A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out‐of‐focus background when scanned in light‐sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth‐order Bessel beam and another type of propagation‐invariant beam, complementary to the zeroth‐order Bessel beam, which greatly reduces the out‐of‐focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light‐sheet to 1.07 μm by subtraction of the two scanned images across a whole field‐of‐view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP‐labeled mouse brain neurons.      

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

EPR imaging for in vivo analysis of the half-life of a nitroxide radical in the hippocampus and cerebral cortex of rats after epileptic seizures.

Recently, we developed an in vivo temporal electron paramagnetic resonance (EPR) imaging technique to be applied to the brain of a rat, into which a blood-brain barrier (BBB)-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally. This imaging technique made it possible to measure decay rates of a nitroxide radical in multiple regions of the brain simultaneously. Using this technique, the half-life of PCAM was estimated from the exponential decay of the signal intensity derived from the temporal EPR images in the hippocampus and cerebral cortex of rats after a kainic acid (KA)-induced seizure. The hippocampal half-life of PCAM after KA-induced seizures was significantly prolonged (p < .01), whereas the prolongation of the cortical half-life was not significant. These findings suggest that following a KA-seizure, the intrahippocampal ability to reduce the nitroxide radical is impaired, but the ability is intact in the cerebral cortex. This is the first in vivo quantitative EPR imaging study that has a bearing on the pathogenesis of KA-induced seizures in the brain.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. IV. Four New Species in Talpa europaea from England1

ABSTRACT. Moles from England were examined for coccidian oocysts and all 64 Talpa europaea were infected; of 64 infected hosts, 56 (88%) had multiple infections representing two to six coccidian species when examined. Oocysts in 31 of the 64 samples remained unsporulated. Three eimerians and one isosporan were studied from the 33 fecal samples that had sporulated oocysts and these are described as new species; Cyclospora talpae Pellérdy & Tanyi, 1968, and Isospora sofiae (Golemansky, 1978) Levine & Ivens, 1979, are redescribed; and Cyclospora sp., similar to C. talpae, is discussed. Sporulated oocysts of C. talpae are ellipsoidal, 14.3 × 9.6 (12–19 × 6–13) μm with sporocysts ovoid, 9.4 × 5.7 (6–13 × 4–8) μm; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Cyclospora sp. are subspheroidal to ellipsoidal, 12.5 × 8.9 (10–14 × 6–12) μm with sporocysts ovoid, 8.6 × 5.3 (6–10 × 4–6) μm; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Eimeria avonensis n. sp. are elongate-ellipsoidal, 15.0 × 9.6 (13–20 × 7–12) μm with sporocysts ovoid, 6.6 × 3.6 (5–9 × 3–7) μm; it was found in 15 of the 33 (45.5%) sporulated samples. Sporulated oocysts of Eimeria berea n. sp. are subspheroidal, 12.1 × 10.5 (10–15 × 8–14) μm with sporocysts ovoid, 6.3 × 3.9 (5–10 × 2–5) μm; it was found in 8 of the 33 (24.2%) sporulated samples. Sporulated oocysts of Eimeria globula n. sp. are spheroidal, 20.9 × 19.9 (19–24 × 17–21) μm with sporocysts elongate-ovoid, 11.5 × 6.9 (9–16 × 6–9) μm; it was found in 3 of the 33 (9.1%) sporulated samples. Sporulated oocysts of Isospora sporopointaea n. sp. are subellipsoidal to ellipsoidal, 17.1 × 11.4 (13–21 × 8–14) μm with sporocysts ellipsoidal with both ends pointed, 11.9 × 5.9 (9–16 × 4–8) μm; it was found in 27 of the 33 (81.8%) sporulated samples. Sporulated oocysts of I. sofiae are spheroidal to subspheroidal, 12.2 × 11.0 (9–16 × 8–15) μm with sporocysts ovoid, 9.1 × 5.2 (6–13 × 3–8) μm; it was found in 25 of the 33 (75.8%) sporulated samples. To date, the coccidian parasites of talpids include two cyclosporans, 12 eimerians, and six isosporans, exclusive of the four new species described here.     

