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Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

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Abstract

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca2+; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca2+ in biological samples, we demonstrate thatintensity-based measurements of Ca2+ with single-wavelength Ca2+ probes such as Calcium Crimson may falsely report the actual Ca2+ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.

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Authors and Affiliations

  1. Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, CB 7090, 232 Taylor Hall, 27599-7090, Chapel Hill, North Carolina

    Brian Herman, Pawel Wodnicki, Seongwook Kwon, Gerald W. Gordon, Nupam Mahajan & Xue Feng Wang

  2. Cell Biology Program, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, CB 7090, 232 Taylor Hall, 27599-7090, Chapel Hill, North Carolina

    Brian Herman

  3. Department of Biology, University of Virginia, P229 Gilmer Hall, 22903, Charlottesville, Virginia

    Ammasi Periasamy

Authors
  1. Brian Herman
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  2. Pawel Wodnicki
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  3. Seongwook Kwon
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  4. Ammasi Periasamy
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  5. Gerald W. Gordon
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  6. Nupam Mahajan
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  7. Xue Feng Wang
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Herman, B., Wodnicki, P., Kwon, S. et al. Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy. J Fluoresc 7, 85–91 (1997). https://doi.org/10.1007/BF02764581

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  • DOI: https://doi.org/10.1007/BF02764581

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