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Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence

Nature Medicine volume 5, pages 351–355 (1999)Cite this article

Advances in the development of fluorescent probe technology have greatly facilitated the analysis of single cellular function or single-mode microscopy. Existing approaches monitor single probes or follow specific cellular events over time for the purposes of dissecting complex molecular dynamics in living cells1. In addition, fluorescent cellular probes are more frequently used with fixed samples, yielding static information on processes captured only at the time window when fixation of cells or tissues had occurred. Fixation results in protein denaturation, cross-linking and other interactions, with the subsequent loss of dynamic signals that can only be captured in living cells. Techniques that would allow a tandem, coherent approach using multiple probes in living cells would obviously be preferable. An ideal system of compatible probes would allow simultaneous monitoring of various dynamic functions in a manner that was not feasible until recently. Here we describe a coherent system of fluorescent probes for tracking multiple cellular functions in vitro closely spaced within a narrow sampling period. The term 'coherent' applied to this system represents the systematic, coordinated and integral connections between the probes used, their spectral characteristics and the instrumental mode of application to analyze simultaneous target organelles or cellular functions with acquired images stacked on register. Observing different cellular events simultaneously within a narrow time window increases the ability to discern interrelationships of processes affecting cellular organelles from animals and humans. We have studied specific organelle functions in vitro using live liver cells, a useful model system for observing cellular events in real time. By analyzing the sequence of intracellular events closer to their native state, this approach will reveal relevant new insights into normal, pathologic or toxic processes2.

Coherent multiprobe fluorescence probes and cellular functions

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Figure 1: Multimode microscopy.
Figure 2: Photobleaching and probe stability.
Figure 3: Quantitation of multiprobe fluorescence in hepatocytes treated with tacrine.
Figure 4: Multiprobe fluorescence analysis.
Figure 5: Time-lapse images of hepatocytes treated with melittin.

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Authors and Affiliations

  1. Pathology and Experimental Toxicology Department, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, 48105, Michigan, USA

    Douglas R. Plymale, Jeffrey R. Haskins & Felix A. de la Iglesia

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  1. Douglas R. Plymale
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Correspondence to Felix A. de la Iglesia.

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Plymale, D., Haskins, J. & Iglesia, F. Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence. Nat Med 5, 351–355 (1999). https://doi.org/10.1038/6574

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