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PMID: 9788950 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Fluorescence correlation microscopy of cells in the presence of autofluorescence.

Biophysical journal ·Vol. 75 ·No. 5 ·1998-11-00 ·Pages 2547-57

Brock R, Hink MA, Jovin TM

Abstract

Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mol. Biol. 44:847-856), has been implemented for measuring molecular diffusion and association in living cells with explicit consideration of autocorrelations arising from autofluorescence. Autofluorescence excited at 532 nm colocalizes with mitochondria, has flavin-like spectral characteristics, exhibits relaxation times characteristic for the diffusion of high-molecular-weight proteins, and depends on the incubation conditions of the cells. These time- and location-dependent properties preclude the assignment of universal background parameters. The lower limit for detection of microinjected dextran molecules labeled with the carboxymethylindocyanine dye Cy3 was a few thousand molecules per cell, and the diffusion constant of 1.7 x 10(-7) cm2/s agreed well with values measured with other methods. Based on the fluorescence signal per molecule (fpm) and the molecule number derived from autocorrelation analysis, a new method is devised to define intracellular association states. We conclude that FCM is a powerful, noninvasive method for probing molecular interactions in femtoliter volume elements within defined subcellular locations in living cells.

MeSH Terms
3T3 Cells Animals Carbocyanines/metabolism Dextrans/metabolism Diffusion Fluorescent Dyes/metabolism Humans Lasers Mice Microinjections Microscopy, Fluorescence/methods Mitochondria/metabolism Time Factors Tumor Cells, Cultured
Chemicals
Carbocyanines Dextrans Fluorescent Dyes cyanine dye 3
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Brock R
Department of Molecular Biology, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
Hink M A
Jovin T M
References (51)
51 references, click to expand
  1. Fluorescence resonance energy transfer.
    Curr Opin Biotechnol. 1995 Feb;6(1):103-10 PMID: 7534502
  2. Two-photon laser scanning fluorescence microscopy.
    Science. 1990 Apr 6;248(4951):73-6 PMID: 2321027
  3. The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.
    J Physiol. 1996 Oct 1;496 ( Pt 1):111-28 PMID: 8910200
  4. Identification of protein-bound riboflavin in rat hepatocyte plasma membrane as a source of autofluorescence.
    Biochim Biophys Acta. 1989 Jun 6;981(2):303-8 PMID: 2730907
  5. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.
    Biophys J. 1989 Apr;55(4):621-30 PMID: 2720061
  6. Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy.
    Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12811-6 PMID: 8917501
  7. Distribution and lateral mobility of voltage-dependent sodium channels in neurons.
    J Cell Biol. 1988 Jun;106(6):1911-25 PMID: 2454930
  8. On the statistics of fluorescence correlation spectroscopy.
    Biophys Chem. 1990 Oct;38(1-2):49-57 PMID: 2085652
  9. Mitogen-independent activation of Na+/H+ exchange in human epidermoid carcinoma A431 cells: regulation by medium osmolarity.
    J Cell Physiol. 1985 Feb;122(2):178-86 PMID: 3918045
  10. Green fluorescent protein as a marker for gene expression.
    Science. 1994 Feb 11;263(5148):802-5 PMID: 8303295
  11. Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.
    EMBO J. 1984 Aug;3(8):1831-6 PMID: 6207019
  12. Fluorescence correlation spectroscopy. II. An experimental realization.
    Biopolymers. 1974 Jan;13(1):29-61 PMID: 4818131
  13. Dynamics of fluorescence marker concentration as a probe of mobility.
    Biophys J. 1976 Nov;16(11):1315-29 PMID: 974223
  14. Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters.
    Cytometry. 1990;11(3):418-30 PMID: 2340776
  15. Aldolase exists in both the fluid and solid phases of cytoplasm.
    J Cell Biol. 1988 Sep;107(3):981-91 PMID: 2458365
  16. Determination of DNA helical handedness by fluorescence resonance energy transfer.
    J Mol Biol. 1996 Apr 5;257(3):597-617 PMID: 8648627
  17. ER-to-Golgi transport visualized in living cells.
    Nature. 1997 Sep 4;389(6646):81-5 PMID: 9288971
  18. Cellular autofluorescence--is it due to flavins?
    J Histochem Cytochem. 1979 Jan;27(1):44-8 PMID: 438504
  19. Lateral diffusion in planar lipid bilayers.
    Science. 1977 Jan 21;195(4275):305-6 PMID: 831279
