Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Apr 1;100(4):1091–1102. doi: 10.1083/jcb.100.4.1091

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy

PMCID: PMC2113758  PMID: 3980580

Abstract

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18- (3), and the cytoplasmic compartment was stained with fluoresceinyl- dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close- contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close- contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AMBROSE E. J. The movements of fibrocytes. Exp Cell Res. 1961;Suppl 8:54–73. doi: 10.1016/0014-4827(61)90340-8. [DOI] [PubMed] [Google Scholar]
  2. Abercrombie M., Heaysman J. E., Pegrum S. M. The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Exp Cell Res. 1971 Aug;67(2):359–367. doi: 10.1016/0014-4827(71)90420-4. [DOI] [PubMed] [Google Scholar]
  3. Akatov V. S., Kudriavtsev A. A., Lezhnev E. I. Ispol'zovanie éffekta narushennogo polnogo vnutrennego otrazheniia dlia izucheniia adgezii kletok zhivotnykh k steklu. I. Rezul'taty teoreticheskogo analiza. Tsitologiia. 1980 Feb;22(2):230–233. [PubMed] [Google Scholar]
  4. Amato P. A., Unanue E. R., Taylor D. L. Distribution of actin in spreading macrophages: a comparative study on living and fixed cells. J Cell Biol. 1983 Mar;96(3):750–761. doi: 10.1083/jcb.96.3.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arndt-Jovin D. J., Jovin T. M. Analysis and sorting of living cells according to deoxyribonucleic acid content. J Histochem Cytochem. 1977 Jul;25(7):585–589. doi: 10.1177/25.7.70450. [DOI] [PubMed] [Google Scholar]
  6. Axelrod D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol. 1981 Apr;89(1):141–145. doi: 10.1083/jcb.89.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Axelrod D., Thompson N. L., Burghardt T. P. Total internal inflection fluorescent microscopy. J Microsc. 1983 Jan;129(Pt 1):19–28. doi: 10.1111/j.1365-2818.1983.tb04158.x. [DOI] [PubMed] [Google Scholar]
  8. BARER R., ROSS K. F. A., TKACZYK S. Refractometry of living cells. Nature. 1953 Apr 25;171(4356):720–724. doi: 10.1038/171720a0. [DOI] [PubMed] [Google Scholar]
  9. Bereiter-Hahn J., Fox C. H., Thorell B. Quantitative reflection contrast microscopy of living cells. J Cell Biol. 1979 Sep;82(3):767–779. doi: 10.1083/jcb.82.3.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
  11. CURTIS A. S. THE MECHANISM OF ADHESION OF CELLS TO GLASS. A STUDY BY INTERFERENCE REFLECTION MICROSCOPY. J Cell Biol. 1964 Feb;20:199–215. doi: 10.1083/jcb.20.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohen R. L., Muirhead K. A., Gill J. E., Waggoner A. S., Horan P. K. A cyanine dye distinguishes between cycling and non-cycling fibroblasts. Nature. 1981 Apr 16;290(5807):593–595. doi: 10.1038/290593a0. [DOI] [PubMed] [Google Scholar]
  13. Fernandez S. M., Berlin R. D. Cell surface distribution of lectin receptors determined by resonance energy transfer. Nature. 1976 Dec 2;264(5585):411–415. doi: 10.1038/264411a0. [DOI] [PubMed] [Google Scholar]
  14. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  15. Geiger B., Dutton A. H., Tokuyasu K. T., Singer S. J. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol. 1981 Dec;91(3 Pt 1):614–628. doi: 10.1083/jcb.91.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  17. Gingell D. The interpretation of interference-reflection images of spread cells: significant contributions from thin peripheral cytoplasm. J Cell Sci. 1981 Jun;49:237–247. doi: 10.1242/jcs.49.1.237. [DOI] [PubMed] [Google Scholar]
