|
Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in
cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons.
Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of
multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal
mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons.
We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon
laser scanning fluorescence microscopic experiments on brain slices.
The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM).
From a technical and operational point of view, two developments are important:
Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the
electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated
electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing
of microscopic light paths by the experimentalist.
Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled
thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields.
The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around
the microscope stage.
The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.
|
