Two-photon excitation has enabled investigators to image living cells in highly scattering media like the central nervous system (1). We have used a custom-built two-photon microscope to image dendritic spines from living cortical pyramidal neurons. Pyramidal cells form the majority of the neuron in the mammalian cortex and they receive practically all their synaptic contacts through dendritic spines. Dendritic spines are small (<1 μm diameter) appendages that have been practically inaccessible to physiological measurements until the application of two-photon excitation to their study (2). We have concentrated in two questions:

A- Calcium compartmentalization of spines: Mechanisms of calcium decay kinetics.

Dendritic spines can compartmentalize calcium (2). Although the mechanisms of calcium influx into spines have been explored (3), it is unknown what determines the calcium decay kinetics in spines. We investigate calcium dynamics in spines from rat CA1 pyramidal neurons in slices.

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption coefficient of biological specimen in the near infrared (NIR) range. Using a pulsed laser, it is possible to efficiently excite two-photon fluorescence with a high peak power while keeping the average power low to avoid thermal and photochemical damages to the specimen. Currently, mode-locked Ti-sapphire and Cr-Forsterite lasers that generate sub-picosecond pulses are used as the light source for multi-photon fluorescence microscopy. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the application of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes may have impacts on cell physiology and viability in ways much different from low illumination in the linear domain.

Considering its non-linear nature, two-photon excitation may generate very different spectral response in samples when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of samples, so that the two-photon fluorescence microscopic images can be properly interpreted. Fluorescence spectra obtained from bulk samples may not provide useful information for microscopy. For instance, due to the relatively small contribution to the total fluorescence intensity, a small number of fluorescent particles in a generally fluorescing specimen may escape detection when the spectrum of the specimen as a whole is obtained. Under two-photon excitation, the background noise can be greatly reduced due to the naturally limited excitation volume of focused laser beam. In addition, signals resulted from second harmonic generation (SHG) may be mixed with low level broad-band background autofluorescence which is commonly found in biological specimen. Therefore, measuring fluorescence spectrum from a micro-focused volume is essential for the proper interpretation of multi-photon fluorescence images.

Confocal microscopy is an excellent high resolution method to image fluorescently labeled cells. However, the use of confocal microscopy to monitor physiological events, such as membrane potential changes, in living cells is hampered by photobleaching and phototoxicity. To reduce the collateral damage from excitation of fluorescent probes outside the optical slice, Webb and co-workers introduced the use of two-photon excited (TPE) fluorescence in laser scanning microscopy.1 Two-photon absorption depends on the square of the incident light intensity; this has the effect of confining excitation to the plane of focus where the photon flux density is greatest. The wavelength of the exciting light is in the near infrared facilitating penetration of thick tissues. Due to these significant advantages this methodology is rapidly gaining popularity as a tool for live cell and tissue imaging.

To further exploit non-linear optical processes in laser scanning microscopy, we have developed surface second harmonic generation (SHG) as a powerful new imaging modality.

Tightly focused beams of ultrashort optical pulses such as produced with a high numerical aperture optic as found in a microscope exhibit an interesting property: a strong third harmonic signal is produced when the axial focal symmetry is broken through a change in material properties. This change can be manifested as either a change in refractive index and/or a change in the nonlinear susceptibility of the specimen. Conversely, third harmonic production is inhibited in those cases when the axial focal symmetry is unbroken. This property can be effectively exploited to produce volumetric images of biological and non-biological specimens. The third harmonic is localized to interfaces within the specimen making it possible to produce optical sections in much the same manner as in confocal, and two-photon fluorescence microscopy. Unlike these techniques however, no exogenous fluorophore is necessary for labeling - the signal is produced by endogenous interfaces.

CCD sensors are able to image a few megapixels within one frame. The image is scanned electronically within the semiconductor chip (instead of mechanical-optical scanning with moving mirrors, Nipkow discs or acousto-optical devices). Through parallelization. the novel concept yields speed at an unprecedented degree for confocal imaging Routine applications requiring high speed and low costs will profit from this principle. Thus, confocal imaging technology will take the same path television technology has taken—from mechanical scanning to the broadest possible application of electronics

In order to go this way, a pinhole matrix is used on the illumination side. It has as many pinholes and the same pitch as the pitch and number of pixels of the CCD (Figure 1). In front of the receiver, a second pinhole matrix with the same pitch and number of pinholes is used All pinholes of both matrices and the pixels of the CCD are in confocal position.

Bioinformatics is the science of developing computer databases and algorithms for the purpose of speeding up and enhancing biological research. This paper will detail the application of a three-dimensional cell boundary detection algorithm to compute the volumes of osmotically dehydrated apple cells in an effort to better understand the effects of this treatment.

