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Optimal Conditions for Live Cell Microscopy and Raster Image Correlation Spectroscopy

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Reviews in Fluorescence 2010

Part of the book series: Reviews in Fluorescence ((RFLU,volume 2010))

Abstract

Live cell microscopy is now commonplace across all fields of the life sciences, as well as, many of the physical sciences. In order to properly study physiological processing within living cells, tissues, or organisms it is crucial that viability of the sample takes the forefront as the most important aspect of the experiments. If samples are subject to high levels of light, phototoxicity can alter the very physiological processes under investigation. In order to minimize damage to the sample it is crucial to have as sensitive a microscope platform as possible so that light impact on the sample will be minimized. In order to minimize this impact, many aspects have to be kept in mind to maintain the sample and protect it from phototoxicity such as (1) keeping the cells in a favorable environment; (2) using transmitted light techniques when possible and carefully selecting fluorescent dyes; (3) using low light densities of optimal wavelengths to image; (4) optimizing the light path for maximal efficiency; and (5) using sensitive detectors. These aspects are discussed in detail with suggestions how to maximize your sample viability while performing live cell microscopy. Many researchers want to measure submicroscopic molecular dynamics in living samples. One novel technique that has been recently developed for this purpose is raster image correlation spectroscopy (RICS). RICS was developed to measure molecular dynamics, concentrations, and intermolecular interactions. It has the advantage over other dynamic fluorescence measurements in that it only requires very low laser intensities to measure molecular dynamics. Measuring dynamics using other techniques often requires the use of a high intensity of laser light to bleach, activate, or photo-switch fluorescent molecules. Therefore, RICS is ideally suited for live cell microscopy. Two color cross-correlation RICS, ccRICS, is even more powerful determining if two proteins are moving together and determining the concentration and dynamics of the protein complex.

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Acknowledgments

Thank you to the lab of Dr. John Silvius for providing the Cos7 cells expressing mEGFP. Thank you to Cory Glowinski, Alison Ostendorf, and Nan Gray for critically reading the manuscript. Thank you to the McGill Imaging Facility for providing the equipment in order to conduct these experiments.

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Authors and Affiliations

  1. MIA Cellavie Inc, Montreal, QC, Canada

    Judith Lacoste

  2. Department of Biology, Cell Imaging and Analysis Network, McGill University, Montreal, QC, Canada

    Judith Lacoste

  3. Department of Physiology, McGill University, Montreal, QC, Canada

    Charles Vining

  4. Goodman Cancer Centre, McGill University, Montreal, QC, Canada

    Dongmei Zuo

  5. McGill University Life Sciences Complex Imaging Facility, 3649 Promenade Sir William Osler, Bellini Building, Rm137, Montreal, QC, Canada, H3G 0B1

    Aleksandrs Spurmanis & Claire M. Brown

Authors
  1. Judith Lacoste
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  2. Charles Vining
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  3. Dongmei Zuo
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  4. Aleksandrs Spurmanis
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  5. Claire M. Brown
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Corresponding author

Correspondence to Claire M. Brown .

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Editors and Affiliations

  1. Institute of Fluorescence, University of Maryland, E. Pratt St. 701, Baltimore, 21202, Maryland, USA

    Chris D. Geddes

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Lacoste, J., Vining, C., Zuo, D., Spurmanis, A., Brown, C.M. (2012). Optimal Conditions for Live Cell Microscopy and Raster Image Correlation Spectroscopy. In: Geddes, C. (eds) Reviews in Fluorescence 2010. Reviews in Fluorescence, vol 2010. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-9828-6_12

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