High-throughput microscopy must re-invent the microscope rather than speed up its functions.

Knowledge gained from the revolutions in genomics and proteomics has helped to identify many of the key molecules involved in cellular signalling. Researchers, both in academia and in the pharmaceutical industry, now screen, at a sub-cellular level, where and when these proteins interact. Fluorescence imaging and molecular labelling combine to provide a powerful tool for real-time functional biochemistry with molecular resolution. However, they traditionally have been work-intensive, required trained personnel, and suffered from low through-put due to sample preparation, loading and handling. The need for speeding up microscopy is apparent from the tremendous complexity of cellular signalling pathways, the inherent biological variability, as well as the possibility that the same molecule plays different roles in different sub-cellular compartments. Research institutes and companies have teamed up to develop imaging cytometers of ever-increasing complexity. However, to truly go high-speed, sub-cellular imaging must free itself from the rigid framework of current microscopes.

Two-photon microscopy in brain tissue: parameters influencing the imaging depth.

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Imaging transmitter release. I. Peeking at the steps preceding membrane fusion.

Over the recent year we have witnessed considerable advances in the study of neurotransmitter release. This progress has been severalfold as different techniques have allowed us to characterise many steps along the process of exocytosis, membrane fusion, formation of the fusion pore, and have given insight in the kinetics of release and membrane re-uptake. Patch clamping provided quantitative measurements of the capacitance changes as the membrane of the secretory vesicle is added to the surface of the cell during secretion, and the change in the opposite direction when membrane is retrieved back into the cell during exocytosis. Carbon-fibre microelectrodes have measured electrochemically the release of oxidisable transmitters into the extracellular space. Differential interference contrast microscopy has given us spatially resolved images of the cell surface during exocytosis; real-time images that are suggestive of bubbles breaking the surface of a boiling pot of water. The interest in novel techniques stems from the fact that existing approaches can provide only indirect evidence on the steps preceding membrane fusion. The vesicular dynamics just beneath the plasma membrane are out of the reach of capacitance measurements or amperometric detection. What we have needed is a tool that would allow us to look just below the cell surface. This much-needed tool appears to be evanescent-wave microscopy. This review describes how laser microscopy can be used to study exocytosis at single-vesicle resolution. A companion paper deals with the practical aspects of evanescent-wave imaging.

Imaging transmitter release. II. A practical guide to evanescent-wave imaging.

Total internal reflection of a laser beam at an interface between two media of different refractive index sets up an evanescent wave field in the medium with lower refractive index. This near field decays over a distance of approximately lambda/5, lambda denoting the wavelength of light, and thus provides a convenient means for the confinement of fluorescence excitation to the near-interface region. Evanescent-wave excitation thereby permits, for example, the observation of individual fluorophores at the surface despite the presence of high concentrations in bulk solution. Although evanescent-wave excitation of fluorescence and the related technique of surface-plasmon resonance have a long record in the study of chemical reactions at surfaces, adsorption kinetics or spectroscopy, their potential for biomedical studies is only gradually emerging. Evanescent-wave microscopy provides high-contrast images of the near-membrane region of cells grown on a glass substrate at unprecedented resolution. At present, no commercial equipment is available for evanescent-wave microscopy. This review aims at readers who want to modify their fluorescence microscope to include an evanescent-wave illumination mode. Starting from the point that every objective exceeding a certain numerical aperture is generating evanescent waves, we demonstrate how the optical path can be modified to suppress the far-field excitation, and how one can switch easily between these types of illumination. The phenomena resulting from interactions of evanescent waves with cells are reviewed. The ways in which systematic variations of the angle of incident light can be used to obtain quantitative information on fluorophore distance, distribution and concentration are also discussed.

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence.

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.

Tracking chromaffin granules on their way through the actin cortex.

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.

Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release.

Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles.

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied "footprint" region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site.

Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.

We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) in the whole-cell configuration of the patch clamp technique. We compared the loading time-courses of CG-1 and BS, investigated their intracellular distribution patterns and studied the time course of photobleaching. We determined the apparent dissociation constant of CG-1, both optically and by potentiometric titration. Our findings indicate that: (i) with excitation at 420/488 nm, calibrated fluorescence signals could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellular constituents; and (iii) that calibration requires diffusional equilibration between pipette and the accessible volume of the cell. Spatially resolved recordings of fluorescence excitation spectra revealed elevated fluorescence of CG-1 in the nucleus such that reported [Ca2+]i levels seemed 25% higher compared to cytosolic values. Comparing fluorescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca(2+)-insensitive dye.

The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM).

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the 'visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.