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Whole-Object Fluorescence Lifetime Setup for Efficient Non-Imaging Quantitative Intracellular Fluorophore Measurements

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Abstract

In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H2O2. The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.

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Abbreviations

FLT:

fluorescence lifetime

wo-FLT:

Whole-Object Fluorescence Life Time

FI:

Fluorescence intensity

FLIM:

Fluorescence life-time imaging

PMP:

plasma membrane potential

AO:

Acridine Orange

Rh123:

Rhodamine 123

CCCP:

Carbonyl cyanide 3-chlorophenylhydrazone

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Acknowledgements

This study was made possible through the Bequest of Moshe-Shimon and Judith Weisbrodt.

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Authors and Affiliations

  1. The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan, 52900, Israel

    Yaniv Namer & Mordechai Deutsch

  2. Faculty of Engineering and the Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan, 52900, Israel

    Lior Turgeman & Dror Fixler

Authors
  1. Yaniv Namer
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  2. Lior Turgeman
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  3. Mordechai Deutsch
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  4. Dror Fixler
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Corresponding authors

Correspondence to Mordechai Deutsch or Dror Fixler.

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Namer, Y., Turgeman, L., Deutsch, M. et al. Whole-Object Fluorescence Lifetime Setup for Efficient Non-Imaging Quantitative Intracellular Fluorophore Measurements. J Fluoresc 22, 875–882 (2012). https://doi.org/10.1007/s10895-011-1025-x

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  • DOI: https://doi.org/10.1007/s10895-011-1025-x

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