Osmotic dehydration is an efficient pre-treatment technique in food processing. However, the changes that occur in the food material at the cellular level have not been thoroughly understood and this has limited the full industrial application of the process. During osmotic dehydration, plant tissues when placed in hypertonic solutions undergo plasmolysis that can be directly observed under light microscopy. The usual phenomenon observed during plasmolysis is the separation of the cytoplasm from the cell wall, due to the removal of water from the protoplast (Frey-Wyssling and Muhlethaler, 1965). Although readily observed under the microscope, however, the quantitative changes that occur during the process are not easily measurable.

Confocal Microscopy is now an essential, and widely used tool for biological research. Particularly valuable is its ability to image thick sections of intact tissue. A compelling need in this field is to improve the imaging depth, as well as quantitative accuracy, and minimize the possibility of missing important structures/phenomena due to attenuation of the imaging signal. Previous methods work by amplifying the signal from deeper parts of the specimen. In these methods, amplification of noise is an unavoidable artifact. We describe a novel method that not only corrects confocal stacks for attenuation without noise amplification, but also enhances the achievable imaging depth. Interestingly, it does not require explicit estimation of the extinction factor, It relies on a synergistic combination of specimen preparation and image reconstruction algorithms.

The key idea is to image the specimen from two angles (Fig. 1), separated by 180° and then to reconstruct the complete thick image using computational methods.

Fluorescent probes are commonly used in biological fluorescence microscopy for tracking specific structures and sub-cellular compartments, and for indicating cellular ionic conditions. Recent development in multi-photon fluorescence microscopy has greatly expanded the usage of fluorescent probes in biomedical research. Considering its non-linear nature, two-photon excitation may generate very different fluorescence spectral response in the sample when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of various fluorescent probes, so that two-photon fluorescence microscopy may be operated effectively and the images properly interpreted. This report represents the first installment of a continued effort in characterizing the multi-photon fluorescence spectra of commonly used bio-probes.

Two-photon fluorescence spectra excited with near infrared at 780nm were obtained with a SpectraPro-500 spectrophotometer (Acton Research) equipped with a TE-cooled PMT and coupled to a Spectra-Physics Tsunami Ti-sapphire laser pumped by a Coherent Verdi solid-state laser operated at 85MHz, l00fs pulse.

Morphological studies in spider and insect groups (Phylum Arthropoda) have historically relied upon traditional imaging techniques such as light microscopy (LM) and scanning electron microscopy (SEM). It has often been difficult, however, to examine some structural details of certain specimens due to limitations of the techniques. Light microscopy does not always provide adequate depth of focus for visualizing the true three-dimensional structure of a specimen, and out-of-focus light in the image can obscure detail. SEM is not applicable if the specimen is too delicate to undergo critical point drying. Additionally, SEM is a surface technique and does not provide any information about internal structure.

If a specimen is transparent to light, information about the morphology of internal structures, and connections between structures, can easily be obtained using confocal microscopy without the problems associated with traditional LM. In this study, we used confocal laser scanning microscopy (CLSM) to study the morphology of chitinous arthropod structures by taking advantage of their autofluorescent properties.

Automated, large-scale quantitative morphologic analysis of extended three-dimensional (3-D) branched structures such as neurons and vasculature is of broad interest to biomedicine, especially efforts such as the Human Brain Project, and angiogenesis. The present work has resulted in a key enabling technology for such studies - rapid, accurate fully-automatic 3-D tracing of such structures from confocal image stacks. The robustness and efficiency of the proposed method makes it attractive for large-scale applications such as high-throughput assays in the pharmaceutical industry, and initiatives such as the Human Brain Project. Also of interest are attempts to simulate computationally the electrochemical behavior of large collections of neurons for which actual, rather than simulated, neuro-anatomical data, would be valuable. Finally, of long-term interest are emerging studies of the development and growth of live neurons observed over time, for which the present method can provide a powerful morphometric tool.

Near-field scanning optical microscopy (NSOM) is an emerging optical technique capable of probing samples at the nanometric level. With the NSOM technique, high spatial resolution is achieved by scanning a small light source (or collector) close to a sample surface. The light source is usually formed with special fiber optic probes that funnel light down to an aperture that is smaller than the optical wavelength. By positioning the aperture close to a sample, the emerging radiation is forced to interact with the sample before diffracting out. Therefore, the spatial resolution in NSOM is only limited by the size of the aperture and its proximity to the sample, and not the wavelength of the light as in conventional optical microscopy.

Recently, we have been using the single molecule detection limits combined with the unique nature of the electric fields present near the NSOM tip aperture to probe molecular level structure in model lipid membranes.

