Interferometric fluorescence cross correlation spectroscopy

Autoři: Ipsita Saha aff001;  Saveez Saffarian aff001
Působiště autorů: Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah, United States of America aff001;  Department of Physics and Astronomy, University of Utah, Salt Lake City, Utah, United States of America aff002;  Department of Biology, University of Utah, Salt Lake City, Utah, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Measuring transport properties like diffusion and directional flow is essential for understanding dynamics within heterogeneous systems including living cells and novel materials. Fluorescent molecules traveling within these inhomogeneous environments under the forces of Brownian motion and flow exhibit fluctuations in their concentration, which are directly linked to the transport properties. We present a method utilizing single photon interference and fluorescence correlation spectroscopy (FCS) to simultaneously measure transport of fluorescent molecules within aqueous samples. Our method, within seconds, measures transport in thousands of homogenous voxels (100 nm)3 and under certain conditions, eliminates photo-physical artifacts associated with blinking of fluorescent molecules. A comprehensive theoretical framework is presented and validated by measuring transport of quantum dots, associated with VSV-G receptor along cellular membranes as well as within viscous gels.

Klíčová slova:

Cameras – Cell membranes – Fluorescence – Mass diffusivity – Wave interference – Quantum dots – Fluorescence spectroscopy – Quantum interference


1. van Oijen AM, Blainey PC, Crampton DJ, Richardson CC, Ellenberger T, Xie XS. Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder. Science. 2003;301(5637):1235–8. doi: 10.1126/science.1084387 12947199

2. Qian H, Elson EL. Fluorescence correlation spectroscopy with high-order and dual-color correlation to probe nonequilibrium steady states. PNAS. 2004;101(9):2828–33. doi: 10.1073/pnas.0305962101 14970342

3. Yu J, Xiao J, Ren X, Lao K, Xie XS. Probing Gene Expression in Live Cells, One Protein Molecule at a Time. Science. 2006;311(5767):1600–3. doi: 10.1126/science.1119623 16543458

4. Wang J, Li C, Wang E. Potential and flux landscapes quantify the stability and robustness of budding yeast cell cycle network. Proceedings of the National Academy of Sciences. 2010;107(18):8195–200.

5. Li C, Wang J. Landscape and flux reveal a new global view and physical quantification of mammalian cell cycle. Proceedings of the National Academy of Sciences. 2014;111(39):14130–5.

6. Gelles J, Schnapp BJ, Sheetz MP. Tracking kinesin-driven movements with nanometre-scale precision. 1988;331(6155):450–3. doi: 10.1038/331450a0 3123999

7. Schmidt C, Horwitz A, Lauffenburger D, Sheetz M. Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated. J Cell Biol. 1993;123(4):977–91. doi: 10.1083/jcb.123.4.977 8227153

8. Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, et al. Robust single-particle tracking in live-cell time-lapse sequences. Nature Methods. 2008;5(8):695–702. doi: 10.1038/nmeth.1237 18641657

9. Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, Danuser G. Two distinct actin networks drive the protrusion of migrating cells. Science. 2004;305(5691):1782–6. doi: 10.1126/science.1100533 15375270

10. Thomann D, Rines DR, Sorger PK, Danuser G. Automatic fluorescent tag detection in 3D with super-resolution: application to the analysis of chromosome movement. Journal of Microscopy-Oxford. 2002;208:49–64.

11. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Meth. 2008;5(2):155–7.

12. Ragan T, Huang HD, So P, Gratton E. 3D particle tracking on a two-photon microscope. Journal of Fluorescence. 2006;16(3):325–36. doi: 10.1007/s10895-005-0040-1 16544202

13. Magde D, Elson EL, Webb WW. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers. 1974;13(1):29–61. doi: 10.1002/bip.1974.360130103 4818131

14. Korlann Y, Dertinger T, Michalet X, Weiss S, Enderlein J. Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy. Opt Express. 2008;16(19):14609–16. doi: 10.1364/oe.16.014609 18794997

15. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring Fast Dynamics in Solutions and Cells with a Laser Scanning Microscope. Biophysical Journal. 2005;89(2):1317–27. doi: 10.1529/biophysj.105.062836 15908582

16. Brown CM, Dalal RB, Hebert B, Digman MA, Horwitz AR, Gratton E. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. Journal of Microscopy. 2008;229(1):78–91.

