INTRODUCTION: The mitochondrial permeability transition (MPT) has been implicated in mediating both necrotic and apoptotic cell death. Opening of the permeability transition pore in the mitochondrial inner membrane causes the MPT. Previously, our laboratory developed a method to detect the MPT in cultured hepatocytes by visualizing redistribution of calcein fluorescence from the cytosol into the mitochondria after permeability transition pore opening, using confocal microscopy. (1). However, a recent paper suggests that unstained mitochondria are unlikely to be detected against bright cytosolic signal, because fluorescence spreading magnifies the size of emitting objects while reducing that of dark spot (2). Rather, it was suggested that dark mitochondrial voids in green calcein images were the result of fluorescent quenching by concurrent use of a red-fluorescing mitochondrial dye, tetramethylrhodamine (TMRM). The AIMS of the present study were to determine 1) whether small voids can be visualized in the absence of any potential quenching agent; 2) whether quenching can produce dark mitochondrial voids and 3) the importance of alignment in visualizing mitochondria as negative contrast by confocal microscopy. METHODS: Overnight cultured rat hepatocytes were used. To load calcein exclusively into cytosolic space, hepatocytes were incubated with 1 μM calcein AM for 15 min at 37°C. To load calcein into both cytosol and mitochondria, cells were incubated with 1 μM calcein AM for 1 hour at 4°C.

State of the art in optical microscopy

Optical microscopy is still and increasingly one of the most valuable tools in biological investigation. In particular, confocal microscopies are capable of achieving best performances in the study of three-dimensional fluorescent and reflecting specimens. Nevertheless, current techniques adopted in confocal microscopy present some drawbacks and limitations that stimulate to devise and set-up further techniques, suited to a wider range of applications.

Advantages of confocal microscopes mainly correspond to an improved spatial resolution, especially in the axial direction. Depending on the narrow-field scanning approach used, there are two main forms of confocal microscopes: single-point (SP) and multi-point (MP) ones. Unfortunately, SP confocal microscopes require the use of lasers as illumination sources with consequent high costs and scarce spectral flexibility. Moreover, specimen photo-damage due to relatively high instantaneous irradiation doses involved, can often limit their investigative capabilities. On the other hand, proposed MP confocal microscopes still rely on the revolving-disk approach and exhibit a relatively low luminous efficiency, substantial constructional complexity, and limited contrast in the study of thick fluorescent objects.

Recent advances in fluorescence microscopy add to the versatility of this optical technique and intensify its significance as an indispensable tool in biological research. Especially the use of multiphoton excitation offers the microscopist many advantages like inherent optical sectioning, reduced out-of-focus bleaching and higher penetration depths into the sample. In this regard, the commercial availability of pulsed solid-state lightsources like the Ti:Sapphire laser, that provide short pulses needed in the nonlinear multiphoton process, have paved the way for the routine implementation of multiphoton microscopy in the biologists laboratory. Although the spectral range of the commonly used Ti:Sapphire laser allows the application of two-photon fluorescence microscopy on chromophores that absorb in the range of 350-450 nm, a lasersource that enables the two-photon excitation of molecular probes at even shorter wavelengths (<350nm) would be highly beneficial.

In this contribution we present a visible femtosecond optical parametric oscillator (OPO, Figure 1) that is ideally suited to excite molecular species at 285-335 nm by means of a two-photon process. Femtosecond pulses with durations as short as 30 fs can be generated within a tuning range from 570 to 670 nm. A cavity dumper incorporated in the laser cavity provides variable pulse repetition rates (single shot to 82 MHz).

A variety of biological labeling techniques has been developed in order to obtain specific chemical and spatial information from cells and tissues. Traditionally theses labeling techniques have been categorized as cytochemistry, immunocytochemistry, and in situ hybridization. Another special category relates to fluorescence analog cytochemistry in which specific fluorescently-labeled molecules become incorporated into the pool of endogenous molecules of the cell. They can thus serve as reporters for analysis of the dynamic properties of the population of molecules of interest. Such molecules are usually introduced into cells by microinjection or expressed within the cell (e.g., green fluorescent protein derivatives).

