ABSTRACT
In vivo neuronal and glial calcium activity was monitored using a novel direct-contact probe that was designed for fluorescence detection deep within biological tissue. A small diameter fibre bundle direct-contact probe was employed with a laser scanning confocal microscope to detect evoked neuronal and glial activity in the posteromedial barrel subfield of the rat somatosensory cortex in vivo. Resolution of the probe allowed discrimination of single cells, and calcium dynamics spanning milliseconds to several seconds were observed. Initial results indicate that the probe has useful practical applications in the imaging of individual cells and monitoring rapid calcium fluctuations within their cell body and large processes.
Subject(s)
Calcium/metabolism , Fiber Optic Technology/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Confocal/methods , Neuroglia/metabolism , Neurons/metabolism , Aniline Compounds , Animals , Brain/cytology , Brain/drug effects , Brain/metabolism , Calcimycin/pharmacology , Calcium Signaling/drug effects , Female , Fluorescent Dyes , In Vitro Techniques , Ionophores/pharmacology , Rats , Rats, Sprague-Dawley , XanthenesABSTRACT
A small diameter (600 micro m) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non-focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM-probe combination is an improvement over conventional fluorescence-probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.
