The human erythrocyte membrane comprises many different biological macromolecules arranged into a cohesive two dimensional structure. It has long been a model system for studying cell membrane component interactions and their related underlying cell biology. Erythrocyte membrane rigidity emanates from its hexagonal semi-solid cytoskeletal network of spectrin polymers joined at junctional complexes by globular proteins. The network supports a 2D fluid double layer of lipid in which is dissolved a large array of receptor proteins some of which completely span the lipid bilayer and link to the underlying cytoskeletal network.

To study membrane component - component interactions, we have used the technique of fluorescence imaged microdeformation (1). This technique combines fluorescence labeling of specific membrane components, single cell microdeformation and state-of-the-art image collection and analysis to map the distribution of labeled components on the surface of the cell.

Primary cultures of mouse cortical astrocytes were used to investigate the changes in intracellular Na+ concentration ([Na+]i) following exposure to the excitatory neurotransmitter glutamate. The fluorescent probe sodium-binding benzofuran isophthalate (SBFI) was used to measure [Na+]i by epifluorescence microscopy imaging. Detection of the low light level fluorescent signal was accomplished using a Gen III+ intensified CCD camera. Fluorescence excitation ratio images of dye-loaded cells were obtained after sequential illumination at 340nnm and 380nm, with an emission observed at >520nm. Ratio images were proportional in intensity to [Na+]i. In situ calibration of the fluorescent signals was obtained for each experiment and each cell under study by equilibrating [Na+]i with external Na+ after treatment with the cation ionophores monensin and gramicidin, and with ouabain, a specific inhibitor of the Na+/K+ ATPase. Cells on glass coverslips were perfused at 35°C in a closed microscope chamber using solutions buffered with 25 mM bicarbonate/5%CO2.

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume. This inherent localization minimizes photobleaching and photodamage - the ultimate limiting factors in fluorescence microscopy of living cells. One of the most powerful applications of two-photon excitation microscopy is imaging from the naturally occurring reduced pyridine nucleotides (NAD(P)H). NAD(P)H is a useful indicator of cellular metabolism, but it is not a “good“ fluorophore (it has a small absorption cross-section and a low quantum yield). Two-photon excitation of NAD(P)H yields minimal photodamage, thus allowing time-resolved threedimensional metabolic mapping of cellular redox state. We have used two-photon excitation microscopy to examine glucose metabolism in pancreatic and muscle cells. As glucose is metabolized by these cells, intermediate metabolism results in an increase in the reduced-tooxidized NAD(P)H/NAD(P)+ ratio, and a concomitant increase in autofluorescence.

Plants sense the light environment using pigment-protein complexes that discriminate light color, intensity, duration and direction. The most well-studied of these photoreceptors are the phytochromes, a family of soluble biliproteins found in plants, algae and cyanobacteria. Owing to the linear tetrapyrrole pigment phytochromobilin (PΦB) or phycocyanobilin (PCB) that is covalently linked to a large polypeptide via a thioether linkage, phytochromes perceive differences in the quality and quantity of light via their ability to photointerconvert between red (λmax660 nm) and far-red (λmax730 nm) light absorbing forms. Due to an efficient Z,E photoisomerization of the double bond between the C and D-ring pyrroles, phytochromes are nonfluorescent proteins with fluorescent quantum yields less than 10“3 at room temperature (Figure 1).

Phytochrome genes have been cloned from a wide variety of photosynthetic organisms.

We have studied the dynamics of certain key processes near the plasma membrane inside two types of chemically-triggerable living cells using total internal reflection fluorescence microscopy (TIRFM). In TIRFM, a laser beam is incident upon the cell/glass-substrate interface from the glass side at an angle greater than the critical angle for total internal reflection. This creates an exponentially decaying evanescent field in the cell medium (with a characteristic depth of > 100 nm) capable of exciting fluorescence selectively from the membrane-proximal regions at cell/substrate contacts. Various ways of setting up the optics for such a system are discussed, involving the use of either prisms or very high aperture objectives.

