Advertisement

Setting the Operating Parameters

Chapter

Abstract

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.

Keywords

Averaging Bit depth Colocalization Dynamic range Laser Look-up-table Photobleaching Pixels Resolution Signal to noise ratio 

References

  1. Barlow, AL, MacLeod, A., Noppen, S., Sanderson, J., and Guerin, JG. 2010. Colocalization analysis in fluorescence micrographs: Verification of a more accurate calculation of Pearson’s Correlation Coefficient. Microsc Microanal 16(6):710–724.Google Scholar
  2. Boltes, S and Cordelieres, FP. 2006. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232.CrossRefGoogle Scholar
  3. Manders, EMM., Verbeek, FJ., and Aten, JA. 1993. Measurement of co-localization of affects in dual-colour confocal images. J Microscopy 169:375–382.CrossRefGoogle Scholar
  4. North, AJ. 2006. Seeing is believing? A beginners guide to practical pitfalls in image acquisition. J Cell Biol 172:9–18.PubMedCrossRefGoogle Scholar
  5. Pawley, J. 2000. The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques. 28:884–888.Google Scholar
  6. Zinchuk, V., Zinchuk, O., and Okada, T. 2007. Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: Pushing pixels to explore biological phenomena. Acta Histochem Cytochem. 40:101–111.PubMedCrossRefGoogle Scholar
  7. Zucker, RM and Price, OT. 1999. Practical confocal microscopy and the evaluation of system performance. Methods 18:447–  458.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Cell and Developmental Biology, School of MedicineUniversity of South CarolinaColumbiaUSA

Personalised recommendations