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice

Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imaging quality within a volume, however, is degraded by involuntary movements and trades off between the imaged volume, imaging speed and quality. To balance such trade‐offs incurred by two‐photon excitation microscopy during intravital imaging, we developed a unique combination of interlaced scanning and a simple image restoration algorithm based on biological signal sparsity and a graph Laplacian matrix. This method increases the scanning speed by a factor of four for a field size of 212 μm × 106 μm × 130 μm, and significantly improves the quality of four‐dimensional dynamic volumetric data by preventing irregular artifacts due to the movement observed with conventional methods. Our data suggest this method is robust enough to be applied to multiple types of soft tissue.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. VI. Six New Species from the Eastern Mole,Scalopus aquaticus1

ABSTRACT Thirteen eastern moles, Scalopus aquaticus, collected in West Texas were examined for coccidian oocysts; 11 (85%) were infected and eight (73%) of these had multiple infections representing two or more species. One cyclosporan, three eimerians, and two isosporans were studied and all are described as new species. Sporulated oocysts of Cyclospora megacephali n. sp. were subspheroidal, 18.5 × 15.7 (14–21 × 12–18) μm; they had sporocysts pointed at one end with Stieda bodies nearly as wide as the sporocysts themselves, and were 15.0 × 7.2 (11–17 × 6–9) μm; C. megacephali was found in four (31%) hosts. Sporulated oocysts of Eimeria scalopi n. sp. were spheroidal to subspheroidal, 13.6 × 12.6 (11–17 × 11–15) μm with sporocysts lemon-shaped, 8.7 × 5.5 (7–10 × 4–7) μm; it was found in six (46%) hosts. Sporulated oocysts of Eimeria aquatici n. sp. were asymmetrically ellipsoidal, 17.0 × 10.6 (14–20 × 9–14) μm, with sporocysts elongately ovoidal, 9.0 × 5.2 (8–11 × 4–6) μm; it was found in two (15%) hosts. Sporulated oocysts of Eimeria motleiensis n. sp. were subspheroidal, 17.0 × 15.3 (15–20 × 13–18) μm with sporocysts ovoidal, 10.7 × 6.8 (10–13 × 6–8) μm; it was found in seven (54%) hosts. Sporulated oocysts of Isospora motleiensis n. sp. were spheroidal to subspheroidal, 13.6 × 12.0 (10–17 × 8–15) μm with sporocysts broadly ovoidal, 9.5 × 6.7 (7–11 × 4–8) μm; it was found in nine (69%) hosts. Sporulated oocysts of Isospora aquatici n. sp. were subspheroidal, 20.9 × 18.4 (15–24 × 13–21) μm with sporocysts ellipsoidal, 11.8 × 9.0 (9–14 × 7–11) μm; it was found in two (15%) hosts.     

Coccidia of Brazilian Mammals: Eimeria corticulata N. Sp. (Apicomplexa: Eimeriidae) from the Anteater Tamandua tetradactyla (Xenarthra: Myrmecophagidae) and Eimeria zygodontomyis N. Sp. from the Cane Mouse Zygodontomys lasiurus (Rodentia: Cricetidae)

Feces from a specimen of Tamandua tetradactyla (Linn.) from Portel, Para State, north Brazil, contained two different coccidial oocysts; one identified as Eimeria tamanduae Lainson 1968, and the other as a new species, described here as Eimeria corticulata n. sp. Oocysts of E. corticulata are ellipsoidal, 37.4 × 30.4 (31.2–43.7 × 23.7–35.0) μm, shape index (length/width) 1.2 (1.0–1.5). Oocyst wall 2.5–3.7 μm thick and composed of two layers; an outer thick, brown-yellow one with radial striations, and a thin inner smooth one: no visible micropyle. Oocyst residuum a large globule of about 10.7 × 10.3 μm, usually accompanied by a number of smaller attached globules. Sporocysts ellipsoidal, 21.0 × 11.0 (20.0–22.5 × 10.0–12.5) μm, with a conspicuous Stieda body: shape index 1.9 (1.6–2.2). Sporocyst residuum a small number of scattered granules: sporozoites 18.7 × 5.0 μm, with a large posterior refractile body. Eimeria zygodontomyis n. sp. is described in feces from Zygodontomys lasiurus (Lund) from the Serra dos Carajas, Para. Oocysts ellipsoidal to cylindrical, 16.5 × 12.0 (13.7–18.7 × 11.2–12.3) μm, shape index 1.4 (1.2–1.5). Wall colorless, smooth, single-layered and about 0.6 μm thick: no micropyle. No oocyst residuum, but a polar granule of about 1.8 × 1.0 μm is sometimes present. Sporocysts ellipsoidal, 8.4 × 5.5(7.5–8.7 × 5.0–6.2) μm, shape index 1.5 (1.4–1.7), with a thin colorless wall and a delicate Stieda body. Sporozoites enclose a compact residuum of about 2.5 × 3.7 μm.     

