Martin Oheim

The last few milliseconds in the life of a secretory granule

Abstract 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.

Tracking chromaffin granules on their way through the actin cortex.

Abstract Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a ∼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 ∼60u2009nm beneath the plasma membrane moved on random tracks (D(3)≈10−10 cm2 s−1) with several reversals in direction before they approached their docking site at angles larger than 45∘. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100u2009ms. 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.

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

Abstract 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.

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.

Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation

The trajectory of secretory vesicles to their fusion sites at the plasma membrane is expected to give insight into the mechanisms that underlie vesicle transport, maturation and the initiation of membrane fusion. Evanescent-wave (EW) microscopy allows the tracking of fluorescently labeled granules and vesicles prior to fusion with nanometer precision in xy-direction. At the same time, the exponential sensitivity of granular fluorescence to experimental parameters can preclude quantitative estimates of the granules approach to the plasma membrane. Thus, it has remained controversial to which extent axial distance can be obtained from simple intensity measurements. We used the information contained in a stack of images acquired at 80-125 nm penetration depth of the EW field to estimate individual granule diameter and axial distance. A population analysis on 90 granules revealed an average diameter of 305 +/- 47 nm, below the diffraction-limited 352 +/- 31 nm obtained from xy measurements at fixed depth penetration. Stimulation of exocytosis by potassium depolarization resulted in the selective loss of the 18 +/- 5% of granules located closest to the plasma membrane, while a second population of granules located 60 nm deeper within the cytoplasm increased by recruitment of granules previously located at > or = 120 nm depth. These measurements extend and corroborate previous observations at fixed penetration depth of functionally distinct granule populations. Parameters influencing the accuracy of the parameter estimation are evaluated in the appendix.

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.

Multiparameter evanescent-wave imaging in biological fluorescence microscopy

Wide-field optical microscopy of live cells with nearfield precision, tracking the dynamics of subcellular organelles, imaging of single-molecule reactions with millisecond time-resolution, or watching synaptic nerve terminals in action-these are some examples of recent work that triggered the renaissance of evanescent-wave (EW) spectroscopy in biological imaging. In these studies, the goal is to markedly reduce background fluorescence from locations in the sample other than the cell/substrate interface. After a brief reminder of EW generation, we discuss how the confinement of fluorescence excitation highlights cellular structure near the plasma membrane with unprecedented detail. We then illustrate how the intensity distribution and polarization of the EW can be used to study dynamic biological processes that have neither been accessible with optical (confocal or two-photon fluorescence) nor electron microscopy, and take a glimpse of what is to come in EW imaging.

Simple optical configuration for depth-resolved imaging using variable-angle evanescent-wave microscopy

The evanescent wave (EW) elicited by total internal reflection of light provides a means to selectively excite fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epi-illumination microscopy, and can offer a 5-fold enhancement of axial resolution compared to confocal and two- photon microscopy. The decay length of the evanescent field is a function of the refractive indices at the interface, the wavelength of the light, and is modulated by the beam-angle. EW microscopy has been used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution on top of the interface. 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. 3-D reconstruction of image stacks by an inverse Laplace transform results in topographical information with an axial resolution of 10s of nanometers. We have labeled subcelluar storage organelles (vesicles) of approximately equal 300 nm diameter and visualized the trajectories of single vesicles in the footprint region of living neuroendocrine cells, grown on the interface.

Non-linear evanescent-field imaging

Summary form only given. The cell surface is a 2-D membrane compartment relaying most cellular signals. We use multiparameter evanescent-field imaging and nonlinear optical methods to image near-membrane molecular distribution and orientation changes associated with these signalling processes.

Strategies for improving depth-penetration of two-photon imaging in vivo

We investigate tissue and instrument parameters affecting the penetration depth in two-photon microscopy. We show that the temporal redistribution of the same average power into fewer pulses of higher peak energy by means of a regenerative amplifier results in an increase in excitation depth by approximately 2-3 scattering mean free paths. We then measure the excitation scattering mean free path in vitro, using rat brain slices, as a function of the excitation wavelength and tissue age. We find that young-animal tissue (< P18) is two-fold less scattering than adult tissue (P90). We quantify the fall-off of the collected fraction of generated fluorescence in a backward detection geometry, in vivo. At large depths, we observe that the collected fraction scales as the angular acceptance squared (related to the effective field-of-view) of the detection optics. Matching the angular acceptance of the detection optics to that of the objective (63X NA-0.90) results in a factor 3-4 of the collected fluorescence. The collection efficiency can be further increased (10X) by using an objective with large field-of-view and high numerical aperture (20X NA-0.95). These gains translate into approximately 120 micrometers additional depth penetration when working in the rat brain in vivo with a standard Ti:sapphire source.