PubMed Health⌕ Search

Biomedical subjects

Martin Oheim

Publications and source records attributed to Martin Oheim.

8 recordsLinked to original sources
PubMed2006

Calcium microdomains in regulated exocytosis.

Katz and co-workers showed that Ca(2+) triggers exocytosis. The existence of sub-micrometer domains of greater than 100 microM [Ca(2+)](i) was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca(2+) 'microdomains' in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca(2+) nano- and microdomains forming around the mouth of voltage-gated Ca(2+) channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca(2+) channels at different central synapses and pointed out the role of Ca(2+) syntillas - localized, store operated Ca(2+) signals - in facilitation and spontaneous release. The coupling between Ca(2+) increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca(2+) comes from different sources, including Ca(2+) influx via the plasma membrane and the mobilization of Ca(2+) from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca(2+) signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca(2+) signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca(2+) sources, leading to a wide spectrum of Ca(2+) signals triggering release.

Action Potentials↗
PubMed2006

Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.

The aim of this article is to review the basic principles of two-photon excitation fluorescence (2PEF) microscopy and to compare the advantages and disadvantages of 2PEF imaging to other microscopy methodologies. 2PEF imaging is a nonlinear approach that generates images of optical sections and that is particularly well suited for deep-tissue and in vivo imaging of live animals. The nonlinear excitation used for 2PEF offers the advantage, too, of being able to generate contrast from second or third harmonic generation as well as coherent anti-Stokes Raman scattering. We also review the recent use of nonlinear excitation to provide image resolution beyond the diffraction limit and discuss the progress in non-scanning (planar) 2PEF microscopy, an approach that holds great potential for large-scale quantitative imaging and plate reading, e.g., in screening applications.

Animals↗
PubMed2006

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing.

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.

Animals↗
PubMed2003

Two-photon imaging of capillary blood flow in olfactory bulb glomeruli.

Analysis of the spatiotemporal coupling between neuronal activity and cerebral blood flow requires the precise measurement of the dynamics of RBC flow in individual capillaries that irrigate activated neurons. Here, we use two-photon microscopy in vivo to image individual RBCs in glomerular capillaries in the rat dorsal olfactory bulb. We find that odor stimulation evokes capillary vascular responses that are odorant- and glomerulus-specific. These responses consist of increases as well as decreases in RBC flow, both resulting from independent changes in RBC velocity or linear density. Finally, measuring RBC flow with micrometer spatial resolution and millisecond temporal resolution, we demonstrate that, in olfactory bulb superficial layers, capillary vascular responses precisely outline regions of synaptic activation.

Animals↗
PubMed2003

Fluorescence imaging with two-photon evanescent wave excitation.

We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.

Animals↗
PubMed2003

Calcium regulates exocytosis at the level of single vesicles.

Ca2+ microdomains that form during the opening of voltage-gated Ca2+ channels have been implicated in regulating the kinetics of hormone and transmitter release. Direct assessment of the interaction between a single Ca2+ microdomain and a single secretory vesicle has been impossible because of technical limitations. Using evanescent field imaging of near-membrane micromolar Ca2+ concentration ([Ca2+]) and fluorescently labeled vesicles, we have observed exocytosis of individual chromaffin dense-core vesicles that was triggered by Ca2+ microdomains. Ca2+ microdomains selectively triggered the release of vesicles that were docked within 300 nm. Not all vesicles exposed to a Ca2+ microdomain were released, indicating that some vesicles are docked but are not ready for release. In addition to its established role as a trigger for release, elevated near-membrane [Ca2+] reduced the distance between docked vesicles and Ca2+ entry sites. Our results suggest a new mechanism for stimulation-dependent facilitation of exocytosis, whereby vesicles are moved closer to Ca2+ entry sites, thereby increasing a Ca2+ microdomain's efficacy to trigger vesicle fusion.

Action Potentials↗
PubMed2002

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 granule's 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.

Animals↗