Observations that polyribosomes are localized near dendritic spines and beneath synapses has led to a hypothesis of synapse-specific gene expression in which local synthesis would provide a mechanism to influence synaptic structure and strength (l).The active transport of specific mRNAs into dendrites and spines may be a regulated mechanism to target synaptic and regulatory proteins to postsynaptic locations and influence synaptic activity. RNA granules labeled with the vital dye, SYT014, were observed to localize into developing neurites in response to the neurotrophin, NT-3 (2). Neurotrophins have been shown to enhance synaptic activity by a process which requires new protein synthesis (3). The identity and source of newly synthesized proteins which are required to enhance synaptic strength in response to NT-3 are unknown. CaMKIIαRNA localization into dendrites has been shown to occur during long term potentiation (4).

The transmembrane receptor for aspartate, Tar, in Escherichia coliand Salmonellais representative of a large class of receptors that generates chemotaxis responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an Nterminal extracellular ligand-binding domain linked through a transmembrane sequence to a C-terminal signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms soluble ternary complexes with CheA and an adapter protein, CheW. The isolated complex is biochemically active, has a molecular weight of 1,400,000 Daltons, and includes approximately 28 receptor signaling domains for 2 CheA dimers.

Electron microscopy of the complexes indicates well-defined bundles, presumably composed of numerous receptor filaments surrounding a core of CheA dimers and CheW. CheA also interacts with CheY, the response regulator of the bacterial flagellar motor. Immunoelectron microscopy has provided a general picture of the domain organization of the complexes.

Bacterial flagellum is a helical filament by means of which bacteria swim. Each filament rotated by the motor at its base works as a screw that propels the cell, but it is not simply a rigid propeller. The filament is normally in a left-handed supercoiled form and several of them form a bundle when bacteria swim. Upon quick reversal of the motor rotation, which occurs every few seconds, the filament switches into a right-handed supercoil, making the filament bundle fall apart and enabling the cell to tumble for its tactic behavior. The filament is a tubular structure formed by helical assembly of single protein, flagellin, whose molecular mass is 51.5 kDa in the case of Salmonella typhimurium, which we study. The supercoiling of the filament is thought to involve two distinct subunit conformations and/or packing, whose mechanism is interesting in terms of conformational distinctness and adaptability of flagellin.

To understand the mechanisms of self-assembly and polymorphism of the filament, electron cryomicroscopy (EM) and X-ray fiber diffraction have been used to analyze the structures of two straight filaments with distinct helical symmetries.

Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.

A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.

Calmodulin is thought to be the most important calcium sensor in signal transduction. When calcium binds to its 4 calcium binding sites, CaMCa4 becomes a multifunctional activator that regulates a large number of critical cell functions. In the nucleus, CaMCa4 controls gene transcription and other important nuclear processes. Here, using confocal microscopy to measure changes in fluorescence resonance energy transfer (FRET) of an indicator composed of two GFP-variants (CFP and YFP) linked by a calmodulin-binding sequence, we show that free cytosolic CaMCa4 concentration increased rapidly after a cell-wide increase in calcium concentration while the initial free CaMCa4 in the nucleus remained much lower and only equilibrated to the cytosolic CaMCa4 concentration after a delay of 1-2 minutes. Thus, immediately after a cell-wide calcium increase, a significant gradient in free CaMCa4 concentration exists between the cytosol and nucleus.

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. The phenomenon is characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA. Apoptosis is distinctly different from necrosis. Unlike necrosis, which is a passive form of cell death, apoptosis appears to be an active physiological process using a controlled genetic “program” of gene expression. Furthermore, apoptosis does not involve severe tissue damage or inflammation. Membrane integrity is preserved, and there is no loss of cellular contents before phagocytosis. Apoptosis is also distinct from necrosis in that it can be triggered or suppressed by tissue-specific hormones and growth factors.

Apoptosis plays a critical role in tissue homeostasis by counterbalancing mitosis. It is an essential component of many physiological processes, including embryonic development, clonal selection in thymocytes, and in protection against disease.

We employed force-distance measurements, carried out by Atomic Force Microscopy (AFM), to explore the local elastic properties of the membrane-skeleton of human erythrocytes. To calculate the elastic constant of the membrane-skeleton we chose that part of the withdraw region in the force-distance curve which corresponds to the stretching of the membrane-skeleton complex. In order to achieve efficient stretching of the membrane we increased the adhesion of the tip to the membrane by polysine coating of the tip.

Fig. 1 is presents the method of stretching AFM on which our measurements are based, and in Fig.2 one can see the theoretical model of the deformation of a circular plate with fixed boundaries by an external force (AFM probe ) used for recalculating the membrane's bending modulus from the experimental data.

Since we obtained different detachment patterns of tip from the membrane, we set selection criteria for the choice of appropriate curves corresponding to membrane stretching.

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.