_{>> Microscopy Section <<}

The transformation of theoretical assumptions into true results requires the experimental testing of the theories. To do so, numerous single informations, recipes and "How-to" hints are necessary.

This page shall You supply with some general considerations about information conserving (how to keep results reliable) and basic infos about microscopy in practice (how to start making specimens).

It is not possible, and even not intended, to give a solution for every single approach. The considerations on the topics mentioned below are collected as a help for the beginner. The set is useful to get images from many different cell specimens. Feel free to modify it according to Your particular needs and to the hints You got from your local advisors.

The buttons guide You to further explanations.

Vital staining means depositing colour inside a living cell. This can be done by microinjection (enormous work), hypo-osmotic loading (a little bit woodoo) or ion trap mechanisms. Besides dyes for the plain staining of the cells enterior there are ion selective stains to detect ions like calcium or pH sensitive dyes for detecting protons. Live/dead tests also exist.

Green Fluorescent Protein (GFP) is another way to stain living cells. The GFP from the jelly fish Aequorea victoria is one beneath several fluorescent proteins mostly from Coelenterata. GFP is produced by the cells and can be coupled with several generic proteins to localize cell structures and functions. Gentechnologic equipment is needed.

Membrane stains are a good alternative to cytosolic stains. They work well in living cells and show (oftenly) a low cell toxicity. By their transition from the aequeous surrounding to the lipophilic membranes their fluorescence increases up to 100-fold. Organell movement can be observed. The application is simple as compared to GFP.

Cytosceleton is conveniently stained with Phalloidin, a poison from the fungus Amanitha phalloides. As actin is found in all cells this offers a good way to start in specimen preparation or to counterstain routinuously. Similar, Paclitaxel is used to stain another cytosceleton scaffold, the tubulin fibres.

Immunostaining with antibodies which selectively bind to cell structures make it possible to show the distribution of one cell component beyond thousends. There two drawbacks: You need an appropriate antibody (perhaps You will have make Your own one) and the method works well only on fixed cells. Vital staining works only on cell surfaces.

Image contrast production is the main problem in the observation of culture cells. The accessible resolution depends on the possible image contrast. Besides the application of staining dyes (see above) there are some methods to use the properties of light in producing contrast. The phase and the polarization of light rays can be modified and exploited.

Fidelity here means to keep the information content of an image unmodified and to conserve it for later evaluations. No signal conservation theories are given to You but a set of rules of thumb to prevent You from loosing information during image acquisition and processing. Some simple considerations can save time and motivation.

Resolution power, not magnification, is the limiting factor in light microscopy. The resolution capabilities of a microscope depend not only on the microscopy method in use but also on the specimen preparation and the image acqusition system connected. Besides the spatial resolution temporal and functional resolution exist.