1.3 New Scientic Insights Through New Experimental Tools
{Misura ciò che è misurabile, e rendi misurabile ciò che non lo è.|
(Measure what can be measured, and make measurable what can not.)
probably Galileo Galilei (1564 - 1642)
1.3.1 Why olfactory research could prot from fast image acquisition Our understanding of the nervous system, and of nature in general, was always advanced by the introduction of novel experimental tools. The advent of molecular biology in the second half of the 20th century has provided powerful tools that enabled the uncovering of the molecular principles underlying the early stages of olfactory coding (Buck and Axel, 1991; Ressler et al., 1994; Vassar et al., 1994).
Signicant improvements in photochemistry on the other hand provided functional dyes, such as voltage or calcium sensitive uorophores (Orbach et al., 1985; Tsien, 1981), which are today ubiquitously used to monitor neuronal activities in large populations of cells. Thanks to confocal and multi-photon microscopy, it is possible to measure uorescent signals with high specicity in all three spatial dimensions, even deep inside living tissue (Stosiek et al., 2003; Yaksi et al., 2007).
These advances allow the simultaneous observation of the activity of a large num-ber of neurons with a high spatial resolution. They suer however from a low tem-poral resolution, usually at the order of a few Hertz for both conventional confocal and wideeld microscopes. The time scale at which neurons communicate is about a hundred times shorter. The investigation of functional aspects of neuronal systems is thus limited to either electrophysiological recordings with high temporal, but ef-fectively no spatial resolution, or to the observation of neuronal populations using imaging systems with a low temporal resolution. Since the olfactory system is char-acterized by population coding, the investigation of a single or a small number of cells can yield only an incomplete understanding of the olfactory coding strategies.
This motivates the investigation of odor-evoked population responses with a high temporal resolution. First attempts in this direction have been made (Spors and Grinvald, 2002; Spors et al., 2006), though with the drawback of a low spatial
res-15
1. Introduction
olution, which restricted the experiments to the observation of olfactory glomeruli, which are about three times larger than neuronal somata. Since the M/T cells are the neurons projecting to higher brain areas, it is of particular interest to investigate the activity patterns of these cells. This requires a microscopic tool with high spatial and high temporal resolution.
1.3.2 Demands on a high-speed uorescent microscope to study neu-ronal populations
A microscope designed for the investigation of neuronal populations with high tem-poral resolution should meet the following requirements:
1. The spatial resolution has to be sucient to distinguish single somata in all three spatial dimensions. This implies the ability of creating so called `op-tical sections', i.e. to exclude out-of-focus light, as realized by the confocal microscope rst introduced by Marvin Minsky (Minsky,1957, 1988).
2. The temporal resolution should be sucient to capture details of neuronal activity patterns. The time scale at which neurons communicate is in the range of milliseconds, which denes the desired resolution for a fast microscope.
3. Since biological systems show large inter-trial variability, it is required to re-peat a given experiment several times in the same preparation, in order to separate systematic from random events.
These considerations motivated the design of a novel confocal microscope op-timized for fast imaging of biological specimen. By focusing the light into a line instead of a point as in conventional laser scanning microscopes, the scanning is re-duced to one dimension, and in combination with a fast detector high frames rates can be achieved. A number of line scanning realizations have been described (Im et al.,2005;Masters and Thaer,1994;Sheppard and Mao,1988), most often though with applications to non-uorescent samples or non-biological specimens. The new microscope was thus designed to (1) maximize eciency in the emission pathway, (2) optimize the trade-o between spatial resolution and signal-to-noise ratio for
imag-1.3 New Scientic Insights Through New Experimental Tools ing of neuronal populations, and (3) provide the possibility to restrict fast image acquisition to short time intervals of interest.
1.3.3 Visualizing neuronal morphology based on fast 3D image acquisi-tion
While the microscope setup was primarily motivated by questions concerning olfac-tory coding, its application is not limited to fast 2D imaging. By extending the image acquisition to the third dimension, considerably larger fractions of a network could be observed, and the analysis could be extended from somata to neuronal processes. In this way, another important aspect of the study of the brain could be approached, namely the investigation of the structure of neuronal networks. The density of biological tissues generally requires a sparse staining in order to generate images with sucient contrast for the visualization of individual neuron's morphol-ogy. Instead of using the uorescent intensities for the generation of image contrast, it is possible to exploit other parameters, as it is commonly done in functional MRI.
In the case of neuronal networks, the complex and diverse temporal structure of neuronal activity, visualized by a calcium indicator dye, could be exploited as a means of intrinsic contrast. By using fast image acquisition, it is possible to ob-serve a large fraction of a network quasi simultaneously. Based on these recordings, the spatial positions exhibiting a given activity pattern can be detected by means of correlation analysis. This approach should enable the detection of functionally synchronous structures in the volume under observation.
This section of the thesis was a collaboration with Tsai-Wen Chen, Department of Neurophysiology and Cellular Biophysics, University Göttingen.
17
1. Introduction