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.

Fluorescence Resonance Energy Transfer (FRET) is a process by which a fluorophore (the donor) in the excited state transfers its energy to a neighboring fluorophore (the acceptor) non-radiatively through dipole-dipole interactions. Since the efficiency of energy transfer varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores, for FRET to occur the distance between the two molecules cannot exceed 10 to 100 angstroms (1 to l0nm). The combination of FRET and optical microscopy allows examination and quantitation of dynamic molecular interactions between cellular constituents at resolutions beyond the Abbe diffraction limit of light microscopy. Through the microscope one may detect FRET by an overall decrease in fluorescence emission of the donor with a concomitant increase in fluorescence emission of the acceptor.

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. In humans, ∼100,000 cells die every second by apoptosis and are replaced by new cells of the same number. Apoptosis was originally described by Kerr, Wyllie, and Currie in 1972 as an event characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA (1). Apoptotic death is a tightly controlled process, and the cellular pathways that regulate apoptosis have been well conserved in multicellular organisms from simple to complex animals.

One of the most reproducible inducers of apoptosis is mild oxidative stress. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. Several incompletely reduced forms of oxygen are highly reactive and quite cytotoxic.

The typical aging processes can be characterized by a gradual alteration and essential breakdown of the functional systems of the body. Numerous factors play important roles in the alterations of these pathways from biochemical individuality and genetic predisposition to environmental insults and deficiency states.1 Clinical research, as but one example, has clearly documented the role of free radicals (ROS) or oxidative injury in disease and aging affecting the body's basic cellular structures.

The objective of functional or health assessments is to be able to detect in vivo stresses and imbalances in the biological systems, while the patient is asymptomatic, so that early therapeutic intervention can resolve the problems prior to disease onset or accelerated aging. The ability to assess risk factors and treat the sub-clinical metabolic toxicities, deficiency states, hormonal imbalances, oxidative injury (ROS), immunodeficiencies, enzyme down regulations, antioxidant status, metal toxicities, cardiovascular stresses, organ reserves/stresses and detox systems in a low-complexity, cost-effective office procedure is true preventive medicine.

Green fluorescent protein (GFP) and its variants have become powerful tools with which to study the dynamics of many fundamental processes in living cells. I will first discuss the advantages of GFP and important parameters that one must consider in performing live-cell microscopy experiments including how to keep cells happy on the microscope stage. I will then go on to discuss work from our laboratory using multiple microscopic approaches to address questions related to gene expression.

Many of the basic processes that are essential for normal cell growth and division are regulated in the nucleus, ie. DNA replication, transcription, RNA processing and transport. The organization of these functions in the nucleus must be highly controlled in order to enable cells and/or the entire organism to function properly. However, relatively little has been known about the relationship of nuclear organization to gene expression.

Autofluorescence , also known as adventitious fluorescence or background fluorescence, ofter poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can swamp the desired signal. Particularly difficult samples to image include many pathology specimen that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most (green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels which can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal.However, in many cases, these approaches are either infeasible or ineffective.

Because the genes in all somatic cells of an individual are identical, it is the differential expression of the proteins encoded by particular genes that determines a cell's phenotype. There is now increasing interest in relating the simultaneous expression patterns of multiple proteins to complex biological processes. The goal is to develop ‘expression profiles’ of the proteins that are active within a cell or tissue during a particular condition. The ultimate control of functional protein expression is quite complex and may involve transcriptional, combinatorial, and postranslational mechanisms, as well as differences in protein degradation rates that vary under different conditions. Although mRNA levels do not always predict functional protein levels, they are a useful first step in determining the expression profile.

The evolution of three key technologies offers the promise of obtaining quantitative analysis of gene expression from restricted amounts of nervous system tissue. Laser capture microdissection can accomplish harvesting of identified cell populations from histologically processed tissue.

Laser capture microdissection (LCM) is a technique that provides homogenous cell populations for molecular and light microscopic analysis. During viewing by a standard wide-field microscope, a specific cell is selected. Heat from a near-infrared laser melts an ethylene vinyl acetate (EVA) transparent film which bonds to the individual selected cell. Several thousand cells can be selected and captured using this method. A homogeneous subpopulation of cells may be collected, one at a time, by histologic characteristics and/or histochemical staining from frozen sections, deparaffinized tissue, cell cultures or a blood smear.

Previously, this technique has primarily been used to capture cells for DNA or RNA analysis. This study was undertaken to investigate the possibility of capturing a subpopulation of cultured cells in order to study their ultrastructure with the transmission electron microscope (TEM). We report here that cultured cells can be processed, captured and embedded for electron microscopy, in such a manner as to maintain ultrastructure.