Del Mar Photonics

Multiphoton Microscopy publications

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Mitochondrial organization and motility probed by two-photon microscopy in cultured mouse brainstem neurons.
Muller M, Mironov SL, Ivannikov MV, Schmidt J, Richter DW.
Zentrum Physiologie und Pathophysiologie, Abteilung Neuro-und Sinnesphysiologie, Georg-August-Universitat Gottingen, Humboldtallee 23, D-37073 Gottingen, Germany. mike@neuro-physiol.med.uni-goettingen.de

Two-photon microscopy of rhodamine 123-labeled mitochondria revealed that mitochondria of neurons cultured from mouse respiratory center form functionally coupled, dynamically organized aggregates such as chains and clusters, while single mitochondria were rarely seen. Mitochondrial chain structures predominate in dendrites, while irregularly shaped mitochondrial clusters are mostly found in the soma. Both types of mitochondrial structures showed chaotic Brownian motions and the mitochondrial chains also revealed well-directed movements. The latter dislocations were arrested upon mitochondrial depolarization or blockade of mitochondrial ATP synthesis. Depolymerization of microtubules by colchicine or nocodazole or inhibition of protein phosphatases by calyculin A disrupted mitochondrial chains and the mitochondria accumulated in the soma. Forskolin and IBMX reversibly blocked directed movements of mitochondria, but did not affect their overall spatial distribution. Thus, protein phosphorylation seems to control both mitochondrial transport and organization. Protein phosphorylation downstream of enhanced cytosolic cAMP levels apparently regulates the transition from motile to non-motile mitochondria, while phosphorylation resulting from inhibition of types 1 and 2A protein phosphatases massively disturbs mitochondrial organization. The complex phosphorylation processes seem to control the close interaction of mitochondria and cytoskeleton which may guarantee that mitochondria are immobilized at energetic hot spots and rearranged in response to changes in local energy demands.

 

Construction and performance of a custom-built two-photon laser scanning system
Michael Müller et al 2003 J. Phys. D: Appl. Phys. 36 1747-1757


Michael Müller1, Jörg Schmidt, Sergeij L Mironov and Diethelm W Richter
Zentrum für Physiologie und Pathophysiologie, Abteilung Neuro- und Sinnesphysiologie, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
E-mail: mike@neuro-physiol.med.uni-goettingen.de

Abstract. Two-photon microscopy, compared with conventional wide-field or laser scanning microscopy, offers several advantages which arise from the near-infrared excitation and the confinement of two-photon excitation to a tiny focal volume. Therefore, there is a considerable interest in optimizing the performance of two-photon laser scanning microscopes (TPLSMs). Despite the existence of several commercially available devices, there are many reasons to start ab initio. Accordingly, we set-up a TPLSM from single components, and in this report construction details of our custom-built system are given. The system was designed for simultaneous optical and electrophysiological recordings and the illumination path was optimized in view of power-delivery and laser pulse-broadening. For this purpose, a solid-state pumped, mode-locked Ti : sapphire laser was directly coupled into a modified upright microscope. The scan unit was built around commercial scanners and a Zeiss scan lens. Fluorescence was detected in non-descanned mode by a photomultiplier tube. Many mechanical parts and the software for system control and image acquisition were developed in our lab and can be readily modified according to special needs of experiments. All components are easily accessible and can be upgraded according to optical requirements. The performance is comparable to available commercial systems, but our TPLSM is superior in many aspects of cost, flexibility and versatility.

 

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