  18. Gingell D., Todd I. Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement. Biophys J. 1979 Jun;26(3):507–526. doi: 10.1016/S0006-3495(79)85268-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heath J. P., Dunn G. A. Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study. J Cell Sci. 1978 Feb;29:197–212. doi: 10.1242/jcs.29.1.197. [DOI] [PubMed] [Google Scholar]
  20. Herman B. A., Fernandez S. M. Dynamics and topographical distribution of surface glycoproteins during myoblast fusion: a resonance energy transfer study. Biochemistry. 1982 Jul 6;21(14):3275–3283. doi: 10.1021/bi00257a005. [DOI] [PubMed] [Google Scholar]
  21. Izzard C. S., Lochner L. R. Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J Cell Sci. 1976 Jun;21(1):129–159. doi: 10.1242/jcs.21.1.129. [DOI] [PubMed] [Google Scholar]
  22. Jacobson K., Elson E., Koppel D., Webb W. International workshop on the application of fluorescence photobleaching techniques to problems in cell biology. Fed Proc. 1983 Jan;42(1):72–79. [PubMed] [Google Scholar]
  23. Johnson L. V., Walsh M. L., Chen L. B. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980 Feb;77(2):990–994. doi: 10.1073/pnas.77.2.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kinnally K. W., Tedeschi H., Maloff B. L. Use of dyes to estimate the electrical potential of the mitochondrial membrane. Biochemistry. 1978 Aug 8;17(16):3419–3428. doi: 10.1021/bi00609a036. [DOI] [PubMed] [Google Scholar]
  25. Kreis T. E., Geiger B., Schlessinger J. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery. Cell. 1982 Jul;29(3):835–845. doi: 10.1016/0092-8674(82)90445-7. [DOI] [PubMed] [Google Scholar]
  26. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McNeil P. L., Murphy R. F., Lanni F., Taylor D. L. A method for incorporating macromolecules into adherent cells. J Cell Biol. 1984 Apr;98(4):1556–1564. doi: 10.1083/jcb.98.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McNeil P. L., Tanasugarn L., Meigs J. B., Taylor D. L. Acidification of phagosomes is initiated before lysosomal enzyme activity is detected. J Cell Biol. 1983 Sep;97(3):692–702. doi: 10.1083/jcb.97.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ross K. F., Gordon R. E. Water in malignant tissue, measured by cell refractometry and nuclear magnetic resonance. J Microsc. 1982 Oct;128(Pt 1):7–21. doi: 10.1111/j.1365-2818.1982.tb00433.x. [DOI] [PubMed] [Google Scholar]
  31. Sims P. J., Waggoner A. S., Wang C. H., Hoffman J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. doi: 10.1021/bi00713a022. [DOI] [PubMed] [Google Scholar]
  32. Tanasugarn L., McNeil P., Reynolds G. T., Taylor D. L. Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH. J Cell Biol. 1984 Feb;98(2):717–724. doi: 10.1083/jcb.98.2.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  35. Thompson N. L., Burghardt T. P., Axelrod D. Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy. Biophys J. 1981 Mar;33(3):435–454. doi: 10.1016/S0006-3495(81)84905-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Waggoner A. S., Grinvald A. Mechanisms of rapid optical changes of potential sensitive dyes. Ann N Y Acad Sci. 1977 Dec 30;303:217–241. [PubMed] [Google Scholar]
  38. Wang Y. L., Taylor D. L. Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin. J Histochem Cytochem. 1980 Nov;28(11):1198–1206. doi: 10.1177/28.11.6107318. [DOI] [PubMed] [Google Scholar]
  39. Wehland J., Osborn M., Weber K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. J Cell Sci. 1979 Jun;37:257–273. doi: 10.1242/jcs.37.1.257. [DOI] [PubMed] [Google Scholar]
  40. Yguerabide J., Foster M. C. Fluorescence spectroscopy of biological membranes. Mol Biol Biochem Biophys. 1981;31:199–269. doi: 10.1007/978-3-642-81537-9_5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES