Biomedical imaging technique has rapidly progressed in recent years. It is impossible that only the more and more sophisticated microscope that makes this happened. The com-bination of the discovery of green fluorescent protein (GFP), the maturity of transgenic technology, and the more sophisticated microscopy make today’s enormously progress.
Contemporary neurologist and biologist modify the gene of their observing target that cause the organ express the specific function and fluoresce at the same time. At the mean-while, by the help of the confocal microscope, the tomograph of the fluorescing region can be derived. Below, we briefly introduce the the UAS-GAL4 system, labeling tachnique, confocal laser scanning microscopy and bi-photon (excitation) microscopy.
1.1.1 The UAS-GAL4 system
The UAS-GAL4 system [1] is a powerful technique for studying gene expression. The system has two parts: the GAL4 gene, encoding the yeast transcription activator protein GAL4, and the UAS (Upstream Activation Sequence), a short section of the promoter region, to which GAL4 specifically binds to activate gene transcription. The responder, the gene ’X’ to be activated or over expressed, is under the control of UAS sequence. In order to activate transcription, the UAS gene ’X’ carrier need to cross to the GAL4 gene carrier which will express in particular cells, then the resulting progeny will over express gene ’X’. Usually the GAL4 gene is placed under the driver gene, while the UAS controls expression of a target gene. Then, GAL4 is only expressed in cells where the driver gene is usually active. As a consequence, GAL4 should only activate gene transcription where a UAS has been introduced. For example, by fusing a gene encoding the GFP the expression pattern of the driver genes can be determined. An additional strength of the system arises from the ability to target expression of any responder in a variety of spatial and temporal fashions by mating it with distinct GAL4 drivers. So far the UAS-GAL4 system has been
applied on many species to study gene expression in organisms, such as fruit fly Drosopila, African clawed frog Xenopus and zebrafish.
1.1.2 Single-neuron labeling
There are many ways to visualize individual neuron with their native environment. In the 19th century Dr. Golgi had developed the classic Golgi staining method which was later largely extent by Dr. Ramón y Cajal. Both methods are still widely used today. From then on, many methods to observe single neuron have been developed including the ge-netic methods developed in current era. Single-neuron labeling using gege-netic methods can be divided into three categories: (1) using highly specific promoters, or insertions of transgenes in chance, to limit marker expression to a very small subset of isolated neuron;
(2) using site-specific recombination within the same piece of DNA, such as ”flip-out” in Drosophila to limit marker expression in isolated neurons; and (3) using mitotic recombi-nation to couple maker expression cell division. For more details about the Single-neuron labeling methods, please refer to Yuste et al, ch. 13 [2]. Labeling single neuron in their native environment can be used for the following applications:
• To trace the axonal projection and dendritic elaboration patterns of individual neu-rons.
• To study molecular mechanisms of dendritic and axonal development and plasticity with high anatomical resolution.
• To study the physiological functions of identified neurons in brain slice or in vivo.
Here, we would like to claim in advance. Our study object, neuron, in this dissertation, if without specification it means the single-neuron.
1.1.3 Confocal microscopy
Confocal laser scanning microscopy is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to
Figure 1.1: Principle of confocal microscopy. From Wikipedia.
acquire in-focus images from selected depths by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. In a confocal laser scan-ning microscope, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within or on the surface of a specimen. A beam splitter separates off some portions of the light into the detection apparatus, which in fluorescence confocal microscopy will also have a filter that selectively passes the fluorescent wavelengths while blocking the original excitation wavelength (Fig. 1.1). After passing a pinhole, the light intensity is detected by a pho-todetection device, transforming the light signal into an electrical one that is recorded by a computer.
1.1.4 Bi-photon confocal microscopy
Multi-photon fluorescence microscopy has similarities to confocal laser scanning microscopy.
Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect. Unlike confocal microscopes, multi-photon microscopes do not contain pinhole apertures, which give confocal microscopes their optical sectioning quality. The optical sectioning produced by multi-photon microscopes is a result of the point spread function formed where the pulsed laser beams coincide. Two-photon exci-tation microscopy is a fluorescence imaging technique that allows imaging living tissue up to a depth of one millimeter. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and
re-Figure 1.2: Scheme of Two-photon excitation microscopy. From Wikipedia.
duced phototoxicity. The most commonly used fluorophores have excitation spectra in the 400-500 nm range, whereas the laser used to excite the fluorophores lies in the 700-1000 nm (infrared) range. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used. The use of infrared light to excite fluorophores in light-scattering tissue has added benefits.
Longer wavelengths are scattered to a lesser degree than shorter ones, which is a benefit to high-resolution imaging. Figure 1.2 shows a brief scheme of Two-photon microscope.