The past few years have witnessed a renaissance in biological optical microscopy. Many of the advances in the elucidation of cell structure-function relationships made through the use of optical microscopy have relied upon fluorescence labeling technology. These advances notwithstanding there remain experimental situations in cell biology that require the higher spatial resolution afforded by electron microscopy. Combining fluorescence and electron microscopy to study the same structures would be very useful in many experimental situations in cell biology. Such an examination of the same structures with more than one imaging modality can be referred to as correlative or integrated microscopy. The number of such studies is relatively small; this is probably due to technical difficulties encountered by various investigators.

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.

[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.

Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.

F(ab’)2 Goat anti-Mouse IgG and F(ab’)2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’)2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo-NHS-Nanogold® at pH 7.5. Nanogold® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).

Fluorescence in situ hybridization (FISH), long the method of choice for chromosomal cytogenetics, is becoming recognized as a powerful method for correlative histochemical detection of multiple messenger ribonucleic acid (mRNA) in cells. The technique is based upon the principle of the binding of a labeled strand of DNA (an oligonucleotide probe) or RNA (a riboprobe) to complementary strands of mRNA. In the traditional in situ hybridization method, nucleic acid probes are labeled with radioactive isotopes and the hybrids are localized by autoradiography. More recently, labeling of the probes with digoxigenin, biotin, or fluorescein has allowed nonisotopic detection of probe-mRNA hybrids with immunoenzymatic techniques that are based on peroxidase or alkaline phosphatase histochemistry, in which the results are observed as brightfield stains. FISH is similar to conventional nonisotopic in situ hybridization except that the labeled nucleic acid probes are detected by fluorescence microscopy.

The FISH technique has advantages for performing correlative histochemistry. The results of a FISH protocol can be observed within a few hours of completing the hybridization protocol instead of waiting for days as is the case with isotopic methods. FISH permits cellular resolution and facilitates multiple labeling, where in situ hybridization probes can be resolved at the level of individual cells (Hahn et al. 1998).

Previous studies have documented NADH oxidase as a source of the oxygen radicals, superoxide (O2) and hydrogen peroxide (H2O2), at sites of disruption of the blood retinal barrier (BRB) in a rat model of noninsulin dependent diabetes (NIDDM). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the complications of diabetes. The effects of NO depend on its microenvironment and one mechanism for its role in complications of diabetes is interaction with oxygen radicals to form the toxic species, peroxynitrite (ONOO−). Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS. The activity of iNOS can be detected by localization of nitrotyrosine, a marker indicative of the formation and activity of ONOO−.

Colloidal gold labeled immunocytochemical studies of iNOS and nitrotyrosine were done on sections of neural retina in eyes of obese, NIDDM BBZ/Wor rats in which NADH oxidase had been localized with cerium perhydroxide. Disruption of the BRB was documented by immunocytochemical localization of extravasated endogenous serum albumin. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.

An animal model for Cystic Fibrosis (CF) has been developed to further comprehend the pathogenesis of the disease as well as the development of therapeutic interventions. This is a mouse line from a phenotype of an inbred congenic strain of CFTR-knockout mouse that spontaneously develops lung disease. Pathology of the mice include severe impairment of the mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening with evidence of fibrosis and inflammatory cell invasion.

Although protein replacement therapy using liposomes containing purified reconstituted functional CFTR has been done on the nasal epithelium of CFTR-knockout mice the strain did not develop lung disease nor were the liposomes applied beyond the nasal cavity. Assessment of the treatments was done by measuring the transepithelial potential difference (PD), immunofluorescent labeling of the CFTR and immunogold labeling of the segments of nasal epithelium which were found to have a correction in their PD readings and CFTR present by immunofluorescence. It was clear after these studies that some form of correlative microscopy technique was needed to be devised in order to improve the efficiency of detection.

