Setting the Operating Parameters
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The goal of this chapter is to systematically look at each decision made in obtaining images from a sample stained with single or multiple fluorochromes. While in this chapter most of the specific examples of choices made in setting instrument parameters are given by using images collected on a Zeiss LSM 510 META confocal microscope equipped with the following lasers: 405 nm blue diode, argon with peaks at 458, 477, 488 and 514 nm, 543 nm HeNe and 633 nm HeNe lasers, the basic choices made are the same regardless of the manufacturer of the system being used and the available hardware. When operating various confocal instruments, it is often simply a matter of identifying the terminology used, for example, pinhole versus iris or the name of a specific look up table (LUT), or finding where a particular software function is hidden in the multiple menus necessary to operate the system. Once the basic principals are understood, it is possible to quickly learn to operate any confocal microscope and to obtain publication quality images. Figure 9.1 illustrates a generalized flowchart that can be used to start an imaging session and to obtain the initial images. Detailed information on each of the steps involved is presented below.
KeywordsAveraging Bit depth Colocalization Dynamic range Laser Look-up-table Photobleaching Pixels Resolution Signal to noise ratio
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