In the last few years there has been a mild explosion in both microscopy and cytometry. in part, this has been a more sensitive understanding of new fluorescent chemicals or probes as well as the ability to transfect fluorescent proteins into cells. in part, this has also been a case of substantial improvement in instruments, electronics, computers, lasers and sensors. in this session, we hope to examine correlative and comparative methods that may improve the quality of data.

This is an introduction to session 29, Correlative Fluorescent Microscopy and Flow Cytometry Techniques, and the first time the Microscopy Society of America has sponsored a session that involves flow cytometry. Flow cytometry offers several statistical advantages over standard microscopies in evaluating populations of cells. Flow cytometry also facilitates a statistical precision in evaluating fluorescence and fluorescence changes in both chemical probes and transfected fluorescent proteins.

Introduction This presentation will follow the pathways to convergence of two very complementary technologies - flow cytometry and imaging. Interestingly, each technology has evolved and matured almost exclusively isolated from the other. in the past several years it has become increasingly clear that these technologies are able to deliver complementary solutions and that when combined the end value of each exceeds the sum of the individual components.

History of development Flow cytometry has a rich history over the past 40 years. Ironically, as a technology, flow cytometry was almost a consolation prize to its imaging cousins. in fact, it seems that it might only have been because imaging solutions proved to be technologically difficult that flow cytometry not only thrived, but left the imaging field well behind for nearly 20 years. Initial interest in evaluating differences between normal and cancer cells was almost exclusively attempted by imaging techniques.

Flow cytometry is an analytical technique in which optical measurements are made of cells or other biological particles as the cells or particles flow, ideally in single file, in a fluid stream past one or more optical measurement stations. Modern optical flow cytometers typically measure light scattered at small (1-5°) and large (15°-135°) angles to an illuminating laser beam, and fluorescence emitted in three or more discrete spectral bands; the most complex instruments employ three or four spatially separated illuminating beams at different wavelengths and can measure twelve fluorescence signals from each cell or particle analyzed.

Flow cytometry was developed in the 1960's to speed up and automate microspectrophotometry and microfluorometry, which had previously been microscope-based, in order to facilitate development of observer-independent clinical tests which could replace the Papanicolaou smear and the differential leukocyte count. At that time, high-resolution scanning was an extremely slow process; high-intensity sources were restricted to arc lamps, and memory in which images could be stored was expensive.

As originally developed in the 1960's, flow cytometry was primarily a technique for the analysis of mammalian cells. Analysis of cellular constituents such as DNA or cell surface antigens made fluorescent by a variety of reagents has been the main stay of flow cytometric applications. Over the years, flow cytometric analysis techniques have been developed that range from multicellular spheroids containing a million or more cells down to single molecule detection. An outgrowth of single molecule detection capability is DNA fragment size analysis.

DNA fragment size analysis starts with a sample of naked DNA that can be derived from a variety of sources including PCR products, double stranded viral genomes, BAC/PAC clones, and bacterial genomes. For genomic or cloned DNA, restriction enzyme digests are analyzed to produce a fingerprint pattern. The fingerprint, i. e., the distribution of fragment sizes produced by the restriction enzyme digestion, is characteristic of the source of DNA and forms the basis for identifying the source.

The plasma membrane defines the outer limit of eukaryotic cells and it is here that cells interact with their environment through receptor proteins. Clearly the management of cell surface receptor concentration is critical for normal cellular function, and this is achieved through two key processes, exocytosis and endocytosis. During exocytosis transport vesicles containing both membrane receptors and soluble cargo fuse with the plasma membrane releasing cargo and delivering receptors to the cell surface. During clathrin mediated endocytosis receptors destined for internalisation become concentrated in clathrin coated pits before coat invagination and membrane fission. Since transferrin receptors are recycled and undergo numerous rounds of exocytosis and endocytosis by labeling transferrin receptor with an appropriate fluorophore it is possible to label both exocytic and endocytic organelles in the same cell. We have exploited this phenomenon to visualise the spatial and temporal organisation of constitutive exocytosis and endocytosis at the surface of single cells using dual wavelength evanescent field microscopy.