Gold labels such as Nanogold® and colloidal gold are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification or silver enhancement, is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone or npropyl gallate. Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA® ; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell. However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.

Multiple labeling for electron microscopy (EM) is typically accomplished by using colloidal gold (cAu) particles of different sizes. The size distribution of each cAu preparation, which may vary by up to 15%, is the principal factor that limits the number of labels which can be used simultaneously. Furthermore, there is no effective way to use multiple labeling for quantitative, high resolution EM studies. Such analysis requires the use of a single Fab antibody fragment conjugated to a single cAu particle in order to ensure that each particle corresponds to only one antigenic site. Whole antibody molecules cannot be used for quantitative analysis because they are at least divalent, and some, such as dimeric IgA or pentameric IgM, have even more antigen-binding sites. Consequently, it is impossible to deduce whether the presence of one whole antibody molecule corresponds to the presence of one, two, or more targets. Particle diameters ranging from 3 to 5nm are optimal for quantitation because one Fab fragment adsorbs to one particle, more than one Fab fragment may adsorb to a single particle larger than 5nm, and, when smaller than 3nm, several particles may bind to a single Fab fragment.

We are evaluating parameters apart from particle size variation to accomplish multiple labeling for both qualitative and quantitative analyses. One method relies on electron energy loss spectroscopy (EELS) to distinguish particles of several metallic compositions, including Au, Ag, Pt, Pd, Rh, and Ru. EELS is performed using a LEO 912 energy filtering transmission electron microscope (EFTEM) with an in-column Omega spectrometer.

Non-imaging microscopy has developed somewhat independently of both traditional light microscopy and laser confocal microscopy. Flow cytometry is the chief commercial and research technology among these microscopies, and, like other nonimaging detection systems, developed around the theme of automation in clinical laboratory medicine. It is an important correlative or parallel microscopy to several image forming microscopical methods. Cell sorting is an important option as well.

The basic structure of the flow cytometer certainly parallels light, laser and electron microscopes. The flow cytometer has a light source, a set of adjustable optics to focus the beam on the specimen, objective optics to collect the light and direct it to appropriate sensors, and the sensors themselves. A real image is not formed because the sensors are not in an even plane with the projection, such as provided by the retina in light microscopy or an image plane or film plate in electron microscopy, and the objective optics may not focus in the image plane.

While early flow cytometers were developed primarily for the automatic counting of cells and particles, modern instruments offer particular advantages for the analysis of fluorescence, fluorescent chemicals and probes and cellular auto fluorescence.

Hard metal is an alloy of tungsten carbide and cobalt along with other components such as chromium carbide, molybdenum carbide, tantalum carbide, and nickel. Chronic exposure to hard metal particles by inhalation causes alveolitis leading to interstitial fibrosis, the pathogenesis of which is still undefined. The initial inflammatory response includes a change in epithelial cell permeability barrier function (1) which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network (2, 3). Therefore, the objective of this study was to evaluate the effect hard metal particles have on F-actin organization of rat lung epithelial cells in an in vitro culture system.

Rat lung epithelial cells (L2: ATCC, CCL-149) were grown to confluence on glass coverslips and exposed to various concentrations of hard metal particles for 24 hours. The effect on F-actin organization was visualized by confocal microscopy following Bodipy-Phallacidin staining, while changes in cell morphology were assessed by phase contrast microscopy. Hard metal particles of cobalt, tungsten carbide, and tungsten carbide/cobalt (6 % cobalt) were tested at concentrations of 1, 3, and 5 μg/ml. There was a dose-dependent change in the F-actin organization in the cells. The actin microfilaments lost their uniform distribution and aggregated into homogeneous masses of F-actin staining. Significant change in F-actin state was observed even at a 1 μg/ml concentration of tungsten carbide/cobalt particles. This is consistent with previous observations that pathological effects of tungsten carbide/cobalt particles are more pronounced compared to either metal alone. Phase contrast microscopy revealed no significant change in the cell morphology at this short incubation time.

