Osmium Tetroxide-Potassium Ferrocyanide Intensification of a Diaminobenzidine Product Obtained by Photoconversion of a Fluorescent Label: a Study of Human Neutrophil Granules

A diaminobenzidine posttreatment employing osmium tetroxide and potassium fer-rocyanide was successfully used to intensify the diaminobenzidine stain formed by photoconversion of immunofluorescent labelling. Lactoferrin labelled granules became visible following the photoconversion process. Adequate diaminobenzidine staining was obtained after irradiating the cytospin preparations with ultraviolet light for 30-40 min. The diaminobenzidine stain had advantages over the fluorescent stain owing to its greater stability, greater density, and ability to be intensified. The enhancement procedure intensified both the color and density of the diaminobenzidine product. Consequently, shorter irradiation times could be used. Osmium tetrozide solutions of 3-4% increased the intensity with minimal background staining. Ultrastructural immunogold cytochemistry on ultrathin sections confirmed the existence of the lactoferrin labelled structures observed by light microscopy of cytospin preparations indicating that these were secondary grannies. The photomicroscopy procedures used in this study were simple to perform and could be applied to studies of other cellular antigens prior to using Immnnoultramicroscopy.     

Visualizing Endocytotic Pathways at Transmission Electron Microscopy via Diaminobenzidine Photo-Oxidation by a Fluorescent Cell-Membrane Dye

The endocytotic pathway involves a complex, dynamic and interacting system of intracellular compartments. PKH26 is a fluorescent dye specific for long-lasting cell membrane labelling which has been successfully used for investigating cell internalization processes, at either flow cytometry or fluorescence microscopy. In the present work, diaminobenzidine photo-oxidation was tested as a procedure to detect PKH26 dye at transmission electron microscopy. Our results demonstrated that DAB photo-oxidation is a suitable technique to specifically visualise this fluorescent dye at the ultrastructural level: the distribution of the granular dark reaction product perfectly matches the pattern of the fluorescence staining, and the electron density of the fine precipitates makes the signal evident and precisely detectable on the different subcellular compartments involved in the plasma membrane internalization routes.Key words: Endocytosis, PKH26 dye, diaminobenzidine photo-oxidation, transmission electron microscopy     

Recent advances in freeze-fracture electron microscop: the replica immunolabeling technique

Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique samples are frozen fractured and replicated with platinum-carbon as in standard freeze fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.     

Immunocytolocalization of Glutamine Synthetase in Green Leaves and Cotyledons of Lycopersicon esculentum

Glutamine synthetase was localized in leaves and cotyledons of young tomato (Lycopersicon esculentum Mill.) plants using immunogold techniques coupled to transmission electron microscopy. The enzyme occurs only in chloroplasts and is most probably a stroma constituent.     

Detection of endogenous and immuno-bound peroxidase — The status Quo in histochemistry

The discovery of synthetic dyes goes back to 1856 and launched the development of the whole chemical and pharmaceutical industry. In life sciences synthetic dyes represent indispensable tools for the microscopic and macroscopic level. Small dyes have the advantage of their easy adaptability to various measuring equipments. By way of structural modification of the chromophore portion, dye labels can be tailored that they absorb and emit light at desired wavelengths ranging from the UV to the near infrared region of the spectrum. Assisted by the development of light measuring techniques and the commercial availability of highly sensitive equipment, today luminescent labels represent most sensitive detection tools in life sciences and dominate over chromogen based techniques. However, for detection of active sites of peroxidase (PO) so far fluorescent labels have been confined to only a few substrates while a broad variety of well-established chromogenic techniques exist. This review covers fluorescent and chromogenic approaches for the permanent detection of immuno-bound and endogenous PO-activity in fixed cells and tissues. Thereby the tailoring of suitable dye labels is additionally challenged by two demands: (1) The applied dye (or its precursor) must act as enzyme substrate specifically and (2) the enzymatic impact must furnish an insoluble dye product from easy soluble starting materials in a very quick reaction. Hence it is not surprising that among PO-substrates (and enzyme substrates generally), dye conjugates represent only an exception while most of these labels represent reactive dyes or suitable precursors. Chromogenic and fluorescent approaches for the permanent labeling of enzymatic sites are compiled. Furthermore, various area-spanning PO-detection principles are discussed ranging from transmission light (TLM) and fluorescence light (FLM) microscopy (chromogenes, flourochromes, fluorescent chromogenes, chromogenes with nonlinear optical properties) to correlated transmission electron microscopy (TEM; photoconversion of specific chromogenic reaction products, electron opaque and/or osmiophilic chromogenic substrates). Also, approaches for reflectance laser microscopy (RLM), polarization microscopy (PM), and correlative TLM, FLM, and multiphoton fluorescence microscopy (MFM) are discussed.     

Ultrafast Photoconversion of the Green Fluorescent Protein Studied by Accumulative Femtosecond Spectroscopy

The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to ΔOD = 10⁻⁷ absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.     

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.     

Photoconversion of some fluorescent markers to a diaminobenzidine product

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.     

