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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 7 — Jun. 25, 2012
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Using fixed fiduciary markers for stage drift correction

Sang Hak Lee, Murat Baday, Marco Tjioe, Paul D. Simonson, Ruobing Zhang, En Cai, and Paul R. Selvin  »View Author Affiliations


Optics Express, Vol. 20, Issue 11, pp. 12177-12183 (2012)
http://dx.doi.org/10.1364/OE.20.012177


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Abstract

To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 µm x 1 µm x 1 µm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are bio-compatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.

© 2012 OSA

1. Introduction

2. Experiment

To make fiduciary markers with a polymer on the coverslip, we employed the soft lithography method [21

21. D. Qin, Y. N. Xia, and G. M. Whitesides, “Soft lithography for micro- and nanoscale patterning,” Nat. Protoc. 5(3), 491–502 (2010). [CrossRef] [PubMed]

]. First, we made a template of the marker pattern, which is a square (1 μm x 1 μm x 1 μm) or a circular (1 μm diameter and 1 μm height) pillar, on Si wafer. For making mask pattern on Si wafer, we spin coated with PMMA (A4, 950K) at 4000 rpm for 1min and baked at 200 °C for 2 min. The coated Si wafer was then exposed e-beam at 50 kV with 3 nA beam current and developed in MIBK:IPA (1:2, organic solvent mixture, Micro-Chem) for 2 min and rinsed in IPA for 30 sec. 50 nm Cr was then deposited with e-beam evaporator and then etched in STS ICP etcher for 1 min. This Si template was coated with silicon nitride (Si3N4), which allows the template to serve as a non-sticky mold. The Si template was then exposed to (Tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane to make the self-assembled monolayer that helps it to be easily peeled off the PDMS mold (Polydimethylsiloxane, Sylgard 186, Dow Corning). We poured the PDMS onto the Si template to make a mold of the fiduciary markers and then baked it to solidify the PDMS. Then the PDMS mold was then peeled off from the Si template. Next, we molded PDMS wells using UV curable glue (UV curable poly urethane, NOA 61, Thorlab) on a glass coverslip to make fiduciary markers and incubate 30 ~ 40 min with UV light for curing. Alternatively, the fiduciary markers can be made using 3% PMMA (Polymethyl-methacrylate-MW 97000, Sigma-Aldrich 370037) dichloromethane solution instead of UV curable glue.

To track the fiduciary marker we use an IR-LED (850 nm, ELJ-850-629, Roithner LaserTechnik) placed opposite to the objective lens without a condenser (Fig. 1
Fig. 1 The optical configuration to track the fiduciary markers using IR scattering with the Total Internal Reflection Fluorescence.
). We use an inverted microscope, although an upright microscope can also be used. The scattered IR light is completely excluded from visible-light fluorescence arising from common fluorophores by using a simple dichroic in the emission path. IR illumination also avoids autofluorescence that may add to the signal background. The IR illumination is collected by the objective lens (Olympus, 100 X, NA = 1.40) and detected by a simple camera (JAI Ltd., CV-A55 IR E). We note that Total Internal Reflection (TIR) for the IR light is not used. However, simultaneously, the objective lens is often used in TIR mode for collecting the visible fluorescence (Fig. 1). The optical configuration is simply designed to observe the visible-light fluorescence from the biological sample and IR scattering for the fiduciary marker, which is place in the same side of the coverslip as the sample, at the same time (Fig. 1).

TTL pulses synchronize the IR and visible cameras with each other so that the IR and visible light images from both cameras can be correlated with each other. We typically detect single molecules at about 10 Hz. (Commercially-available frame rates are 30 or 60 Hz, with 500 Hz also available.) At this frame rate, the IR camera, which is either operating in analog or digital mode, can follow the frame rate of the visible-light fluorescence. Alternatively, at slower rates, one can always integrate the output of the IR-camera.