In one application of TIRFM, the motion of adrenalin-containing secretory granules in the immediate submembrane region of chromaffin cells is examined before and after chemical stimulation that causes the granules to release their contents to the cell exterior.

We have developed a method of numerical holography which allows simultaneous intensity and quantitative phase contrast imaging. Measurement of optical path length provides precise information about the cell morphology. As presented in Fig. 1, the experimental set-up for the recording of digital holograms is basically a Mach-Zender interferometer. At the exit of the interferometer, a CCD camera records a hologram which is made by the interference between a reference wave R and the object wave O that is transmitted by the biological sample. In order to obtain a plane wave as reference, a beam expander (B.E.) including a pinhole is introduced in the reference arm and enlarges the beam diameter to about 2cm. A similar system which produces a smaller beam diameter (≈5mm) is inserted in the objet arm. To perform holography in the off-axis geometry, the mirror in the reference arm is oriented in such a way that the reference wave reaches the CCD camera with a small incidence angle.

We explored the ability of a novel method of nano-scale movement detection to analyze the effects of chemical crosslinking and thiol reduction on the fluctuations of the human erythrocyte membrane. The method employs line images of light intensity distribution across the red blood cell, obtained by laser scanning phase contrast microscope. The essential feature of these images is a noiseless region corresponding to edge scene. This enables- one to carry out very accurate fitting procedure between the theoretical intensity distribution and the experimental one. One of the free parameters of fitting is the edge position. Measuring line scan intensity distributions with a certain sampling rate and defining the edge positions in consequent moments of time, yields the time-dependent displacement of cell's edge with up to nanometer scale precision.

The significant feature of the suggested procedure is that it implicitly takes into account the halo effect.

Optical microscopy has been at the center of biological research, and neuroscience in particular, for a long time. For example, over one hundred years ago advances in microscopical techniques directly led to the discovery that neurons are in fact cells (the so-called ‘neuron doctrine’), and subsequently to the description of the high-resolution structure of the nervous system. More recently, the development of fluorescent indicators of cellular function, including Ca2+, Na+, and pH indicators, has made it possible to image not only the structure of neurons but also aspects of their function. The most important of these has been the measurement of intracellular free calcium concentration, [Ca2+]. Calcium is an important messenger molecule because it couples electrical excitation to biochemical signaling cascades. In addition, in neurons [Ca2+] is also a good indicator of local electrical excitation and thus [Ca2+] imaging allows the measurement of cellular excitability in subcellular compartments that are inaccessible to other methods.

A major challenge for fluorescence imaging of living cells is maintaining viability during and following prolonged exposure to excitation illumination, especially when imaging over hours or even days, as when studying mammalian embryonic development. The use of specific fluorescently labeled components in living embryos promises to reveal the roles of organelles and molecules in a native and reproducible context. However, to gain a thorough understanding of dynamic biological systems, events of interest must be recorded as they occur, while limiting perturbations caused by the observation technique. Therefore, establishing long-term fluorescence imaging methods that maintain viability is critical for advancing our understanding of cell and developmental biology.

One promising technique for imaging living cells is two photon laser scanning microscopy (TPLSM). The lower energy per photon and the restriction of fluorophore excitation to the imaged focal plane should reduce the total photodamage to thick specimens when compared to conventional laser scanning confocal microscopy (LSCM).

One current limitation of two-photon technology is the lack of optimized fluorochromes for Two-Photon Laser Scanning Microscopy (TPLSM). Recently, Albota et al. (1998) have reported the design and synthesis of a number of bis(styryl)benzene derivatives that have large fluorescence quantum yields and greater two-photon absoption cross sections than any of fluorochromes that are currently used for TPLSM. As a first step in understanding how such molecules could be used in biological experiments, we have tagged a donor-πdonor molecule with biotin. We have successfully used this dye to detect quail nuclei in 2- 4 day old fixed embryos that have been reacted with QCPN, a quail specific monoclonal antibody. We have observed that this molecule gives a very intense signal, among the most intense signals we have been able to measure in our immunoassay. In particular, the brightness was about a factor of four higher than Rhodamine B-labelled streptavidin (containing 3.3 moles of dye per streptavidin tetramer).