Intracellular bacterial pathogens such as Salmonella typhimurium secrete proteins into the host cell after infection. These proteins alter the normal structural and metabolic machinery of the host cell and benefit the bacterium by facilitating replication and avoidance of host immune surveillance. Since the host cytoplasmic localization of these proteins infers access to the class-I MHC antigen processing and presentation machinery of the host cell, we collectively refer to these proteins as Class- I Accessible Proteins (CAPs).

The design of vaccines for new and emerging bacterial pathogens is often constrained by the selection of appropriate and specific antigens. While vaccine design is being greatly aided by whole genome analysis of bacterial pathogens, it has been of limited use in the assignment of function and host subcellular localization of a large percentage of bacterial proteins. in addition, analysis of the bacteria/host interaction is further complicated by the complex lifestyle of the pathogen.

Flow cytometry offers several advantages in evaluating cellular fluorescence and separating values from chemical probes or transfected fluorescent proteins from autofluorescence. Autofluorescence can introduce artificial values that cannot be separated by examination of the fluorescence histogram. Often a comparison of fluorescence in two detectors can separate autofluorescence from positive fluorescence. A region can be defined around the cells of interest for further statistical evaluation or for subsequent sorting.

Autofluorescence is also correlated with apoptosis in that apoptotic cells often increase autofluorescence values. A clear separation of living cells from dying cells can be achieved by the introduction of a DNA stain, such as 7AAD, which will penetrate cells with disrupted or damaged cellular membranes. Intact living cells will not show the 7AAD fluorescence.

This separation is important in transfected fluorescent protein studies, as the transfection process is often toxic to cells.

While morphological analysis at both the light and electron microscopical levels has been fully exploited in academic research, its potential in biopharmaceutical industry has only been fully appreciated in recent years. The biotechnology industry has been developing highly specialized therapeutic molecules, particularly proteins and peptides that are directed to specific molecular targets. The pace of the development of new therapeutic molecules is expected to increase dramatically in the close future, as the biopharmaceutical community will capitalize on the new targets identified by the Human Genome Project.

An efficient drug discovery and development effort should not only include the characterization of the tissue and intracellular distribution of potential targets, but also establish unambiguously that the therapeutic entities reach their intended targets. It has become apparent in recent years that a comprehensive view of the pharmacological action of drugs must include not only the pharmacokinetic parameters but also the tissue distribution and the cellular localization.

Digital microscopy, the integration of digital and microscopy technologies, was initiated for quantitative microscopic image analysis, but it is now for almost all microscopy applications. During the past decade, with the advance of digital technologies, digital microscopy imaging is becoming an indispensable technology in drug discovery.

We started establishing state-of-the-art digital microscopy imaging for drug discovery with the investigation of bioimaging applications at our US research site. Our results shown that all the top 5 bioimaging needs require computer-aided microscopy. Based on this investigation and our review of the microscopy imaging applications in the pharmaceutical industry, we determined four directions for microscopy in drug discovery: multidimensional/multimodal microscopy, digitalization, automation, and bioimage informatics.

Multidimensional/multimodal microscopy imaging is required by the nature of biological research, which is fundamental in drug discovery. From genomic imaging to pathology observation, we require biological details and compound activities at the levels from subcellular organelles to organ tissues, from cellular signaling to anatomical locations of compounds.

In spite of improved equipment and software, the routine application of quantitative microscopy to practical problems in drug development has been limited. This is due to a perceived and often real increase in project complexity and time added by quantitation of tissue changes, without an offsetting return of information. A major barrier to quantitative microscopy is the degree of difficulty in collecting data. The more steps in this process, the less attractive the task. Therefore, it follows that the more automated the task, the more likely quantitative methods will be applied. The purpose of this presentation will be to illustrate an approach taken to automate the morphometric quantification of muscle atrophy which follows dennervation.

The traditional morphometric approach to assess myofiber atrophy is based upon laborious planometric measurement of individual muscle fiber cross-sections. Examples of the type of data obtained using the traditional method are shown in text table 1.

Non-invasive imaging technologies such as magnetic resonance imaging are commonly used in clinical practice. in an experimental setting, it is possible to decrease field of view and pixel size, thereby increase image resolution. We tested this magnetic resonance microscopy (MRM) technique in a theopln lline-induced rat model of reproductive toxicity. The goal of this work was to study the sensitivity of MRM to detect or predict changes in tissue proton (i.e. water) characteristics, and confirm those findings with histology.