The Western Mosquitofish, Gambusia affinis affnis, a sexually dimorphic internal fertilizing species of the family Poeciliidae, shows distinct sexual dimorphism in the anal fin and its axial and appendicular support.

The development of the regional vertebral differences (i.e. precaudal [vertebra bearing ribs] and caudal [vertebra bearing hemal spines]) in female and male G. a. affinis is generated through a development program of first posterionzing more anterior vertebra (formation of hemal spines more anteriorly) then antenonzing more posterior vertebra (loss of hemal spines and formation of parapophysis with pleural ribs) . This developmental program is reminiscent of effects obtained by disturbing developmental programs by exogenous treatment with retinoic acid.

Recently we generated data suggesting that all-trans retinoic acid (tRA), a non-steroidal lipophilic molecule may play a role in the regulation and patterning of sexual dimorphic phenotype(s), an area considered the domain of steroid hormones.

We wanted to identify the specific neural cell type(s) synthesizing endogenous tRA within the developing spinal cord which might coordinate sexually dimorphic patterning of spinal cord levels in G. a. affinis. To achieve this, we employed retrograde transport studies using 3,000 molecular weight (MW) dextran amine and biocytin combined with immunogold and/or lmmunohistochemistry to identify the specific neural type(s) which express tRA synthesizing enzymefs] (i.e. retinaldehyde dehydrogenase [RALDH]).

This study was conducted to compare the immunohistochemical and morphometric characteristics of blood vessels in brain metastasis with blood vessels of the adjacent uninvolved brain tissue. This is based on the hypothesis that new vessels formed in the tumor express antigens that are not expressed in blood vessels of the adult brain parenchyma. Thus, a panel of antibodies known to react with mouse endothelial cells at various stages of differentiation or stimulation was tested on frozen sections of experimental brain metastasis. Intratumoral microvessel density which has been reported to reflect the intensity of tumor angiogenesis was inapplicable to experimental brain metastasis therefore other parameters were measured.

Brain metastasis was produced in nude mice following intracarotid injection of tumor cells. Three human tumors and three murine tumors were injected into the carotid artery of nude mice. The animals were sacrificed when they were moribund, the brains were embedded in OCT and frozen in liquid nitrogen. Some animals were injected with bromodeoxyuridine (BrdU) 1 h before they were necropsied. Immunohistochemistry was performed using antibodies directed to the following antigens: CD31, CD34, MECA-32, endoglin, ICAM-1, ICAM-2, and BrdU. Antibodies to leukocyte antigens, F4/80 and CD3 were also tested to determine the amount of host cells infiltrating the tumors. SEM of immunogold labeled preparations determined the cellular localization of some antigens.

Determination of the fine organization of actin networks is important for understanding different functional aspects of the central nervous system (CNS) such as plastic events. Early studies investigating the localization of actin filaments at the ultrastructural level relied on structural methods or antibodies raised against actin isoforms. More recently, many investigators have employed the actin-binding peptide phalloidin conjugated to various fluorescent molecules. By conjugating phalloidin to the fluorophore eosin, we have been able to localize f-actin at the electron microscopic level using photooxidation of diaminobenzidine (DAB) by eosin. The goal of this study is to provide a better description of the cellular and subcellular localization of actin filaments at the light and electron microscopy level based on the high binding affinity of phalloidin for actin and utilizing this novel method.

Tissues were obtained from male Sprague Dawley rats that were perfused transcardially under deep anesthesia with normal rat Ringer's solution at 35°C followed by fixative. The fixative contained 4 % paraformaldehyde and different concentrations of glutaraldehyde, ranging from 0.1- 2.5%. Sections of cerebellum, striatum and hippocampus which were cut at a thickness of 50-80 μm with a Vibratome were incubated with phalloidin-eosin in a solution of 0.05% in 0.5% cold water fish gelatin/50mM glycine-PBS (“working buffer”) for 2 hrs. As a control, phalloidin-eosin was omitted for one set of tissues.

