Biomedical subjects

Matthias Fricke

Publications and source records attributed to Matthias Fricke.

4 recordsLinked to original sources

PubMed2006

Application of multiline two-photon microscopy to functional in vivo imaging.

High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motion-sensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode.

Animals↗

PubMed2005

Two-dimensional imaging without scanning by multifocal multiphoton microscopy.

We describe multifocal multiphoton microscopy giving images without laser scanning. A multitude of 8 x 8 laser beams is focused into a sample yielding two-photon excitation in a plane. The focal spots are arranged in a rectangular array with close spacing between individual points (approximately 0.5 microm). The fluorescence emission from the sample is recorded with a CCD camera, but, owing to the close distance between the beams, they can no longer be regarded as individual points but rather as an illumination of the plane that is covered by the array of focal points. The axial sectioning capability is comparable with an ordinary single-beam two-photon microscope. Interference between the beams that could compromise the axial sectioning capability does not occur in our setup owing to small temporal delays between the individual beams. The axial sectioning capability of the setup is discussed in detail by means of the step response in which the foci are scanned axially into a uniformly fluorescent medium.

Equipment Design↗

PubMed2004

The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP.

Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 [Mol. Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group Ia, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg(2+) on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg(2+). Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4.

Amino Acid Sequence↗

PubMed2004

In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons.

We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca(2+) dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca(2+) concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca(2+)-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca(2+) imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca(2+) dynamics between individual neurites.

Animals↗