Light and electron histochemistry of phosphoamidase with P-chloranilidophosphonic acid and with cyclophosphamide (Endoxan)

Summary The original metal-salt technique of Gomori (1948a) employing p-chloranilidophosphonic acid as a substrate for the demonstration of the activity of phosphoamidase has been used with varying success by a number of investigators for light microscopy, Cyclophosphamide (endoxan) which is a cytotoxic drug known to activate phosphoamidase and other lysosomal enzymes in neoplasm (Grillo, 1971) is proposed as another substrate for the enzyme for both light and electron microscopy.     

Characterization and cAMP inhibition of a lysyl-(N-epsilon-5'-phospho) adenosyl phosphoamidase in Dictyostelium discoideum

A lysyl-(N-epsilon-5'-phospho) adenosyl phosphoamidase activity has been identified in Dictyostelium discoideum. Conjugates, formed by coupling AMP via a phosphoamide bond to the epsilon amino group of lysine in avidin and tuftsin, served as substrate. Lysyl-N-epsilon-5'-phosphoadenosine and adenosine phosphoramidate (AMPNH) were substrates as well. The phosphoamidase liberated AMP from all four compounds but did not degrade cAMP. Approximately 90% of the phosphoamidase activity was inhibited competitively by 100 microM cAMP with an apparent Ki of 35 microM for all substrates.     

Isolation and characterization of the protein phosphoamidates formed by a membrane bound adenylyl transferase reaction in Dictyostelium discoideum.

1. In this paper we report the results of studies on an adenylyl transferase (AyTase) activity from Dictyostelium discoideum. 2. Previous studies suggested that this activity catalyzed the transfer of AMP from ATP to a membrane protein to form a phosphoamidate reaction product. 3. In the present study we have isolated and characterized the product of the AyTase reaction and surprisingly found two AMP labeled products by SDS-gel-filtration HPLC. 4. The apparent molecular weights of these phosphoamidates were 13.5 and 1.5 kDa. 5. In addition, because the reaction product was rapidly degraded by a phosphoamidase, experiments were undertaken using AVAMP, a synthetic phosphoamidate, as an alternative phosphoamidase substrate, to trap the reaction products. 6. As the phosphoamidase activity could be inhibited by cAMP, cyclic formycin monophosphate, an analog of cAMP resistant to hydrolysis by cAMP phosphodiesterase, was also used to trap the products. 7. Both attempts at trapping failed. 8. A model for the AyTase reaction was developed to account for the failure to trap the products and the formation of two phosphoamidates.     

Study of single-molecule dynamics and reactions with classic light microscopy.

Single-molecule studies in the life sciences often deal with observation or spectroscopy. Studies of reactions are rare, and the light microscope has been used for such experiments only occasionally. In an experimental environment, for example, as is required for most nearfield scanning or electron microscopies, it is difficult to study single-molecule reactions of biological relevance. Therefore, we have developed techniques to study single-molecule reactions with classic (nonscanning) farfield light microscopy. The conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH (a reduced form of NAD+), pyruvate, and H+ catalyzed by a few LDH-1 enzyme molecules has been studied in substrate solutions with different viscosity using the NADH autofluorescence. It is even possible to monitor the progress of the reaction by phase-contrast microscopy via scattering or absorption by product molecules. As an example for a single-molecule reaction with a macromolecule as substrate, the handling and enzymatic cutting of fluorescently stained lambda-DNA is studied. In solutions containing 10 mM magnesium and 66 mM potassium ions at pH 7.9, an individual DNA molecule tends to collapse into a globular structure. When moved through an aqueous solution, it becomes stretched by viscosity drag. After stopping the motion, the molecule collapses and the dynamics of this process can be quantified. When a restriction enzyme is present, sequence-specific cutting can be directly observed in the light microscope. The theoretical restriction pattern, as predicted from the sequence of the molecule, can be generated directly under visual inspection.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Fine structure and distribution of epidermal projections associated with taste buds on the oral papillae in some loricariid catfishes (Siluroidei: Loricariidae)

Two morphologically distinct structures occur on the surfaces of the oral papillae in several loricariid catfish species; namely, (1) typical vertebrate taste buds composed of receptor and sustentacular cells and (2) brushlike projections, termed epidermal brushes, that represent specialized epidermal cells containing keratin. Both of these structures were studied with the combined use of light microscopy and scanning and transmission electron microscopy. The general body surface, fins, and rostral cutaneous processes of some loricariid catfishes are covered with taste or terminal buds but lack the epidermal brushes. It is suggested that the epidermal brushes found on the oral papillae serve as protective devices for the taste buds and as abrasive surfaces for substrate scraping during feeding. The taste buds on the oral papillae may detect any gustatory stimuli from the resulting substrate disturbance. Comparative studies reveal many differences in the number and spatial arrangement of these two structures on the oral papillae among the several species of the Loricariidae examined. These differences may represent functional adaptations to the various modes of life in the Loricariidae.     

Fluorescence-based staining for tartrate-resistant acidic phosphatase (TRAP) in osteoclasts combined with other fluorescent dyes and protocols.

Osteoclasts are the only bone-resorbing cells. In addition to other specific properties, osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), which is usually detected using a histochemical method for light microscopy. Using ELF97 phosphatase substrate, this study describes a new fluorescence-based method for TRAP detection. The fluorescence-based ELF97 TRAP stain not only results in a better resolution of the TRAP-positive granules, because confocal microscopy can be applied for image acquisition and analysis, but it reveals additional and more specific information about osteoclasts because it can be combined with other fluorescence-based methods.     