17. Digman MA, Gratton E. Imaging Barriers to Diffusion by Pair Correlation Functions. Biophysical Journal. 2009;97(2):665–73. doi: 10.1016/j.bpj.2009.04.048 19619481

18. Di Rienzo C, Gratton E, Beltram F, Cardarelli F. Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes. Proceedings of the National Academy of Sciences. 2013;110(30):12307–12.

19. Hinde E, Cardarelli F, Digman MA, Gratton E. In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow. Proceedings of the National Academy of Sciences. 2010;107(38):16560–5.

20. Ries J, Schwille P. Fluorescence correlation spectroscopy. BioEssays. 2012;34(5):361–8. doi: 10.1002/bies.201100111 22415816

21. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proceedings of the National Academy of Sciences. 2009;106(9):3125–30.

22. Sochacki KA, Shtengel G, van Engelenburg SB, Hess HF, Taraska JW. Correlative super-resolution fluorescence and metal-replica transmission electron microscopy. Nature Methods. 2014;11:305. doi: 10.1038/nmeth.2816 24464288

23. Hess ST, Webb WW. Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy. Biophys J. 2002;83(4):2300–17. doi: 10.1016/S0006-3495(02)73990-8 12324447

24. Saffarian S, Elson EL. Statistical Analysis of Fluorescence Correlation Spectroscopy: The Standard Deviation and Bias. Biophysical Journal. 2003;84(3):2030–42. doi: 10.1016/S0006-3495(03)75011-5 12609905

25. Jacobson K, Ishihara A, Inman R. Lateral Diffusion of Proteins in Membranes. Annual Review of Physiology. 1987;49(1):163–75.

26. Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM. Differential Transmission of Actin Motion Within Focal Adhesions. Science. 2007;315(5808):111–5. doi: 10.1126/science.1135085 17204653

27. Qian H, Saffarian S, Elson EL. Concentration fluctuations in a mesoscopic oscillating chemical reaction system. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(16):10376–81. doi: 10.1073/pnas.152007599 12124397

28. Saffarian S, Qian H, Collier I, Elson E, Goldberg G. Powering a burnt bridges Brownian ratchet: A model for an extracellular motor driven by proteolysis of collagen. Physical review E. 2006;73(4):041909.

29. Chattopadhyay K, Saffarian S, Elson EL, Frieden C. Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(22):14171–6. doi: 10.1073/pnas.172524899 12381795

30. Hanson KM, Davis SK, Bardeen CJ. Two-photon standing-wave fluorescence correlation spectroscopy. Opt Lett. 2007;32(15):2121–3. doi: 10.1364/ol.32.002121 17671556

31. Buchholz J, Krieger J, Bruschini C, Burri S, Ardelean A, Charbon E, et al. Widefield High Frame Rate Single-Photon SPAD Imagers for SPIM-FCS. Biophysical Journal. 2018;114(10):2455–64. doi: 10.1016/j.bpj.2018.04.029 29753448

32. Buchholz J, Krieger JW, Mocsár G, Kreith B, Charbon E, Vámosi G, et al. FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series. Opt Express. 2012;20(16):17767–82. doi: 10.1364/OE.20.017767 23038328

33. Doose S, Tsay JM, Pinaud F, Weiss S. Comparison of Photophysical and Colloidal Properties of Biocompatible Semiconductor Nanocrystals Using Fluorescence Correlation Spectroscopy. Analytical Chemistry. 2005;77(7):2235–42. doi: 10.1021/ac050035n 15801758

34. Willert CE, Gharib M. Digital particle image velocimetry. Experiments in Fluids. 1991;10(4):181–93.

35. Grant I. Particle image velocimetry: A review. Proceedings of the Institution of Mechanical Engineers, Part C: Journal of Mechanical Engineering Science. 1997;211(1):55–76.

36. Hart DP. PIV error correction. Experiments in Fluids. 2000;29(1):13–22.

37. Martial Agueh BK, Louis-Philippe Saumier Optimal transport for particle image velocimetry. Communications in Mathematical Sciences. 2015;13(1):269–96.

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2019 Číslo 12
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