Visualizing astrocytes in the deep mouse brain in vivo

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3‐photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength‐dependent 3‐photon action cross section (ησ ₃) measurement of SR101. 3‐photon fluorescence imaging of the SR101‐labeled vasculature enabled an imaging depth of 1340‐μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal‐to‐background ratio limit encountered in 2‐photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2‐photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101‐labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.      

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. II. Six New Species from Japanese Shrew Moles (Talpidae)1

ABSTRACT. Thirty-eight of 51 (74.5%) shrew moles collected in Japan were infected with from one to four species of Eimeria and/or Isospora including six of six Dymecodon pilirostris and 32 of 45 (71.1%) Urotrichus talpoides. Four eimerians and two isosporans were identified and all are described as new species. Sporulated oocysts of Eimeria amorphospora n. sp. were subspheroid/ellipsoid, 21.1 × 17.9(18-25 × 16-21) μm. Sporocysts were amorphous, gelatinoid envelopes 20.3 × 7.5 (17–24 × 7–9) μm. Sporozoites were enclosed together within a membrane in each sporocyst. This species was found in 9 of 45 (20%) U. talpoides. Sporulated oocysts of Eimeria gonocilia n. sp. were subspheroid/ellipsoid, 28.8 × 24.4 (25–30 × 21–28) μm; a highly ornate outer oocyst wall gave the appearance of a ciliated ball. Sporocysts ovoid, pointed at both ends, were 17.0 × 9.0 (15–19 × 7–11) μm; this species was found in 4 of 45 (8.9%) U. talpoides. Sporulated oocysts of Eimeria talpoidei n. sp. were asymmetrical ovoid, 20.6 × 13.3 (18–23 × 12–15) μm, with sporocysts lacrimiform, 12.0 × 5.8 (10–14 × 5–7) μm. This species was found in 7 of 45 (15.6%) U. talpoides. Sporulated oocysts of Eimeria honshuensis n. sp. were ellipsoid, 15.5 × 11.4 (13–18 × 10–13) μm, with sporocysts ovoid, 9.1 × 5.2 (8–10 × 4–6) μm. This species was found in 10 of 45 (22.2%) U. talpoides and in 5 of 6 (83.3%) D. pilirostris. Sporulated oocysts of Isospora dymecodi n. sp. were subspheroid/ellipsoid, 15.8 × 12.6 (13–17 × 11–13) μm, with sporocysts ellipsoid, 10.9 × 6.9 (10–13 × 6–8). This species was found in six of six D. pilirostris. Sporulated oocysts of Isospora urotrichi n. sp. were spheroid/subspheroid, 13.4 × 12.4 (11–16 × 9–14) μm, with sporocysts ovoid, 9.2 × 6.3 (8–11 × 5–7) μm. This species was found in 27 of 45 (60%) U. talpoides. Only 14 of 38 (36.8%) infected hosts (one D. pilirostris, 13 U. talpoides) were seen to be naturally infected with only one coccidian species when sampled.     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.      