Multiphoton microscopy allows us to follow specific cellular structures (ie. nuclei, mitochondria, and the plasma membrane) in a high resolution three dimensional and time resolved fashion. We have used multiphoton confocal microscopy to evaluate the cellular effects of potential cancer therapeutics. Cancer cells were plated onto coverslips and labelled with either the vital DNA dye Hoechst to image the nuclei and chromosomes or the lipid dye DiA to label the plasma membrane. Both of these dyes can be imaged with several sections without affecting cell viability as assessed by cell motility and the ability to undergo mitosis and cytokinesis.

We have used this technology to show that one compound (DDE 131) rapidly induces in cancer cell lines the hallmarks of apoptosis; DNA hypercondensation, nuclear fragmentation and rapid membrane blebbing. In a leukemic cell line these changes take place within 20 minutes of treatment.

The determination of fluorescence lifetime requires only relative measurements of intensity and so is especially useful for biomedical samples in which the heterogeneous nature of tissue and autofluorescence cause significant problems. Since fluorescence lifetime is dependent upon both radiative and non-radiative decay rates, it may be used to distinguish between different fluorophore molecules (with different radiative decay rates) and to monitor local environmental perturbations that affect the non-radiative decay rate. Fluorescence lifetime probes have been demonstrated for many biologically significant analytes including [O2], [Ca2+] and pH. Fluorescence lifetime imaging (FLIM) can be applied to almost any optical imaging modality, including microscopy and potentially to non-invasive optical biopsy. Fluorescence lifetime data may be acquired in the frequency or time domain. The recent development of user-friendly and relatively portable ultrafast laser technology and the availability of ultrafast gated optical image intensifiers (GOI’s) enable the development of potentially inexpensive time domain FLIM instruments that may be deployed outside specialist laser laboratories.

Several modalities of microscopic analysis have been employed in the investigation of development in the embryos of the sea urchins Strongylocentrotus purpuratusand Lytechinusvariegatus. These include scanning electron microscopy, confocal optical microscopy and multiphoton laser scanning microscopy. These techniques all provide information on embryonic morphogenesis in three-dimensions and may be of use in the analysis of other developing organisms.

Scanning Electron Microscopy. Scanning electron microscopy bridges the gap between light and transmission electron microscopy. Operation in the secondary electron image mode with stereo-pair images combines great depth of field in focus from 10-50,000x with true 3D images of topographical features from whole embryos to macromolecules. The SEM's backscatter electron imaging mode allows one to see and map spatial geographies of colloidal gold labeled probes (e.g. antibodies, lectins) that bind to exposed surfaces of embryos, cells and extracellular matrices.

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. To investigate the effects of spaceflight on calcium-dependent secretion and cytoskeletal formation in a less complex system we utilized sea urchin eggs and embryos which were fertilized and cultured under spaceflight conditions during the STS-77 shuttle mission. Sea urchin eggs were fertilized and cultured in the newly developed aquatic research facility (ARF) which allowed culture of eggs and embryos in microgravity and in a 1g centrifuge in space. This allowed analysis of the comparison of microgravity and 1g spaceflight treatments with samples cultured on ground. Eggs and embryos were maintained in Standard Container Assemblies (SCAs) with identical sets prepared for culture in microgravity, and at 1g in the middeck compartment of the shuttle Endeavor, as well as for ground observations at the Kennedy Space Center.