3. Results

We also examined the biocompatibility of cells with coverslip containing the fiduciary markers (Fig. 5
Fig. 5 Bright field (left) and fluorescence (right) images of the HEK cells cultured on the fiduciary-marked coverslip with quantum dots (605 nm emission; Invitrogen) labeling. In the bright-field image, there are cultured cells as well as regular patterned dots (represented by arrows) which are the fiduciary markers. The fluorescence image has no autofluorescence, showing clear emission spots of the qdots.
) even though there is a report about the biocompatibility of polyurethane [28

28. K. M. Zia, M. Zuber, I. A. Bhatti, M. Barikani, and M. A. Sheikh, “Evaluation of biocompatibility and mechanical behavior of polyurethane elastomers based on chitin/1,4-butane diol blends,” Int. J. Biol. Macromol. 44(1), 18–22 (2009). [CrossRef] [PubMed]

]. HEK cells were cultured on a fiduciary-marked coverslip, using fibronectin to fix the cells in place. We tested viability of cells through the dye exclusion test using trypan blue and it showed the number of live cells on the fiduciary marked coverslip was comparable to that of normal coverslips. The cells expressed a biotinylated AMPA receptor, which was then labeled with a quantum dot emitting at 605 nm (Invitrogen, Inc.) [29

29. M. Howarth, K. Takao, Y. Hayashi, and A. Y. Ting, “Targeting quantum dots to surface proteins in living cells with biotin ligase,” Proc. Natl. Acad. Sci. U.S.A. 102(21), 7583–7588 (2005). [CrossRef] [PubMed]

,30

30. M. Howarth, W. H. Liu, S. Puthenveetil, Y. Zheng, L. F. Marshall, M. M. Schmidt, K. D. Wittrup, M. G. Bawendi, and A. Y. Ting, “Monovalent, reduced-size quantum dots for imaging receptors on living cells,” Nat. Methods 5(5), 397–399 (2008). [CrossRef] [PubMed]

]. The bright-field field image (Fig. 5, left), shows the fiduciary markers. The fluorescence image (Fig. 5, right) clearly shows the q-dot emission with no background autofluorescence coming from the fiduciary markers. Thus, we think this technique is useful for in vivo studies since these studies generally are performed over long-time periods and the fiduciary markers do not have any adverse effect on cells and are very stable over time.

Finally, we have also tested the fiduciary markers using 2-photon excitation. We used a Tai-Sapphire laser (Mai Tai, Spectra-Physics) to excite upconversion nano-particles [31

31. F. Wang, D. Banerjee, Y. S. Liu, X. Y. Chen, and X. G. Liu, “Upconversion nanoparticles in biological labeling, imaging, and therapy,” Analyst (Lond.) 135(8), 1839–1854 (2010). [CrossRef] [PubMed]

], with 980 nm light, focused by an oil (or water) objective. The nano-particles were immobilized using poly-lysine, and emitted at 640 nm. We used a 690 nm LED (ELJ-690-629, Roithner LaserTechnik) for fiduciary markers, so as to not interfere with excitation (or emission) light. Over the course of 6 minutes, the immobilized particles drifted 400 nm, the same as the fiduciary markers. Hence this method is applicable for the 2-photon microscopy.

4. Conclusion

In summary, we developed a simple method of correcting stage drifts for super-resolution and super- accuracy studies with only minimal and inexpensive additions. Using soft lithography, fiduciary markers were made on the coverslip and then tracked using IR light scattering; the image was then fit using Gaussian and Airy functions. The average traces of four fiduciary markers showed a precision of 1.25 nm and 1.47 nm along the x- and y-axes, respectively, and 16.9 nm along the z-axis for wet samples and 4.6 nm for dry samples. We applied this method in a walking assay with kinesin, which showed excellent agreement with the step size shown previously, while the uncorrected value showed a slightly larger value. In addition, the fiduciary markers are biocompatible in vitro, in vivo, and for 1- and 2-photon excitation. There is a potential compatibility of this scheme with microscopes that have built-in NIR sources and sensors for focus drift correction.

Acknowledgments

This work was funded in part by NIH (GM068625) and NSF (DBI- 02-15869, EAGER 0968976, and 082265).

References and links

1.

E. Toprak and P. R. Selvin, “New fluorescent tools for watching nanometer-scale conformational changes of single molecules,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 349–369 (2007). [CrossRef] [PubMed]

2.