Four New Species of Isospora from the Small Tree Finch (Camarhynchus parvulus) from the Galapagos Islands1

ABSTRACT. Four isosporan species are described from the small tree finch, Camarhynchus parvulus from Isabela Island on the Galapagos Archipelago. Isospora exigua n. sp. oocysts subspheroidal, one-layered, smooth, yellow-brown color, 20.4 × 20.1 (20-23 × 18-23) μm, with no micropyle, residuum, or polar body. Sporocysts ovoidal, 14 × 9.5 (13-15 × 8-10) μm, with small Stieda and substieda bodies and irregular-shaped residuum. Isospora rotunda n. sp. oocysts subspheroidal, single-layered, smooth, yellow-brown wall with large polar body and no micropyle or residuum, 20.9 × 20.8 (20-24 × 19-23) μm. Sporocysts ovoidal, 15 × 9.7 (13-16 × 9-10) μm with knob-like Stieda bodies, prominent substieda bodies and round residuum. Isospora fragmenta n. sp. oocysts subspheroidal with no micropyle or residuum but with many splinter-like polar granules and a smooth, colorless, single-layered wall, 25.3 × 24.2 (24-27 × 23-25) μm. Sporocysts piriform 15.4 × 11.5 (14-17 × 11-12) μm with knob-like Stieda bodies, prominent substieda bodies, and irregular-shaped residuum. Isospora temeraria n. sp. oocysts ellipsoidal with one polar body, no micropyle or residuum, and wall of a single layer, smooth, yellow-brown color, 25.4 × 21.1 (21-30 × 17-23) μm. Sporocysts piriform, 15 × 10 (14-15 × 9-11) μm with knob-like Stieda bodies, prominent substieda bodies, and a round residuum. One woodpecker finch, Cactospiza pallida, was found to be infected with I. exigua, and a warbler finch, Certhidea olivacea was infected with I. fragmenta.     

A flagellate protozoan from Mythimna separata (Lepidoptera: Noctuidae)

Abstract

A trypanosomatid flagellate, Herpetomonas sp., is recorded from an adult Mythimna separata (Walker) (Lepidoptera: Noctuidae). Promastigote, paramastigote, and opisthomastigote stages measured 3.4–12 × 1.3–4.3 μm, Giemsastained, with flagella measuring 13.8–18.7 μm. A few amastigotes (3.9–8.3 × 3.7–6.7 μm) were also observed. Fourteen larvae fed with a suspension of flagellates were found later to be infected with the protozoan. No flagellate infections were found in the hymenopteran parasitoid, Apanteles ruficus (Haliday), or the hyperparasitoid, Trichomalopsis sp.     

Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Tannin

The effect of the oral administration of mimosa tannin (MMT) on the rat intra-hippocampal antioxidant ability was examined. Wistar rats at the age of 6 weeks were reared for 8 weeks with the rodent diet (RD) consisting of 0.1 g/kg of MMT (RD–MMT). The antioxidant ability of rat brain was evaluated from the decay of a brain–blood-barrier permeable stable nitroxide, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) measured by the microdialysis-electron spin resonance system under a freely moving state. The decay rate of PCAM in the brain of rats fed RD–MMT was significantly larger than that of rats fed control rodent diet, which indicates the increase of the antioxidant ability in the brain of rats fed RD–MMT. In vitro study showed that MMT did not reduce PCAM directly but enhanced the reduction of PCAM by ascorbic acid. These results indicate that MMT is a potent antioxidant in vitro and in vivo.     

First record of Fusarium vascular wilt on grapevine in Egypt

During the summer season of 2003 and 2004, wilt syndromes of grapevine leaves (Cv. crimson) and vascular discolouration of roots have been observed in 2-year-old grapevine plants in the field at two sides in Gharbeia Governorate, Egypt. First, symptoms of wilt began on bottom leaves borderline as chlorosis and then these turned to necrotic spots and the leaves died. Wilt symptoms were spread to apical associated with vascular discolouration of roots and stem basal. Routine isolations of discoloured root tissue from diseased plant yielded eight isolates of Fusarium oxysporum Schlechtend only where no other fungi were developed. Microscopic examination revealed the presence of three shapes of microconidia, first is avoid shape non-septate measuring 2.5–3.0 μm × 6–10 μm, second is cylindrical with one septa measuring 2.6 μm × 17.0 μm and third shape also cylindrical with two septate measuring 3.0 μm × 20.0 μm. Macroconidia was rarely with three septate measuring 3.5– 4.0 μm × 35.0–38.0 μm, and chlamydospores were found singly or in pairs or chains. F. oxysporum isolates attacked grapevine plants (Cv. crimson) causing vascular wilt (66.7%) and root-rot syndrome (33.3%). In vitro isolates of F. oxysporum causing wilt of grapevine (Cv. crimson) varied for producing lytic enzymes, i.e. polygalacturonase (PG) and cellulase. The reactions of several grapevines (Cvs.) with a virulent isolate of F. oxysporum indicated the presence of two different symptoms, i.e. vascular wilt only on grapevine plants (Cv. crimson) and root-rot on the other grapevine (Cvs.), i.e. superior, Thompson, King robi and flame seedless. All F. oxysporum isolates caused vascular wilt of grapevine Cv. crimson, successfully reisolated from symptomatic vascular infected tissue and complete identification on the basis of colony, conidia morphology and host range at formae speciales level as F. oxysporum f. sp. herbemontis (Tochetto) Gordan. This is the first report of Fusarium wilt on grapevine in Egypt.     