A dye, Phloxine B, a common food coloring and one of the active components of a photoreactive insecticide was recently approved by Food and Drug Administration as D&C Red #28 for use in drugs and cosmetics. Phloxine B is also one of the most widely use stain in fluorescence microscopy. However, in spite of the widespread interest in multi-photon spectroscopy and imaging, no information is available on the electronic transitions properties of Phloxine B with red edge fluorescence excitation and with multi-photon excitation.

In the present report we described the steady state and time-resolved fluorescent properties of Phloxine B with up-convert photon excitation and with two-photon excitation. We examined the electronic transitions properties of Phloxine B when was excited by femtosecond pulses from a mode-locked titanium sapphire laser or a mode-locked optical parametric oscillator laser. Phloxine B under excitation wavelengths above 775 nm was found to display two-photon excitation fluorescence with a spectrum maximum at 580 nm, which emission is consistent with one-photon excitation fluorescence spectrum. At the red edge excitation of Phloxine B, from 590 nm to 650 nm, we observed one-photon excitation fluorescence indicating that Phloxine B behaves like upconverting dye with one-photon excitation process.

Dental plaque is the biofilm which forms on the surface of teeth, comprising a multitude of bacterial species embedded in microbe- and host-derived polymers. Confocal microscopy (CLSM) studies revealed an open architecture in which palisades of biofilm biomass were shown to be interspersed with water channels of lower density. However, the penetration depth of CLSM in these samples is limited to around 20-40 μm, depending on the density of the samples. Therefore, thick biofilms associated with, for example, fissures in the tooth surface, can not be investigated using this technique. The limited depth penetration of CLSM can be overcome by employing two-photon excitation microscopy (TPEM). Due to the use of the longer wavelength excitation light in TPEM, a two to four fold higher penetration into the sample is obtained than in CLSM, see fig. 1. This, together with the reduced photobleaching of the fluorescent probes, makes TPEM well suited for in-depth imaging studies of strongly scattering samples such as biofilm.

The biofilm nature of plaque allows the development of gradients of key biochemical parameters such as ion concentrations. These gradients may result in extreme conditions at certain sites within the plaque that are often associated with the development of the diseased state. Conditions in the oral cavity are overtly aerobic, and the pH of saliva is near neutral.

Historically, high-resolution optical microscopy has played a vital role in biomedical research. The resolution of conventional optical microscopy is limited by the diffraction effect to about 200 nm and 500-900 nm in the radial and axial directions, respectively. This modest resolution is a major limitation in using optical microscopy for demanding applications. New techniques such as near field optical microscopy, scanning tunneling microscopy, and atomic force microscopy have been developed to circumvent this problem and have achieved atomic resolution in solid state specimens. Unfortunately, the applications of these new techniques have been difficult in biological systems. First, scanning probe techniques require mechanical interaction with the specimens. The mechanical forces exerted by the probe tip may cause sample deformation. This interaction further degrades the achievable image resolution. Resolutions in the range of 50 to 100 nm are often reported for near field optical systems. Second, scanning probe technique has a relatively low frame rate since the required high precision position control imposes severe bandwidth limitations. Scanning probe techniques are more suitable for studying static sample but often have difficulties in addressing many inherently dynamical phenomena in biology. Third, these are surface imaging techniques and are inherently limited in their ability to investigate the 3-D structures of biological specimens.

Chemical sensing based on fluorescence is presently undergoing rapid development. As one example, fluorescence lifetime measurements are now being used for measuring cation and anion concentrations in clinical chemistry, cellular imaging and flow cytometry. Additionally, there is an increasing use of proteins as sensors because of their high affinity and specificity for ligands.

In this report we describe some recently developed approaches to fluorescence sensing. These new methods are based on the use of a fluorescence reference either within the sample or external to the sample, but within the optical path. Two types of references are used; microsecond lifetime metalligand complexes (MLCs) or oriented films of ns decay time fluorophores. A protein sensor for glucose was developed using the glucose-galactose binding protein (GGBP) from E.coli (Figure 1).