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3385_Ch19 - Chapter 19 Optical Microscopy Studies of[Ca2]i...

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19 © 1999 by CRC Press LLC Chapter Optical Microscopy Studies of [Ca 2+ ] i Signaling Lynda M. Pierini and Frederick R. Maxfield Contents I. Introduction II. Is Optical Microscopy the Right Choice for Your Studies? III. Designing Single-Cell [Ca 2 + ] i Experiments A. Wide-Field or Confocal Microscopy? B. Radiometric or Single-Wavelength Measurements? C. Choosing a [Ca 2+ ] i Indicator D. Dye Loading and Calibration E. Instrumentation IV. Protocols A. Sample Chambers B. Loading Cells With Fura-2/AM C. Instrumentation D. Image Acquisition E. Calibration F. Image Analysis V. Conclusion References
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© 1999 by CRC Press LLC I. Introduction Optical microscopy provides one of the most powerful techniques for studying intracellular second messengers in living cells. In contrast to flow cytometry and spectrofluorometry, optical microscopy can be used to make measurements on individual cells, providing subcellular spatial information in addition to temporal and quantitative information. Unfortunately, there are currently only a modest num- ber of fluorescent indicators of physiologically important intracellular regulators (e.g., Ca 2+ , H + , Na + , and cAMP) that can be used for microscopy studies. 1 Nonethe- less, the advent of even this small group of indicators has been instrumental in broadening our understanding of intracellular signaling. Studies in our laboratory have utilized various fluorescent indicators of intracellular Ca 2+ ([Ca 2+ ] i ) to investigate the role of this ubiquitous second messenger in cell migration. Specifically, we have used optical microscopy to determine the spatial and temporal characteristics of changes in [Ca 2+ ] i in motile polymorphonu- clear leukocytes (neutrophils), 2,3 to correlate these characteristics with motility parameters such as speed and persistence, 4 and to illustrate a possible mechanism for the regulated adhesion necessary to support motility. 5 In this chapter, we will present a practical guide to designing and setting up optical microscopy experiments for studying [Ca 2 + ] i signaling during cell migration. We will discuss available [Ca 2+ ] i indicators, methodological approaches to single-cell measurements, imaging instru- mentation, and quantification using digital image analysis. II. Is Optical Microscopy the Right Choice for Your Studies? Optical microscopy experiments can be expensive and time consuming, so it is worthwhile to consider carefully whether this is the best method to use. Methods such as flow cytometry and spectrofluorometry are relatively easy to use to monitor changes in [Ca 2+ ] i . For certain experiments, flow cytometry has several advantages over fluorometry. First, in flow cytometry, measurements are made on suspended cells, and only cell-associated fluorescence and the fluorescence of a very small volume of buffer contribute to the measurement. For this reason, the need for extensive, time-consuming washing of the sample is eliminated. This feature of the flow cytometer is important for experiments in which cells are loaded with an indicator dye, as for intracellular calcium ([Ca 2+ ] i ) or pH measurements. The contribu-
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