Serial Sectioning of Insects with Hard Exoskeleton by Dissolution of the Exocuticle

The method reported here was designed to produce paraffin serial sections as thin as 5 Mm of insects or other arthropods with a hard cuticle. Heads and abdomens of Apis mellifera, Eristalomyia tenax and Tenebrio molitor were fixed with Schaffer's liquid, dehydrated with 80% ethanol, 90% ethanol, two changes of 100% isopropanol (2 hr each) and 12 hr in a 1:1 mixture of paraffin (58 C melting point) at 60 C. They were molded in paraffin after 12 hr of infiltration under a partial vacuum at 60 C. Large body openings of objects were sealed with paraffin to prevent infiltration of solvents.

Thereafter, the outer paraffin was removed manually and with xylene (15 min); the cuticle was rehydrated with 100% isopropanol and 100% ethanol (15 min each). The objects were then treated with Sputofluol (Merck; a mixture of NaOH and NaCIO) until they became white or their colorless endocuticle was stainable with aniline blue WS (C.I. 42755) after rinsing in a 50% acetic acid solution (v/v). They were then dehydrated with 100% ethanol and 100% isopropanol (15 min each) and subsequently re-embedded in paraffin. They were molded, sectioned, stained and mounted as usual.     

Applying Multiphoton Imaging to the Study of Membrane Dynamics in Living Cells

The endomembrane system of a cell is a highly dynamic, ephemeral structure that is difficult to visualize. Reconstructions from sections of fixed material can provide high-resolution information on intercellular membrane architecture, but such techniques are fraught with artifacts and are of little help in understanding the dynamics of intracellular membrane traffic. Recently, the availability of fluorescent membrane probes and the development of techniques for optically sectioning intact specimens have allowed glimpses of membrane dynamics to be visualized in living tissue. In this review we discuss the potential of a new optical sectioning technique, multiphoton imaging, for visualizing membrane dynamics in living cells. Multiphoton microscopy offers an unparalleled ability to obtain images from deep within specimens while minimizing the effects of phototoxicity.     

介绍一种半薄切片定位样品的方法

介绍了一种半薄切片定位样品的方法。此法与前人的方法不同之处在于位于载玻片之上被重新包埋于塑料环内的切片与载玻片的分离真人处步骤是“切片被包埋聚合好后,立即从６０￣６５℃温箱内被转入冰箱冷冻室中（－１８℃）放置５￣１０分钟。然后,将载玻征从冷冻室中取出,轻推塑料环,即可使包埋在塑料环内的切片与载玻片分离。这一方法成功地解决了样品中靶细胞的发育时期确定和样品丢失问题。而且还有简单、易操作和成功率高等优     

New perspectives for wood anatomical analysis in dendrosciences: The GSL1-microtome

The variability of wood anatomical features in the rings of trees and shrubs is known to be dependent on multifaceted environmental parameters. The ability to determine anatomical variations over longer time periods as decades or centuries is a step forward in dendroecological and dendroclimatological research. In this regard micro sectioning is still one of the basic requirements but sectioning devices (microtomes) designed for wood anatomical purposes are rarely available. We present an affordable heavy duty, but light-weight microtome operated with removable blades. This portable device enables the production of large sections for dendroclimatological reconstructions as well as various types of common sections for wood anatomical studies.     

Light Sheet Fluorescence Microscopy: A Review

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.     

‘Go with the flow ’: A review of methods and advancements in blood flow imaging

Physics has delivered extraordinary developments in almost every facet of modern life. From the humble thermometer and stethoscope to X‐Ray, CT, MRI, ultrasound, PET and radiotherapy, our health has been transformed by these advances yielding both morphological and functional metrics. Recently high resolution label‐free imaging of the microcirculation at clinically relevant depths has become available in the research domain. In this paper, we present a comprehensive review on current imaging techniques, state‐of‐the‐art advancements and applications, and general perspectives on the prospects for these modalities in the clinical realm. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)^{7, 5, 9}, developed and hosted in the authors'' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)^{1, 4, 8}, vascular networks (India ink)^{1, 4}, and cell body distribution (Nissl)³. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain⁶, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research¹⁰; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.     

Microsporogenesis of Cycas and its systematic implications

Taxonomists are divided over the infrageneric classification and species delimitation within the genus Cycas.The division is largely determined by whether a broad or narrow species concept is adopted,t...     

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall. Received: 17 May 1999 / Accepted: 16 September 1999     

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.