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.