Abstract
A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.
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- Bright G. R., Fisher G. W., Rogowska J., Taylor D. L. Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J Cell Biol. 1987 Apr;104(4):1019–1033. doi: 10.1083/jcb.104.4.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeBiasio R. L., Wang L. L., Fisher G. W., Taylor D. L. The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts. J Cell Biol. 1988 Dec;107(6 Pt 2):2631–2645. doi: 10.1083/jcb.107.6.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeBiasio R., Bright G. R., Ernst L. A., Waggoner A. S., Taylor D. L. Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells. J Cell Biol. 1987 Oct;105(4):1613–1622. doi: 10.1083/jcb.105.4.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ehrenberg B., Montana V., Wei M. D., Wuskell J. P., Loew L. M. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J. 1988 May;53(5):785–794. doi: 10.1016/S0006-3495(88)83158-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farkas D. L., Wei M. D., Febbroriello P., Carson J. H., Loew L. M. Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J. 1989 Dec;56(6):1053–1069. doi: 10.1016/S0006-3495(89)82754-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fay F. S., Carrington W., Fogarty K. E. Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope. J Microsc. 1989 Feb;153(Pt 2):133–149. [PubMed] [Google Scholar]
- Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
- Loew L. M. Confocal microscopy of potentiometric fluorescent dyes. Methods Cell Biol. 1993;38:195–209. doi: 10.1016/s0091-679x(08)61003-1. [DOI] [PubMed] [Google Scholar]
- Loew L. M., Tuft R. A., Carrington W., Fay F. S. Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria. Biophys J. 1993 Dec;65(6):2396–2407. doi: 10.1016/S0006-3495(93)81318-3. [DOI] [PMC free article] [PubMed] [Google Scholar]