Male Sprague-Dawley rats were fed either a control or theophylline (1.3-dimethylxanthine. 8000 ppm) diet. Experimental groups of days 8. 16. 24. and 32 animals were imaged with MRM (Figure 1) and tissue morphology confirmed with histology (Figure 2).

Tl-weighted images (with and without gadolinium-DTPA contrast. Magnevist®) and T2-weighted images were acquired at 2 T (spin-echo multislice images. 100 μm2 pixel. 300 μm slice thickness).

The gram positive bacillus, Listeria monocytogenesis a contaminant of the food we eat and although it is a common pathogen in farm animals L. monocytogenesdoes not normally infect humans. However, immunocompromised individuals, infants and the elderly are susceptible to infection. The bacteria, taken into membrane bound phagosomes, use a thiol-activated cytolysin listeriolysin O to disrupt the membrane (LLO) and gain access to the cell cytoplasm. Subsequent actin polymerizations on the bacterial surface induce motility in the cell and long cytoplasmic protrusions at the cell surface that spread bacteria to neighboring cells.

L. monocytogenesenters cells by phagocytosis. By using short infection times of 5 min or less, it was possible to examine early interactions between J774 cells and L. monocytogenes.This study shows that during the early stages of internalization the bacteria are able to modify the protein composition of the forming phagosome membrane.

Efavirenz (Sustiva™) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) for the treatment of HrV-infected patients and has been in the market since 1998. Efavirenz exists as three different polymorphs (Forms I, II, III) and a nonstochiometric heptane solvate (Form IV). The current synthetic process for Efavirenz yields Form IV which is converted to the desired Form I polymorph by heating. One of the concerns for any drug substance is whether a consistent crystal form is produced in order to insure consistent physical and chemical properties, as well as to safeguard pharmacokinetic profiles. The sensitivity and resolution of single-crystal x-ray diffraction, the traditional method employed to determine unit cell parameters of drug compounds in development, are limited for Efavirenz Form IV because of the small crystallite size of the material. Furthermore, Efavirenz Form IV materials, depending on the process conditions, have slightly different powder diffraction patterns in terms of intensity when collected on standard in-house diffractometers.

Microscopy, microdissection, and analytical chemistry techniques were used to localize and identify drug and drug metabolites in specimens from drug-treated rats. During a toxicology study in rats of a drug candidate, concretions (stones) were found in the common bile duct, and pigmented material was found in hepatocytes and liver macrophages after long-term exposure to the drug. Because a recent study showed the effective use of microscopy and mass spectrometry (MS) to identify drug metabolite in tissue sections, we used similar methods to confirm that pigmented material in liver cells was drug metabolite. Energy dispersive x-ray spectroscopy (EDS) suggested that drug and/or metabolite were also present in stones.

Pigmented hepatocytes and macrophages in frozen sections of unfixed liver exhibited fluorescence similar to that of parent drug. When excited by UV illumination, these cells fluoresced the same color blue as the bulk drug (Fig. 1).

Cellular oxidative stress, a common mechanism of drug-induced toxicity, is a result of the formation of reactive oxygen species (ROS) in response to chemical stimuli. An endpoint of ROS production is lipid peroxidation, which can in turn lead to disruption of cellular membranes, loss of mitochondrial function, protein oxidation and DNA damage. This toxicity can be organ-specific due to the varying capacities of tissues to handle oxidative events. Liver is particularly sensitive to the effects of oxidative stress, and hepatic toxicity is seen clinically. HepG2 cells are an immortalized human hepatoma cell line used as an in vitro model for mammalian hepatotoxicity studies. The purpose of this study was to characterize the effects of chemically induced oxidative stress in this cultured cell model.

HepG2 cells were grown to subconfluence in poly-L-lysine coated 4-well LabTek™ II chambered coverglasses (Nalge Nunc International).

The insulin-sensitizing agent troglitazone (Rezulin) has been shown to cause severe hepatotoxicity and liver failure. While the toxic mechanism is still unknown, a component may be oxidative stress. We have demonstrated that increased lipid peroxidation, decreased glutathione levels, and collapse in mitochondrial membrane potential occurred following exposure of cultured hepatocytes to troglitazone. These hallmarks of oxidative stress can manifest morphologically in several ways including damage to cellular membranes, mitochondrial abnormalities, and cell death. The purpose of this study was to assess morphologic changes caused by exposure to troglitazone or cumene hydroperoxide (CH), a compound known to induce oxidative stress.

