore to play a major role in feeding-site selection.

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Résumé De précédentes études ont montré que les propriétés internes (physiques et/ou chimiques) d'une plante peuvent induire la sélection du site de nutrition de la mouche blanche de serres, Trialeurodes vaporariorum (Westw.). Afin d'étudier plus avant le processus de sélection du site de nutrition, les activités de piqûre et le chemin suivi par le stylet dans les tissus de la plante-hôte ont été étudiés par une méthode d'enregistrement électrique en courant continu ainsi que par microscopie à transmission d'électrons (TEM). Des aleurodes en activité de piqûre attachées à un fil d'or ont été incluses dans un circuit électrique pour enregistrer des graphes de pénétration électriques (EPG). Sept motifs d'EPG ont été distingués, dont cinq peuvent être corrélés aux composantes du processus de pénétration du stylet: 1) un avec pénétration de la surface foliaire, 2) un avec pénétration interecellulaire et sécrétion d'une gaine de salive, 3) un avec pénétration du phloème, 4) un avec courte pénétration d'une cellule, et 5) un avec pénétration du xylème. Le parcours du stylet est presque entièrement intercellulaire avant que le phloème soit atteint. Contrairement aux pucerons, les ponctions brèves dans le symplasme sont rares. Généralement, T. vaporariorum met plus d'une demi-heure, à partir du début d'une piqûre, pour atteindre un vaisseau. Le rejet des sites de nutrition par les aleurodes adultes se passe quelques minutes après la pénétration du stylet; pendant ce laps de temps, le stylet pénétrerait juste sous l'épiderme. Le rôle des propriétés de l'apoplaste près de la surface foliaire semble donc majeur dans la sélection des sites de nutrition.

     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Membrane potentials as an indication for plant cell penetration by aphid stylets

Electrical penetration graphs (EPGs) of aphids on plants demonstrate distinct periods of lowered potential level: the potential drop (pd). Such pds are produced frequently during the stylet pathway to the phloem. Experimental evidence supports the hypothesis that the pd corresponds with a stylet puncture of a (epidermal or mesophyll) cell membrane. The intracellular potentials of-100 to-180 mV are thought to be measured thereby with the stylets acting as microelectrodes. The short pd of 5–15 s can be described as a sequence of three distinct phases. Besides the short pd, occurring during pattern B and C, long pds are produced during the complete pattern D+E. Pattern D+E may occur on the potential level of the pd (abbr.: D+E (pd)) and on the potential level of the preceding pattern C (abbr.: D+E(c)). Pattern D+E(pd) seems to be related to sieve element penetration, at least in a number of cases. Both pds, short and long, are produced on host and non-host plants, on susceptible and resistant cultivars, using several aphid species.

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Potentiels membraneux comme indication pour des pénétrations intracellulaires des plantes par les stylets des aphides

Résumé L'enrégistrement électrique de la pénétration du stylet des aphides dans les tissus de la plante se caracterise par des périodes distinctes de chute de potentiel (c.p.). Ces c.p. existent fréquemment au cours du trajet du stylet vers le phloème. Une évidence expérimentale soutient l'hypothèse de la correspondance entre la c.p. et la piqûre de la membrane des cellules épidermales et parenchymateuses. Les potentiels intracellulaires de-100 à-180 mV pourraient être mesurés par l'intermédiaire des stylets comme microélectrodes. Les. c.p. brèves, de 5 à 15 s peuvent être décrites comme des séquences composées de 3 phases distinctes. Outre les c.p. brèves pendant les ondes B et C, les pucerons produisent des c.p. durables qui se maintiennent au cours des ondes D+E. Ou bien l'onde D+E peuve apparaite au niveau de potentiel de c.p. (D+E(cp)), ou au niveau de potentiel de l'onde C précédente (D+E(c)). L'onde D+E(cp) semble être reliée à la pénétration des vaisseaux conducteurs, du moins dans certain cas. Les c.p. brêves et durables sont produits sur les plantes hôtes et non-hôtes chez plusiers espèces d'aphids.

     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Ultrastructure of in vivo fertilization in the goat

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

Ultrastructure of Sporozoites and Zoites of Hammondia heydorni

The ultrastructure of sporozoites and zoites of Hammondia heydorni was studied in cultured bovine cells. In addition to ultrastructural features typical of coccidian parasites, H. heydorni sporozoites and zoites contain rhoptries that are located posteriorly as well as anteriorly. Also, sporozoites contain a posteriorly located crystalloid body (1.2 μm in diameter); a small crystalloid body (0.5 μm in diameter) was occasionally seen in the anterior end. Zoites resulting from the 1st division of endodyogeny contain a posteriorly located crystalloid body, which is absent in zoites formed by subsequent divisions. Zoites contain posteriorly located amylopectin granules and a relatively large anterior vacuole which is not present in sporozoites. During penetration, the host cell plasmalemma ballooned laterally around the sporozoite creating a large cavity, which later disappeared. Sporozoites and zoites undergoing cell penetration usually exhibit partially empty anterior rhoptries; no changes occur in posterior rhoptries. Lysosomes fuse with the par-asitophorous vacuole surrounding killed sporozoites but not live sporozoites.     

Depth profiling in membranes by fluorescence quenching

Membrane penetration depth is an important parameter in relation to membrane structure and organization. A methodology has been developed to analyze the membrane penetration depths of fluorescent molecules or groups utilizing differential fluorescence quenching caused by membrane embedded spin-label probes located at different depths. The method involves determination of the parallax in the apparent location of fluorophores, detected when quenching by phospholipids spin-labelled at two different depths is compared. By use of relatively simple algebraic expressions, the method allows calculation of depth in å. This method has been used to determine the location of fluorophores in NBD-labelled lipids and anthroyloxy-labelled fatty acids in model membranes and of the membrane embedded tryptophan residues in the reconstituted nicotinic acetylcholine receptor.     

Deep soil heterogeneity and fine root distribution in forests and pastures of eastern Amazonia

Little is known about deep soil heterogeneity, or its relationship with fine root distribution. Beneath a mature, closed-canopy forest of eastern Amazonia, and the pastures and secondary forests that are derived from this forest, soil soft spots and hollow chambers occur to at least 9 meters depth. We measured the vertical distribution of these soil patches, and compared chemical characteristics, mycorrhizal infection, and root density of soil soft spots with the surrounding matrix of more homogeneous soil. Soil soft spots and chambers varied little with depth, but occupied the greatest soil volume (0.8 to 1.2%) from 4 to 6 m depth in the mature forest. Soft spots had lower pH, P availability and arbuscular mycorrhizal infection, and higher K availability than surrounding soil. Root length density was 2 to 15 times higher in soft spots than in surrounding soil. In the pastures, roots were found only in soil soft spots at depths of >3 m. Pastures and secondary forest had more soil chambers in the upper meter of soil than mature forest, but were otherwise indistinguishable in their patterns of deep soil heterogeneity. Soil soft spots may be vestiges of cutter ant nest chambers, while hollow chambers are cutter ant chambers and root channels. Chambers may act as conduits for root penetration and water penetration to deep soil.Abbreviations AM arbuscular mycorrhizae - RLD root length density (root length per unit of soil volume)