A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

Preparació de puntes de tungstè per a microscòpia d'efecte túnel (STM)

Es presenten els resultats experimentals obtinguts durant l’estudi sistemàtic realitzat de la preparació electroquímica de puntes de tungstè per al Microscopi d’Efecte Túnel (STM), fent servir dos electròlits: KOH i NaOH. L’estudi sobre la morfologia, longitud de la punta i radi de curvatura de la punta en funció del voltatge aplicat i les concentracions de l’electròlit es descriu al capítol 3. La caracterització de les puntes es va dur a terme, per una part, mitjançant un microscòpic electrònic de rastreig (SEM) i per l’altre banda, amb el ús de les puntes obtingudes al STM. En resumen, els resultats mostren que ambdós electròlits permeten obtenir puntes que es poden fer servir amb èxit per l’obtenció d’imatges amb l’STM. Les millors puntes són aquelles que s’obtenen dins de rangs de concentracions d’electròlit baixes, entre valor de 10 a 15% en pes pel NaOH i entre 10 i 20% pel KOH i rangs de voltatge entre 3 a 7 V pel NaOH i 4 a 8 V pel KOH. S’observa que es requereixen temps d’atac electroquímic menors fent servir com a electròlit NaOH. S’estudia, en el capítol 4, el tractament que requereix la punta per tal d’eliminar les impureses de la seva superfície. Es realitzen diferents proves amb tres mètodes de neteja: (1) tractament químic, (2) bombardeig iònic i (3) tractament tèrmic de recuit. En el capítol 5 del projecte s’analitzen les imatges d’una mostra d’or, Au(110), d’estructura coneguda, amb el microscopi d’efecte túnel STM) del laboratori fent servir les puntes obtingudes sota les condicions considerades òptimes. El resultat confirma el bon comportament de les puntes obtingudes sota les condicions descrites en els capítols anteriors i establert una pauta a seguir per obtenir puntes d’una manera senzilla i reproduïble.

Scanning Electron Microscopy of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae)

Lagochilascaris minor Leiper, 1909 is a parasitic nematode with its biological cycle still unknown, even though it was found in humans, domestic and silvatic animals. Adult worms, collected by surgical drainage from a human patient from the State of Pará, Brazil, were micrographed using a scanning electron microscope. Morphological aspects of males and females such as cephalic structures, caudal papillae and cuticular patterns were analyzed and compared with the previous descriptions adding new data for the identification of this species.

Description of the Oocysts of Three New Species of Eimeria (Apicomplexa: Eimeriidae) from Iguanid Lizards (Sauria: Iguanidae) of Central and South America

Three new species of Eimeria are described from iguanid lizards of Central and South America. The oocysts of each species have no micropyles or residua and the sporocysts lack Stieda bodies, but all have a sporocyst residuum. Eimeria sanctaluciae n.sp. was found in the St. Lucia tree lizard, Anolis luciae, collected from the Maria Islands, Lesser Antilles. The oocysts are spherical to subspherical, averaging 17.3 x 16.5 µm, with a single layered colourless wall; about 60% contain polar granules. The sporocysts are ellipsoidal and average 7.7 x 5.5 µm. Eimeria liolaemi n.sp. was recovered from the blue-gold swift, Liolaemus taenius, from Chile. The oocysts are spherical to subspherical, measuring 21 x 20.1 µm with a single-layered colourless wall. The sporocysts are subspherical and average 7.4 x 6.8 µm. Eimeria caesicia n.sp. is described from the Brazilian collared iguanid, Tropidurus torquatus. The oocysts measure 27.4 x 23.7 µm, are spherical to subspherical, with a bilayered wall, the outer surface of which appears pale blue in colour, the thin, inner wall appearing brown, when viewed by direct light under the optical microscope. The sporocysts are subspherical and average 9.4 x 7.2 µm. Unnamed polysporocystid oocysts with dizoic sporocysts are reported from the faeces of the lesser St. Vincent tree lizard, Anolis trinitatis and the possibility of spurious parasitism briefly discussed. In addition, oocysts of an unnamed Isospora sp. with a smooth oocyst wall which closely resembles I. reui were recovered from A. trinitatis.

Morphological study of adult male worms of Schistosoma mansoni Sambon, 1907 by confocal laser scanning microscopy

Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss). The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.

Scolex Morphology of Proteocephalid Cestodes Parasites of Neotropical Freshwater Fishes

The morphology of the scolices and metascolices of 29 proteocephalid species, parasites of freshwater fishes from Brazil was compared by using scanning electron microscope, light and laser microscopy. The following species were evaluated: Proteocephalus vazzolerae, P. piramutab, Robertiella paranaensis, Travassiella avitellina, Monticellia loyolai, M. spinulifera, M. belavistensis, Houssayela sudobim, Zygobothrium megacephalum, Gibsoniela mandube, Cangatiella arandasi, Nomimoscolex sudobim, N. lopesi, N. admonticellia, N. piraeeba, N. pirarara, Harriscolex kaparari, Crepidobothrium eirasi, Spatulifer rugosa, Megathylacus brooksi, Choanoscolex abscisus, Amphoteromorphus peniculus, A. piraeeba, Paramonticellia itaipuensis, Peltidocotyle rugosa, Othinoscolex lenha, Rudolphiella rugata, R. piranabu, Jauella glandicephalus. Some features as overall shape of scolex, suckers, apical sucker, frontal glands and several kinds of metascolex were analyzed. The taxonomic importance of scolex and metascolex is discussed.