HepG2 and Novikoff hepatoma cells were grown on poly-L-lysine coated 6-well plates overnight, and exposed to 10, 25, 50 or 100 μM concentrations of troglitazone or CH in the growth media for 2 hours. Cells were fixed with 2.5% glutaraldehyde in phosphate buffer, harvested, and pelleted. Pellets were post-fixed in 1% osmium tetroxide, and processed to epoxy resin.

Oxidative stress and inducible nitric oxide synthase (iNOS) have been implicated in endothelial cell dysfunction in diabetic retinopathy by formation of peroxynitrite, a cytotoxic product of superoxide and high levels of nitric oxide. Spontaneous diabetic models do not allow for separating the oxidative stress as a result of hyperglycemia from that of the nitric oxide system. in addition chemicals such as streptozotocin which can induce diabetes in animal models cause oxidative stress and induce iNOS before the onset of hyperglycemia. Galactose has been used to induce non-insulin dependent diabetes in mice and rats while gene knockout mice provide a means to study the role of specific NOS components. This study used cytochemical localization of hydrogen peroxide produced by NADH oxidase, a marker for superoxide production, and immunocytochemical localization of nitrotyrosine, a marker for nitric oxide formed radical peroxynitrite, to investigate the role of oxidative stress and the nitric oxide system in galactose induced diabetic retinopathy in iNOS knockout (iNOS%) mice.

Although nuclear channels (NC) have been described in a variety of cells, their function has not been fully investigated. This investigation used immunofluorescent techniques to examine the formation and function of NC in HeLa cells, a human cervical cancer cell line. NC are defined as invaginations of the nuclear envelope (NE) that traverse the nucleoplasm, thus forming a ring-like nucleus. The resulting tunnel is devoid of nucleic acid and may contain cytoplasmic elements. to date, all the reports of the presence of NC are from electron microscopic investigations, which clearly demonstrate the occurrence of NC in the presence of what appear to be interphase nuclei. This investigation was initiated because the NC that we observed in HeLa cells could be seen with fluorescent microscopy and only appeared during telophase and early interphase.

HeLa cells, obtained from American Type Culture Collection, were cultured on glass coverslips in minimum essential medium (MEM) containing 5% calf serum (CS) and a mixture of 5% CO2 in air at 37°C.

The levels of plasma high density lipoproteins (HDL) are inversely related to the incidence of atherosclerosis and coronary heart disease. This protective effect of HDL is thought to be involved in the reverse transport of cholesterol from peripheral tissue to the liver. The scavenger receptor class B type 1 (SR-BI) is the first HDL receptor that can mediate the selective uptake of HDL cholesterol in steroidogenic tissues and in liver. The expression of SR-BI in liver appears to be responsive to changes in the hormonal status and in the levels of HDL cholesterol. However, long-term effects of high-cholesterol diet on the SR-BI expression in liver parenchymal cells and KupfFer cells have not been studied. to investigate the in vivo effects of liver-specific SR-BI expression on lipid metabolism, we fed male Sprague-Dawley rats with high-cholesterol or normal diet for 1 and 12 months.

It is well-known that high-density lipoprotein (HDL) mediates the transport of cholesterol from peripheral tissues to liver through “reverse cholesterol transport” for metabolism. However there is considerable debate about the mechanisms by which HDL removes excess cholesterol from cells. Two different pathways have been suggested: (i) a docking receptor promots cholesterol translocation, or (ii) a receptor mediates intracellular endosomal pathway termed “retroendocytosis“. Our previous studies have indicated that the removal of cholesterol from aortic endothelial and smooth muscle cells in the presence of HDL is facilitated by plasmalemmal vesicles. in this study, our emphasis is on the ultrastructural changes of cholesterol-loaded endothelial cells after incubated with HDL at different time intervals. Furthermore, the mechanism of HDL-mediated cholesterol efflux by cultured aortic endothelial cells is investigated by immunoelectrophoresis.

Confluent monolayers of endothelial cells were incubated in cholesterol DMEM medium for 48 hr.