Cytopathology of Lettuce Mosaic Virus-infected Lettuce Seeds and Seedlings

The ultrastructure of lettuce mosaic potyvirus (LMV)-infected lettuce seeds and seedlings was studied by transmission electron microscopy. Conventional thin-section electron microscopy and immunogold cytochemistry were both successfully employed to study the location of LMV in embryonic and non-embryonic seed parts. LMV particle aggregates and cytoplasmic “pinwheel” inclusions characteristic of potyviruses were observed throughout the embryonic tissues (radicle, hypocotyl and cotyledon) of infected lettuce seeds and seedlings, and also in the non-embryonic endosperm layer. LMV particles, but not inclusions, were also located in the non-embryonic pericarp layer.     

On the origin and development of vacuoles in promeristematic cells ofCystoseira stricta sauvageau (Phaeophyta,Fucales)

Summary Promeristematic cells of theCystoseira thallus have been studied by transmission electron microscopy and cytochemistry in order to observe vacuole formation and development.Vacuolation seems to occur by different processes. Those here concerned involve the intervention of provacuolar formations, originating probably from G.E.R.L., in the sequestration of cytoplasmic materials. The sequestrated portion then undergoes a degradation. These observations agree with the processes recently reported in root meristematic cells of two higher plants:Euphorbia characias andHordeum sativum.     

Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 μA/cm² for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.     

Sub-cellular localization of PELPK1 in Arabidopsis thaliana as determined by translational fusion with green fluorescent protein reporter

PELPK1, a novel Arabidopsis thaliana gene was earlier annotated to encode a protein of sub-family, PELPK under hydroxyproline-rich glycoprotein (HRGP) super-family of proteins. Previous bioinformatics and computational analyses predicted PELPK1 to contain an amino-terminal signal peptide destined towards the secretory pathway. In the present study, transgenic plants were developed harboring a translational fusion construct comprising of PELPK1 coding sequence (PELPK1-CDS) and green fluorescent protein (GFP) reporter to determine the localization of PELPK1 in Arabidopsis plants. By employing the techniques of confocal laser scanning microscopy, immunolabeling of GFP with quantum dot (Q-dot), and transmission electron microscopy (TEM), it is shown that the translational fusion product is predominantly deposited to the cell wall. These results are in agreement with the earlier bioinformatics prediction that the PELPK1 is transported via the secretory pathway.     

Cytochemical Localization of Urease in a Rumen Staphylococcus sp. by Electron Microscopy

We describe a technique whereby intracellular urease activity can be localized by transmission electron microscopy. The ammonia produced from the enzymatic hydrolysis of urea is first precipitated with sodium tetraphenylboron and then replaced with silver to produce electron-dense silver tetraphenylboron. This direct reaction product deposition procedure was used to demonstrate the presence of membrane-bound urease of Staphylococcus sp. H3-22, a gram-positive ruminal bacterium.     

Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.     

Luminescent gelatin nanospheres by encapsulating CdSe quantum dots

Quantum dots (QDs) have been encapsulated within gelatin nanoparticles (GNPs), which gives GNPs fluorescent properties and improves the biocompatibility of QDs. Hydrophilic CdSe QDs were produced through thermodecomposition following the ligand‐exchange method, and were then encapsulated in GNPs. The results of high‐resolution transmission electron microscopy and transmission electron microscopy show that CdSe QDs and QDs‐encapsulated GNPs (QDs‐GNPs) have average diameters of 5 ± 1 and 150 ± 10 nm, respectively. Results of both high‐resolution transmission electron microscopy and confocal laser scanning microscopy indicate that CdSe QDs are successfully encapsulated within GNPs. The QDs‐GNPs have distinctive fluorescent properties with maximum emission at 654 nm, with a 24 nm red‐shift comapred with hydrophilic mercaptoundecanoic acid (MUA)‐modified QDs. In addition, an in vitro cytotoxicity test shows that QDs‐GNPs do not have any toxic effect on cells. It is expected that QDs‐GNPs might be an excellent candidate as a contrast agent in bio‐imaging. Copyright © 2013 John Wiley & Sons, Ltd.     

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.     

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.     

Synthesis and characterization of a new fluorescent phospholipid

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2′-thio-1′-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and β-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.     

Use of light microscopic immunotechniques in selecting preparation conditions and immunoprobes for ultrastructural immunolabelling of lactoferrin

Summary Successful postembedding immunolabelling for electron microscopy is sometimes difficult to achieve. We propose that light microscopy can be used (1) to detect quickly processing steps which have an adverse effect on the tissue antigenicity and (2) to check the specific reactivity of the immunogold detecting system normally employed at the ultrastructural level.The individual steps of fixation, dehydration and embedding were tested for their ability to preserve antigenicity by light microscopic peroxidase-anti-peroxidase cytochemistry. Steps that severely reduced antigenicity were replaced by less destructive alternatives compatible with reasonable ultrastructural preservation. The specific reactivity of the immunogold detecting system was assessed by using the light microscopic immunogold-silver staining method.We studied the antigen lactoferrin in human neutrophilic granulocytes from patients with chronic myeloid leukaemia. We obtained strong immunolabelling of specific granules and good ultrastructural preservation using routine methods at room temperature. For lactoferrin the method of choice was to fix in 3% paraformaldehyde/0.1% glutaraldehyde followed by 1% OsO₄, dehydrate in 70% ethanol, embed in LR White resin and polymerize at 40°C for 40h. These conditions may not be suitable for all antigens and we emphasize that for each new antigen a similar study should be carried out.