Resolving surface details with reflection and fluorescence video-confocal profilometry.

Video-confocal profilometry has been exploited to characterize reflecting and non-reflecting surfaces in materials with tilted and corrugated areas. An alternative method based on fluorescence detection has been developed and tested to characterize metal surfaces modified by intense laser irradiation. Combined representations of surface topography have been obtained on the basis of both reflectance and fluorescence signals. A discussion of results and problems encountered in reflection and fluorescence based techniques is provided.

Towards many colors in FISH on 3D-preserved interphase nuclei.

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy.

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins.

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

A heterotrimeric G protein of the Gi family is required for cAMP-triggered trafficking of aquaporin 2 in kidney epithelial cells.

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells.

Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the signal transduction pathways of this process.

From the histophotometer to the confocal microscope: the evolution of analytical microscopy.

With new ideas and with the aid of modern technology, novel microscopical and micro-analytical methods are practically implemented, as a fruit of the co-operative effect of theoretical and experimental advances achieved in distinct fields. The results obtained in the development of new micro-analytical instruments and in their applications, increasingly help to solve many investigative problems in materials science and in biology. Among the oldest forms of analytical microscopy, some encountered histophotometers while, among the latest, some are working with confocal microscopes. Given by one that had the opportunity of attending to both of the developments, an overview encompassing these two instruments can hopefully serve to illustrate the progress of analytical microscopy, a discipline evolving during the second half of this century.

Different neurologic outcomes in two patients with neonatal hyperinsulinemic hypoglycemia.

The authors report the cases of two patients with neonatal onset of hypoketotic hypoglycemia with hyperinsulinism and a poor response to diazoxide. Pancreatic venous sampling showed a diffuse pancreatic hyperplasia in one patient and a focal lesion in the other. The second patient, diagnosed after a significant delay, suffered severe long-term neurological sequelae, despite having more limited hyperplasia; the brain MRI also showed severe pathological changes in cortex and white matter, predominantly in the parieto-occipital region. Early and accurate diagnosis is critical in these patients in whom hypoglycemia is compounded by a lack of the ketone bodies which represent a vital alternative source of energy for the central nervous system.

A saporin-insulin conjugate: synthesis and biochemical characterization.

Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.

A chimeric saporin-transferrin conjugate compared to ricin toxin: role of the carrier in intracellular transport and toxicity.

Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.

Hardware and software requirements for quantitative analysis of comparative genomic hybridization.

Recommendations are made for hardware and software capabilities that will permit a level of performance of comparative genomic hybridization (CGH) analysis on metaphase chromosomes that is comparable to the best current practice. Guidelines for interpreting the results of CGH analysis in terms of chromosomal gains or losses are also presented.

Intracellular dynamics of ricin followed by fluorescence microscopy on living cells reveals a rapid accumulation of the dimeric toxin in the Golgi apparatus.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.

Confocal-line microscopy.

The confocal-line (CL) technique combines some of the characteristics of confocal-scanning microscopy with those of conventional-imaging methods. It is based on the introduction of line-shaped illumination and linear image detection, as an alternative to the current confocal-point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non-biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.

Microspectroscopy of red blood cells.

Spectroscopic techniques have been widely employed to analyze properties of macromolecules and dynamics of intracellular events on bulk preparations of cells. The development of computer controlled microspectrophotometers has made possible the study of the same events in single cells, often providing significant and unexpected results. This paper briefly reviews experimental works carried out in our laboratories on single red blood cells. Microspectrophotometric techniques were applied which make use of the fact that ligand binding to intracellular haemoglobin is associated with optical changes. Information on the relative abundance of different haemoglobin components inside single erythrocytes of trout blood was obtained from spectra of air equilibrated samples, taking advantage of the extreme pH sensitivity of one of the four haemoglobin components. The kinetics of oxygen and carbon monoxide binding to haemoglobin has been followed and demonstrated to correspond to a zero order process, with a rate much slower than that characteristic for haemoglobin in solution. These results demonstrate that the process is diffusion limited; computer simulations suggest that ligand uptake is limited by the time required for the diffusion from the extracellular space of enough ligand molecules for total saturation of intraerythrocytic haemoglobin. Finally, oxygen dissociation curves in single red blood cells can be obtained by means of particular flow cell, with promising results for the study of physiological and pathological processes (namely red cell sickling in drepanocytosis).

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a 'memory' of previous sickling cycles.

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a 'memory' of its previous cycle(s), possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

A microspectroscopic analysis of ligand binding in single erythrocyte.

The kinetics of ligand uptake by erythrocytes has been analyzed by single cell microspectroscopy measurements carried out in parallel with mathematical simulations. The main experimental feature is the linear shape of the ligand binding progress curves, whose slope is dependent on the free ligand concentration in the speciman. From the computer simulation a predominant role has been shown to be played by the ligand diffusion from the extracellular space.

Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity.

The kinetics of the reaction of CO with intraerythrocytic haemoglobin has been studied in single red blood cells (RBC) using a scanning microspectrophotometer and a photochemical perturbation method. Measurements have been carried out using red blood cells from man and camel (Camelus dromedarius), the latter at different osmotic pressures. Camel RBC, which are smaller and different in shape compared to human RBC, are known to remain intact even at an osmolarity 6-times lower than physiological (280-290 mosm/l), swelling up to twice their normal volume. The results show that the recombination time course is affected by diffusion of CO through a stagnant layer of solvent around the cell membrane, but that it is also influenced by other parameters such as intracellular diffusion of ligand and haemoglobin.

[Microspectrophotometry in the study of physiological and pathological events in erythrocytes]. / L'uso della microspettrofotometria nello studio di eventi fisiologici e patologici nel globulo rosso.

Microspectrophotometry turns out to be the ideal method for an accurate study of single cells, namely of red blood cells, from the biochemical and physiological standpoint. Hereafter several possible applications are reported: a. the kinetics of gas-exchange (O2 and CO) in human erythrocytes has been made possible by the photosensitivity of the Hb-CO complex. In this way, it has been possible to establish that both ligands recombine with intraerythrocyte hemoglobin following a zero-order kinetic process. This suggested that diffusion of a ligand across an unstirred layer of buffer (approximately 5.10 mu thick) all around the cell could be the rate-limiting step of this recombination. Moreover, an intracellular facilitated oxygen diffusion has been observed and possible physiological implications have been shortly mentioned; b. the kinetics of intracellular polymerization of sickle cell hemoglobin in single red blood cells has been measured by quickly (approximately 1 msec) flashing-off the CO employing a cw Argon ion laser. The polymerization of deoxyhemoglobin has been followed by detecting the increased light scattering of the laser beam itself. The distribution of delay times (td) in several patients has been found significantly correlated to the severity of the disease, supporting the hypothesis that the td is a determinant factor in the pathophysiology of the disease. Possible therapeutic applications of this method are briefly discussed; c. the intraerythrocyte distribution of different hemoglobins of trout (Salmo irideus) among several erythrocytes has been observed by means of different physiochemical properties of single components. These turned out to be homogeneously present in all red blood cells studied.