Two‐photon focal modulation microscopy for high‐resolution imaging in deep tissue

Two‐photon microscopy (2PM) is one of the most widely used tools for in vivo deep tissue imaging. However, the spatial resolution and penetration depth are still limited due to the strong scattering background. Here we demonstrate a two‐photon focal modulation microscopy. By utilizing the modulation and demodulation techniques, background rejection capability is enhanced, thus spatial resolution and imaging penetration depth are improved. Compared with 2PM, the transverse resolution is increased by 70%, while the axial resolution is increased to 2‐fold. Furthermore, when applied in conventional 2PM mode, it can achieve inertial‐free scanning in either transverse or axial direction with in principle unlimited scanning speed. Finally, we applied 2PFMM in thick scattering samples to further examine the imaging performance. The results show that the signal‐to‐background ratio of 2PFMM can be improved up to five times of 2PM at the depth of 500 μm. Fluorescent imaging in the mouse brain tissue. 3D Thy1‐GFP hippocampal neurons imaged by (A) 2PM compared with (B) 2PFMM; (C‐H) xy maximum‐intensity projection imaged by 2PM compared with 2PFMM. Scale bar 50 μm.      

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

First record of dill powdery mildew caused by Erysiphe heraclei DC in Egypt

In Egypt, powdery mildew was observed for the first time on dill plants, during annual disease surveys of March–May 2003 and 2005. Typical symptoms of powdery mildew of dill plant (Anethum graveolens L.) were observed in Gharbeia Governorate. Symptoms of powdery mildew became common on leaves, stems inflorescences and fruits as white irregular areas. These symptoms appeared at vegetative and early flowering stages then gradually increased through fruiting and pre-maturity stages. Samples of infected leaflets, stem, inflorescences and fruits were collected for examination by light and scanning electron microscope (SEM). Microscopic examination revealed that conidiophores were short, erect–69 × 6–10 μm in dimension, conidia were observed without conspicuous fibrosin bodies singly, elliposid to ovoid 25–33 × 10–16 μm in dimension, and the length to width ratio of conidia ranged from 1.7 to 2.0 and were produced singly. Cylindrical foot cells (22.0 × 8.0 μm) were followed by one or two shorter cells (12.5 × 7.5 μm). In spring, the sexual stage (cleistothecia) appeared on infected leaves and stems in spherical, gregarious measures 105–117 (111) × 100– 87.5 μm in diameter. Each cleistothecium contained (2–4) round to ovoid asci, 45–55 (50) × 45–25 (35) μm in dimension. The ascus contained (3–4) ellipsoid to ovoid ascospores, 20–17.5 × 15–10 (13.2) μm. Cleistothecia appendages are simple myceloid branched tips measuring 80–200 (140) μm in length and 3–5 (4) μm in diameter. Based on the observations of the morphology of its anamorph and teleomorph stages, the causal agent of dill powdery mildew was identified as Erysiphe heraclei which is reported for the first time in Egypt.     

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commerically available 2 or 5 μm particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C₁₈ reversed-phase column (condition 1: 100×4.6 mm I.D., particle size 2 μm, TSK gel Super-ODS; condition 2: 100×4.6 mm I.D., particle size 5 μm, Hypersil ODS-C₁₈). The mobile phase was composed of methanol-5 mM NaH₂PO₄ (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (conditions 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 μm was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.