B. Huang, M. Bates, and X. W. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009). [CrossRef] [PubMed]

3.

M. P. Gordon, T. Ha, and P. R. Selvin, “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A. 101(17), 6462–6465 (2004). [CrossRef] [PubMed]

4.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise Nanometer Localization Analysis for Individual Fluorescent Probes,” Biophys. J. 82(5), 2775–2783 (2002). [CrossRef] [PubMed]

5.

X. H. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004). [CrossRef] [PubMed]

6.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multicolor colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. U.S.A. 97(17), 9461–9466 (2000). [CrossRef] [PubMed]

7.

L. S. Churchman, Z. Okten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. U.S.A. 102(5), 1419–1423 (2005). [CrossRef] [PubMed]

8.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

9.

A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, “Stabilization of an optical microscope to 0.1 nm in three dimensions,” Appl. Opt. 46(3), 421–427 (2007). [CrossRef] [PubMed]

10.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). [CrossRef] [PubMed]

11.

P. D. Simonson, E. Rothenberg, and P. R. Selvin, “Single-Molecule-Based Super-Resolution Images in the Presence of Multiple Fluorophores,” Nano Lett. 11(11), 5090–5096 (2011). [CrossRef] [PubMed]

12.

D. T. Burnette, P. Sengupta, Y. H. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011). [CrossRef] [PubMed]

13.

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007). [CrossRef] [PubMed]

14.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005). [CrossRef] [PubMed]

15.

A. R. Wade and F. W. Fitzke, “A fast, robust pattern recognition system for low light level image registration and its application to retinal imaging,” Opt. Express 3(5), 190–197 (1998). [CrossRef] [PubMed]

16.

M. Guizar-Sicairos, S. T. Thurman, and J. R. Fienup, “Efficient subpixel image registration algorithms,” Opt. Lett. 33(2), 156–158 (2008). [CrossRef] [PubMed]

17.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef] [PubMed]

18.

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010). [CrossRef] [PubMed]

19.

L. Nugent-Glandorf and T. T. Perkins, “Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection,” Opt. Lett. 29(22), 2611–2613 (2004). [CrossRef] [PubMed]

20.

M. J. Mlodzianoski, J. M. Schreiner, S. P. Callahan, K. Smolková, A. Dlasková, J. Santorová, P. Ježek, and J. Bewersdorf, “Sample drift correction in 3D fluorescence photoactivation localization microscopy,” Opt. Express 19(16), 15009–15019 (2011). [CrossRef] [PubMed]

21.

D. Qin, Y. N. Xia, and G. M. Whitesides, “Soft lithography for micro- and nanoscale patterning,” Nat. Protoc. 5(3), 491–502 (2010). [CrossRef] [PubMed]

22.

M. Speidel, A. Jonás, and E. L. Florin, “Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging,” Opt. Lett. 28(2), 69–71 (2003). [CrossRef] [PubMed]

23.

E. Toprak, H. Balci, B. H. Blehm, and P. R. Selvin, “Three-dimensional particle tracking via bifocal imaging,” Nano Lett. 7(7), 2043–2045 (2007). [CrossRef] [PubMed]

24.

S. F. Gibson and F. Lanni, “Diffraction by a Circular Aperture as a model for three-dimensional optical microscopy,” J. Opt. Soc. Am. A 6(9), 1357–1367 (1989). [CrossRef] [PubMed]

25.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004). [CrossRef] [PubMed]

26.

E. Toprak, A. Yildiz, M. T. Hoffman, S. S. Rosenfeld, and P. R. Selvin, “Why kinesin is so processive,” Proc. Natl. Acad. Sci. U.S.A. 106(31), 12717–12722 (2009). [CrossRef] [PubMed]

27.

S. M. Block, L. S. B. Goldstein, and B. J. Schnapp, “Bead Movement by Single Kinesin Molecules Studied with Optical Tweezers,” Nature 348(6299), 348–352 (1990). [CrossRef] [PubMed]

28.

