IN SITU PRESERVATION OF THE TRANSVERSE FLAGELLUM OF PERIDINIUM CINCTUM (DINOPHYCEAE) FOR SCANNING ELECTRON MICROSCOPY1

Three-dimensional structure of the transverse flagellum was studied by means of scanning electron microscopy (SEM) in Peridinium cinctum (O.F.M.) Ehrenberg. Several fixation and dehydration procedures were compared. Cells fixed, rapidly in 1% osmium tetroxide and critical-point dried showed accurate preservation of structural features seen in living cells; in particular, both transverse and posterior flagella were retained in the furrows on the cell surface. Osmium-fixed and freeze-dried, samples were prone to cellular collapse and loss of flagella. Glutar-aldehyde-fixed and critical-point dried cells showed a layer of material covering the thecal plates, most conspicuously over the girdle region; flagella were absent. Stereo-pair micrographs illustrate the 3-dimensional configuration of the transverse flagellum. Modifications of previous structural models are proposed.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

Osmium tetroxide-zinc iodide reactive sites in the photoreceptor cells of the retina of the rat

Summary The osmium tetroxide-zinc iodide fixative of Champy-Maillet has been used to study the rat's retina at the electron microscope level. Electron opaque deposits were observed all along the photoreceptor cells and concentrated in the outer segments of rods and cones and in the nerve endings. In the outer segments that deposits are located in the inter and intra disk spaces as well as between the disk and outer membranes. In the outer plexiform layer reactive sites include synaptic vesicles and mitochondria; other minor reactive sites are described in the inner segment and inner plexiform layer.Electron opaque deposits were not seen if potassium iodide substitutes zinc iodide in the fixative. However, if osmium tetroxide-potassium iodide fixed retinae are immersed in osmium tetroxide-zinc iodide the characteristic electron-dense material is evidenced at those same sites. The effect of other several fixatives were studied with a similar double fixation procedure. Our finding points to the histochemical demonstration of an unidentified component (s) of the retina which shows a striking specificity of localization and which is made evident when zinc iodide is used in the Champy-Maillet mixture.This work has been supported by grants of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.We are greatly indebted to Miss Haydée Agoff and to Mr. Alberto Saenz for their skillful technical assistance.     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

Something worth dyeing for: Molecular tools for the dissection of lipid metabolism in Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) has during the last decade emerged as an invaluable eukaryotic model organism to understand the metabolic and neuro-endocrine regulation of lipid accumulation. The fundamental pathways of food intake, digestion, metabolism, and signalling are evolutionary conserved between mammals and worms making C. elegans a genetically and metabolically extremely tractable model to decipher new regulatory mechanisms of lipid storage and to understand how nutritional and genetic perturbations can lead to obesity and other metabolic diseases. Besides providing an overview of the most important regulatory mechanisms of lipid accumulation in C. elegans, we also critically assess the current methodologies to monitor lipid storage and content as various methods differ in their applicability, consistency, and simplicity.     

Raman and infrared spectroscopic and theoretical studies of dinuclear rhenium and osmium complexes, M2(O2CCH3)4X2 (M = Re, Os; X = Cl, Br)

Infrared, far-infrared and Raman spectra of Re₂(O₂CCH₃)₄X₂ (X = Cl, Br) and Re₂(O₂CCD₃)₄Cl₂ have been recorded. Assignments of the vibrational spectra of Os₂(O₂CCH₃)₄Cl₂ and its deuterated derivative have been completed together with the Re complexes on the basis of normal-coordinate analysis. Force constant calculation was made for the acetate ion as well as for a four-atomic unit (with the CH₃ and CD₃ groups considered as point masses) using optimized masses of 16.7, 17.8, 20.5 and 21.6 for ¹²CH₃, ¹³CH₃, ¹²CD₃ and ¹³CD₃ groups, respectively. The force constants of the acetate ion have been adopted to the starting force field of the M₂(O₂CCH₃)₄X₂ type complexes. The metal-halide (0.889, 0.997 and 1.286 N cm⁻¹) and metal-metal stretching (3.32, 3.34 and 3.57 N cm⁻¹) force constants were obtained for Re₂(O₂CCH₃)₄Cl₂Re₂(O₂CCH₃)₄Br₂ and Os₂(O₂CCH₃)₄Cl₂ complexes, respectively. It was shown that the so-called diatomic approximation in most cases overestimates the M-M stretching force constants by 30-40%. Much better correlation has been obtained to fit these force constants, which produced values very close to those obtained by full normal-coordinate calculations. The Re-Re stretching force constants showed a reasonable correlation with the Re-Re bond distances for 18 rhenium complexes.     

Preparation of benzophenone imine complexes of transition metals

Benzophenone imine [M(η¹-NHCPh₂)(CO)_nP_5-n]BPh₄ [M = Mn, Re; n = 2, 3; P = P(OEt)₃, PPh(OEt)₂, PPh₂OEt, PPh₃] complexes were prepared by allowing triflate M(κ¹-OTf)(CO)_nP_5-n compounds to react with an excess of the imine. Hydride-imine [MH(η¹-NHCPh₂)P₄]BPh₄ (M = Ru, Os), triflate-imine [Os(κ¹-OTf)(η¹-NHCPh₂)P₄]BPh₄ and bis(imine) [Ru(η¹-NHCPh₂)₂P₄](BPh₄)₂ [P = P(OEt)₃] derivatives were also prepared. The complexes were characterized spectroscopically (IR, ¹H, ³¹P, ¹³C NMR) and a geometry in solution was also established. Hydride-benzophenone imine [IrHCl(η¹-NHCPh₂)L(PPh₃)₂]BPh₄and [IrHCl(η¹-NHCPh₂)L(AsPh₃)₂]BPh₄ [L = P(OEt)₃ and PPh(OEt)₂] complexes were prepared by reacting hydride IrHCl₂L(PPh₃)₂ and IrHCl₂L(AsPh₃)₂ precursors with an excess of imine. Dihydride IrH₂(η¹-NHCPh₂)(PPh₃)₃ complex was also obtained and a geometry in solution was proposed.     

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: Correlation of stain and phosphorus

Summary— Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.