Tarsal movements in flies during leg attachment and detachment on a smooth substrate

In order to understand the attachment mechanism of flies, it is important to clarify the question of how the adhesive pad (pulvillus) builds and breaks the contact with the substrate. By using normal and high-speed video recordings, the present study revealed that pulvilli are positioned on the surface in a particular way. The pulvilli are apparently loaded or pressed upon the substrate after leg contact, as evidenced by splaying of the claws. Detachment of pulvilli from the substrate may be achieved in four different modes depending on the leg (fore-, mid- or hindleg): pulling, shifting, twisting, and lifting. Lifting is the only detachment mode depending on the claws' action. Kinematics of the tarsal chain is studied in leg preparations, in which the tendon of the claw flexor muscle was pulled by tweezers and video recorded. The morphological background of tarsal movements during attachment and detachment is studied by scanning electron microscopy, fluorescent microscopy, and bright field light microscopy followed by serial semithin sectioning of pretarsal structures. Several resilin-bearing springs are involved in the recoil of the tarsal segments to their initial position, when the tendon is released after pull.     

Slicing embryos gently with laser light sheets

Light sheet microscopy is an easy to implement and extremely powerful alternative to established fluorescence imaging techniques such as laser scanning confocal, multi-photon and spinning disk microscopy. By illuminating the sample only with a thin slice of light, photo-bleaching is reduced to a minimum, making light sheet microscopy ideal for non-destructive imaging of fragile samples over extended periods of time. Millimeter-sized samples can be imaged rapidly with high resolution and high depth penetration. A large variety of instruments have been developed and optimized for a number of different samples: Bessel beams form thin light sheets for single cells, and selective plane illumination microscopy (SPIM) offers multi-view acquisition to image entire embryos with isotropic resolution. This review explains how light sheet microscopy involves a conceptually new microscope design and how it changes modern imaging in biology.     

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.     

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10⁻⁵ M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm²). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24–72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.     

Morphology of fibroblastic cells cultured on poly(HEMA-co-AA) substrates

Fibroblastic cells in culture are characteristically elongated and grow in monolayers. This growth pattern can be modified by different factors, such as substrate interaction. It is characteristic of hydrogels made of poly(2-hydroxyethylmethacrylate) (polyHEMA) that they inhibit cellular attachment and spreading. Vero cells were cultured on porous samples of polyHEMA and the copolymer poly(HEMA-co-AA) with 7.5% (w/w) and 15% (w/w) acrylic acid. Cultures were maintained for 2 and 10 days in HAM F10 medium with 10% foetal calf serum. Hydrogel samples were processed for light microscopy and scanning electron microscopy. The round Vero cells proliferated on the hydrogels and were principally located inside the pores. Some cells were aggregated, but no extracellular matrix was found. The copolymer with 15% (w/w) acrylic acid was the most suitable substrate and should be used in future tests of morphological differentiation and induction of cellular function.     

IMMUNOGOLD LABELLING OF FIBROBLAST FOCAL ADHESION SITES VISUALISED IN FIXED MATERIAL USING SCANNING ELECTRON MICROSCOPY, AND LIVING, USING INTERNAL REFLECTION MICROSCOPY

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.     

Effects of sandy substrate and light on hypermelanosis of the blind side in cultured Japanese flounder Paralichthys olivaceus

Rearing experiments were carried out to clarify the effects of sandy substrate and light irradiation on hypermelanosis of the blind side (the staining type of ambicoloration) in cultured Japanese flounder. Fish were reared in three experimental conditions: (1) no sandy substrate into which fish could bury themselves and with upward light irradiating their blind sides, (2) no sandy substrate and no upward light, and (3) sandy substrate (transparent glass sand) with upward light irradiation. Pigmented areas on the blind side were measured by an image analyzing system. Flounder from the third condition (sandy substrate with light irradiation) showed the lowest pigmentation on the blind side. In contrast, fish from the second condition (no sandy substrate and no light irradiation), showed the highest pigmentation coverage. These results indicate that sandy substrate on the bottom in culture tanks is more important than light irradiation as a factor affecting hypermelanosis of the blind side in cultured Japanese flounder.     

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.     

Rapid induction of apoptosis in human keratinocytes with the photosensitizer QLT0074 via a direct mitochondrial action

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.     

Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

Chrysophyte stomatocyst assemblages associated with periphytic, high arctic pond environments

Chrysophycean stomatocysts associated with three different periphytic substrates (wet mosses, submerged mosses and rock scrapes) were investigated from ponds on Cape Herschel (78°37'N, 74°42'W), Ellesmere Island in the Canadian High Arctic. The goal of this study was to determine whether a distinct assemblage of periphytic chrysophyte cysts existed and, if so, whether assemblage composition varied with substrate and between ponds. One hundred and thirty-seven different cyst morphotypes were observed with light microscopy from 68 periphytic samples taken from 35 ponds. Twenty-six of these cysts were new morphotypes, of which 16 were identified and described using scanning electron microscopy. Significantly more cyst types with collars and hooked projections in the collar region (i.e. 'hooked'), and fewer unomamented morphotypes were recorded in the periphytic habitats as compared to the surface sediments. Wet moss stomatocyst assemblages were particularly distinct, with a high number of heavily silicified and hooked morphotypes. The morphotype richness was far greater in periphytic environments, with 86, 100 and 95 morphotypes observed in the wet mosses, submerged mosses and rock scrapes, respectively, as compared to only 35 types in the surface sediments of the ponds. Canonical correspondence analysis indicated that measured water chemistry did not account for the variation in the species data (p_{axic 1} < 0.01, 999 Monte Carlo permutations). This study suggests that distinct periphytic assemblages exist, and that cyst morphotype composition and richness varies with substrate.