Existing optic fibre-bundle based imaging probes have been successfully used to image
biological signals from tissue in direct contact with the probe tip (Hirano et al. 1996).
These fibre-bundle probe systems employed conventional fluorescence microscopy and
thus lacked spatial filtering or a scanned light source, two features used by laser
scanning confocal microscopes (LSCMs) to improve signal quality. Improving the
methods of imaging tissue in its natural state, deep in-vivo and at cellular resolution is
an ever-present goal in biological research. Within this study, a novel (580 μm
diameter) optic fibre-bundle direct-contact imaging probe, employing a LSCM, was
developed to allow for improved imaging of deep biological tissue in-vivo. The new
LSCM/probe system possessed a spatial resolution of 10 μm, and a temporal resolution
of 1 msec. The LSCM/probe system was compared to a previously used direct-contact
probe system that employed a conventional fluorescence microscope. Quantitative and
qualitative data indicated that the LSCM/probe system possessed superior image
contrast and quality. Furthermore, the LSCM/probe system was approximately 16 times
more effective at filtering unwanted contaminating light from regions below the
imaging plane (z-axis). The unique LSCM/probe system was applied to an exploratory
investigation of calcium activity of both glial and neuronal cells within the whisker
portion of the rat primary somatosensory cortex in-vivo. Fluorescence signals of 106
cells were recorded from 12 female Sprague Dawley rats aged between 7-8 weeks.
Fluo-3(AM) fluorophore based calcium fluctuations that coincided with 10 - 14 Hz
sinusoidal stimulation of rat whiskers for 0.5-1 second were observed in 8.5% of cells (9
of 106). Both increases and decreases in calcium levels that coincided with whisker
stimulation were observed. Of the 8.5 % of cells, 2.8% (3 cells) were categorized as
glial and 5.7% (6 cells) as neuronal, based on temporal characteristics of the observed
activity. The remaining cells (97 of 106) displayed sufficient calcium-based intensity
but no fluctuations that coincided with an applied stimulus. This was partially attributed
to electronic noise inherent in the prototype system obscuring potential very weak cell
signals. The results indicate that the novel LSCM/probe system is an advancement over
previously used systems that employed direct-contact imaging probes. The miniature
nature of the probe allows for insertion into soft tissue, like a hypodermic needle, and
provides access to a range of depths with minimal invasiveness. Furthermore, when
combined with selected dyes, the system allows for imaging of numerous forms of
activity at cellular resolution.
| Identifer | oai:union.ndltd.org:ADTP/216590 |
| Date | January 2005 |
| Creators | Dubaj, Vladimir, n/a |
| Publisher | Swinburne University of Technology. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.swin.edu.au/), Copyright Vladimir Dubaj |
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