Reducing the learning curve for the treatment of morphoeic (sclerosing) basal cell carcinoma of the face.

The treatment of morphoeic (or sclerosing) basal cell carcinoma (mBCC) of the face is associated with high rates of incomplete excision and recurrence. A principal risk factor for incomplete resection is the grade of surgeon. We did a prospective, randomised study of 40 consecutive patients with mBCC of the face. The extent of the tumour was assessed under standard conditions by consultant surgeons and compared with assessments by resident surgeons with the help of the Varioscope, a combination of microscope and loupe glasses with strong illumination and a maximal magnification of 7x. The data from a former retrospective study of all excisions of mBCC of the face during a five-year period at the hospital served as control. Residents with the support of the Varioscope achieved a rate of incomplete excisions similar to that of consultants under standard conditions. There was a significant reduction of the rate of incomplete resections by resident surgeons thanks to high magnification and good lighting (p=0.02). High magnification and good lighting were useful in learning how to recognise skin changes associated with mBCC of the face and achieving a low rate of incomplete excisions.

Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Direct methods for detecting picorna-like virus from dead and alive Triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Purely Endoscopic Keyhole Supraorbital Approaches for Anterior and Middle Skull Base Tumors

Anterior and middle skull base tumors, mainly meningiomas, are usually operated on using a sub-frontal route with a microscope. With modern radiotherapy, the goal of skull base surgery moves from a radical surgery with high rate of side effect to a functional concept that aims to remove as much as possible of the tumor without compromising the neurological status of patients. Minimally skull base surgery benefits from keyhole and endoscopy techniques. For 3 2 decades, the development of endoscopy helps to imagine innovative approaches for skull base tumors such as the endonasal route. Nonetheless, CSF leak issue and the absence of direct control of the tumor margins may limit the interest of such a route. Keyhole craniotomies have been developed with microscope but vision issue limits their use. Combining advantages of both techniques appears therefore natural and gave birth to intracranial assisted and more recently to fully endoscopic keyhole surgery. For anterior or middle skull base tumors, Keyhole supraorbital approaches can be done either by a trans-eyebrow or trans-eyelid routes. A step-by-step description of these fully endoscopic alternative routes summarizing advantages and drawbacks compared to others (traditional sub-frontal or more recent endonasal approaches) is reported in this chapter by the authors.

Some Morphological Aspects of Cylicocyclus brevicapsulatus (Nematoda: Cyathostominae) Parasite of Horse

This paper describes some morphological aspects of Cylicocyclus brevicapsulatus (Ihle, 1920) (Nematoda: Cyathostominae) from Equus caballus in Brazil. The worms were studied using an optical microscope (measurements and illustrations) and a scanning electron microscope for a more detailed examination of the external morphology. The buccal capsule is very short, with a very thin wall, and the dorsal gutter is absent. Other morphological aspects are described including measurement of the spicules and gubernaculum.

Ultrastructural alterations in adult Schistosoma mansoni caused by artemether

Progress has been made over the last decade with the development and clinical use of artemether as an agent against major human schistosome parasites. The tegument has been identified as a key target of artemether, implying detailed studies on ultrastructural damage induced by this compound. We performed a temporal examination, employing a transmission electron microscope to assess the pattern and extent of ultrastructural alterations in adult Schistosoma mansoni harboured in mice treated with a single dose of 400 mg/kg artemether. Eight hours post-treatment, damage to the tegument and subtegumental structures was seen. Tegumental alterations reached a peak 3 days after treatment and were characterized by swelling, fusion of distal cytoplasma, focal lysis of the tegumental matrix and vacuolisation. Tubercles and sensory organelles frequently degenerated or collapsed. Typical features of subtegumental alterations, including muscle fibres, syncytium and parenchyma tissues, were focal or extensive lysis, vacuolisation and degeneration of mitochondria. Severe alterations were also observed in gut epithelial cells and vitelline cells of female worms. Our findings of artemether-induced ultrastructural alterations in adult S. mansoni confirm previous results obtained with juvenile S. mansoni and S. japonicum of different ages.

X-ray mosaic nanotomography of large microorganisms.

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.

Advantages of digital holographic microscopy for real-time full field absolute phase imaging

Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast, to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA microscope objectives is numerically extended. For example, two different biological cells, floating at different depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full- field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase images of biological cells have been demonstrated. ©2008 COPYRIGHT SPIE

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.