  20. Visualization of protein transport along the secretory pathway using green fluorescent protein.
    FEBS Lett. 1995 Aug 7;369(2-3):267-71 PMID: 7649270
  21. Primary structure of the Aequorea victoria green-fluorescent protein.
    Gene. 1992 Feb 15;111(2):229-33 PMID: 1347277
  22. Volume and surface area measurement of viable chondrocytes in situ using geometric modelling of serial confocal sections.
    J Microsc. 1994 Mar;173(Pt 3):245-56 PMID: 8189447
  23. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.
    Curr Biol. 1996 Feb 1;6(2):178-82 PMID: 8673464
  24. Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart.
    Basic Res Cardiol. 1984 Jan-Feb;79(1):49-58 PMID: 6233965
  25. Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.
    Biophys J. 1992 Oct;63(4):1000-10 PMID: 1420920
  26. Characterization of the autofluorescence of rat liver plasma membranes.
    Biochim Biophys Acta. 1988 Apr 22;939(3):441-8 PMID: 3355826
  27. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.
    Biophys J. 1993 Nov;65(5):2021-40 PMID: 8298032
  28. Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy.
    Biochem Biophys Res Commun. 1995 Dec 5;217(1):21-7 PMID: 8526912
  29. Distribution of molecular aggregation by analysis of fluctuation moments.
    Proc Natl Acad Sci U S A. 1990 Jul;87(14):5479-83 PMID: 2371284
  30. Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell.
    Cell Mol Biol (Noisy-le-grand). 1998 Jul;44(5):847-56 PMID: 9764751
  31. Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy.
    Biophys J. 1987 Aug;52(2):257-70 PMID: 3663831
  32. Nerve growth factor receptors on human melanoma cells in culture.
    Proc Natl Acad Sci U S A. 1977 Feb;74(2):565-9 PMID: 265522
  33. Probing the structure of cytoplasm.
    J Cell Biol. 1986 Jun;102(6):2015-22 PMID: 2423529
  34. Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor.
    Biophys Chem. 1996 Jan 16;58(1-2):3-12 PMID: 17023344
  35. Effect of UVA and blue light on porphyrin biosynthesis in epidermal cells.
    Photochem Photobiol. 1993 May;57(5):825-9 PMID: 8337255
  36. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells.
    Proc Natl Acad Sci U S A. 1987 Jul;84(14):4910-3 PMID: 3474634
  37. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.
    Nature. 1997 Aug 28;388(6645):882-7 PMID: 9278050
  38. Probing the mobility of membrane proteins inside the cell.
    Trends Cell Biol. 1996 Aug;6(8):321-4 PMID: 15157441
  39. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.
    Curr Biol. 1997 Mar 1;7(3):176-83 PMID: 9276758
  40. Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry.
    J Histochem Cytochem. 1992 Oct;40(10):1573-8 PMID: 1527376
  41. Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR.
    Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12805-10 PMID: 8917500
  42. Lateral transport of a lipid probe and labeled proteins on a cell membrane.
    Science. 1977 Jan 21;195(4275):307-9 PMID: 556653
  43. Autofluorescence of viable cultured mammalian cells.
    J Histochem Cytochem. 1979 Jan;27(1):36-43 PMID: 220325
  44. Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies.
    Biophys Chem. 1997 Jun 30;66(2-3):211-28 PMID: 9362560
  45. Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation.
    Biophys J. 1996 Jul;71(1):410-20 PMID: 8804624
  46. Fluorescence correlation spectroscopy: diagnostics for sparse molecules.
    Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11753-7 PMID: 9342306
  47. Fluorescence microphotolysis. Diffusion measurements in single cells.
    Naturwissenschaften. 1983 Jun;70(6):294-302 PMID: 6877390
  48. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits.
    J Microsc. 1995 Nov;180(Pt 2):109-16 PMID: 8537958
  49. Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites.
    EMBO J. 1988 Oct;7(10):3045-52 PMID: 3263271
  50. Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment.
    Biophys J. 1995 Feb;68(2):694-701 PMID: 7696520
  51. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.
    Biophys J. 1997 Apr;72(4):1878-86 PMID: 9083691
Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1016/S0006-3495(98)77699-4
Published
1998-11-00
Pages
2547-57
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1299929
Subset
IM
External Links
PubMed source DOI: 10.1016/S0006-3495(98)77699-4 PMC Full Text View journal details
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