A combination of microscopical methods (TEM, SEM, freeze fracture, confocal, immunoEM) are utilized to demonstrate functional interactions between somatic cells and the germline in the model organism, C. elegans.A variety of signaling mechanisms are required to coordinate activity of maturing oocytes and the somatic sheath of the gonad. Communication via gap junctions allows direct coordination of sheath cell motility and oocyte development. Gap junctions link adjoining sheath cells, and link the sheath to the most mature oocyte. Yolk protein produced in the intestine passes from the pseudocoelom through the basal lamina of the proximal gonad and then through fenestrations (sheath pores) in the gonad sheath before being endocytosed into the most mature oocytes. Myoepithelial specializations anchored by hemi-adherens junctions in the proximal sheath act to squeeze the primary oocyte out of the gonad to the spermatheca for fertilization.

A persistent problem in elucidating the anatomy of the peripheral nervous system has been an inability to stain both myelinated and unmyelinated nerve fibers. To overcome this problem, our laboratory developed two workmg protocols for reliably and differentially labeling and staining the peripheral nervous system and combined them with an enzyme clearing and staining procedure for the simultaneous visualization of bone and cartilage.

One protocol uses anti-acetylated α-tubulin immunohistochemistry to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature and a second uses anterograde and retrograde transport of selectively applied 3000 molecular weight (MW) biotin dextran amines and/or biocytin to identify specific afferent and efferent projections and their cell bodies. Both procedures can be combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations.

Because of their great size, availability, and capacity to develop autonomously in culture, amphibian eggs have become a productive system for a wide variety of experimental studies of animal cells. Their use has advanced our understanding of fundamental molecular and cellular processes such as transcription and translation, regulation of the cell cycle, and the function of the cytoskeleton. The amphibian egg, particularly that of the frog Xenopus laevis,has further served as an excellent system for studying complex developmental phenomena, including the roles of growth factors, signal transduction, and transcription regulation in cell determination, morphogenesis, and tissue differentiation.

Despite these advantages, the frog egg's great size and opacity have made it a challenging subject for microscopy. Thus, many problems in early development, such as how maternal cytoplasmic determinants become localized during cell division, have resisted traditional optical approaches.

The early vertebrate embryo develops a characteristic tube-within-a-tube body plan. This plan is realized through a series of cell movements and cell-cell interactions that collectively result in tissue shaping and the formation of the three-dimensional body plan. Tissue shaping is a highly choreographed process that is under the control of the organizer--a specialized region of the embryo that is both sufficient and required for formation of the body plan. Recent technical advances have greatly increased our understanding of the role of the organizer in vertebrate embryogenesis. Such advances include the use of new cellular, molecular, genetic, and embryological approaches.

A hallmark of embryogenesis is its dynamic nature. Classically, embryos were studied in three major ways. 1) With morphological/descriptive analysis, initially involving histological procedures (stained whole mounts and serial sections cut in the three cardinal axes) and more recently electron microscopy.

The embryonic lens contains two populations of cells, lens epithelial cells and lens fiber cells. As the growth of the human lens is linear from the age of 10 to 90, lens epithelial cells divide and differentiate into fiber cells at a uniform rate during adulthood. Any defects that arise during fiber formation will remain in the lens. We postulate that regulation of early lens growth is critical for the onset of cataracts that usually occur later in life. Slowing the growth of the lens could therefore provide a strategy to suppress the formation of cataracts. However, the factor(s) that control lens growth in vivo have not been identified.

We have identified 18 receptor and cytoplasmic kinases present in lens epithelial cells. Using these as potential targets for factors controlling growth, we tested corresponding ligands in a lens epithelial cell proliferation assay. Platelet-derived growth factor (PDGF) proved to promote a growth activity similar to, but distinct from, that of embryo serum.

Over the past several years the central roles of several different growth factors in the normal development of the embryonic heart have been identified through a variety of techniques involving microscopy. Initially, most studies consisted of descriptions of gross changes in cardiac morphology associated with naturally occurring mutations that affected growth factor function. More recently the development of specific probes for growth factor receptors that can be used in confocal microscopy have aided in the identification of changes in the temporal and spatial distributions of receptors at various stages of development. The correlation of these changes with developmental events such as valve formation and trabeculation in the heart, in conjunction with biochemical studies and blocking agents for the growth factors have significantly increased our understanding of growth factor function in cardiac development.