K. M. Zia, M. Zuber, I. A. Bhatti, M. Barikani, and M. A. Sheikh, “Evaluation of biocompatibility and mechanical behavior of polyurethane elastomers based on chitin/1,4-butane diol blends,” Int. J. Biol. Macromol. 44(1), 18–22 (2009). [CrossRef] [PubMed]

29.

M. Howarth, K. Takao, Y. Hayashi, and A. Y. Ting, “Targeting quantum dots to surface proteins in living cells with biotin ligase,” Proc. Natl. Acad. Sci. U.S.A. 102(21), 7583–7588 (2005). [CrossRef] [PubMed]

30.

M. Howarth, W. H. Liu, S. Puthenveetil, Y. Zheng, L. F. Marshall, M. M. Schmidt, K. D. Wittrup, M. G. Bawendi, and A. Y. Ting, “Monovalent, reduced-size quantum dots for imaging receptors on living cells,” Nat. Methods 5(5), 397–399 (2008). [CrossRef] [PubMed]

31.

F. Wang, D. Banerjee, Y. S. Liu, X. Y. Chen, and X. G. Liu, “Upconversion nanoparticles in biological labeling, imaging, and therapy,” Analyst (Lond.) 135(8), 1839–1854 (2010). [CrossRef] [PubMed]

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Microscopy

History
Original Manuscript: March 12, 2012
Revised Manuscript: May 7, 2012
Manuscript Accepted: May 7, 2012
Published: May 14, 2012

Virtual Issues
Vol. 7, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Sang Hak Lee, Murat Baday, Marco Tjioe, Paul D. Simonson, Ruobing Zhang, En Cai, and Paul R. Selvin, "Using fixed fiduciary markers for stage drift correction," Opt. Express 20, 12177-12183 (2012)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-20-11-12177


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References

  1. E. Toprak and P. R. Selvin, “New fluorescent tools for watching nanometer-scale conformational changes of single molecules,” Annu. Rev. Biophys. Biomol. Struct.36(1), 349–369 (2007). [CrossRef] [PubMed]
  2. B. Huang, M. Bates, and X. W. Zhuang, “Super-Resolution Fluorescence Microscopy,” Annu. Rev. Biochem.78(1), 993–1016 (2009). [CrossRef] [PubMed]
  3. M. P. Gordon, T. Ha, and P. R. Selvin, “Single-molecule high-resolution imaging with photobleaching,” Proc. Natl. Acad. Sci. U.S.A.101(17), 6462–6465 (2004). [CrossRef] [PubMed]
  4. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise Nanometer Localization Analysis for Individual Fluorescent Probes,” Biophys. J.82(5), 2775–2783 (2002). [CrossRef] [PubMed]
  5. X. H. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.101(31), 11298–11303 (2004). [CrossRef] [PubMed]
  6. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, “Ultrahigh-resolution multicolor colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. U.S.A.97(17), 9461–9466 (2000). [CrossRef] [PubMed]
  7. L. S. Churchman, Z. Okten, R. S. Rock, J. F. Dawson, and J. A. Spudich, “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Natl. Acad. Sci. U.S.A.102(5), 1419–1423 (2005). [CrossRef] [PubMed]
  8. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]
  9. A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, “Stabilization of an optical microscope to 0.1 nm in three dimensions,” Appl. Opt.46(3), 421–427 (2007). [CrossRef] [PubMed]
  10. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006). [CrossRef] [PubMed]
  11. P. D. Simonson, E. Rothenberg, and P. R. Selvin, “Single-Molecule-Based Super-Resolution Images in the Presence of Multiple Fluorophores,” Nano Lett.11(11), 5090–5096 (2011). [CrossRef] [PubMed]
  12. D. T. Burnette, P. Sengupta, Y. H. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A.108(52), 21081–21086 (2011). [CrossRef] [PubMed]
  13. S. W. Hell, “Far-field optical nanoscopy,” Science316(5828), 1153–1158 (2007). [CrossRef] [PubMed]
  14. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005). [CrossRef] [PubMed]
  15. A. R. Wade and F. W. Fitzke, “A fast, robust pattern recognition system for low light level image registration and its application to retinal imaging,” Opt. Express3(5), 190–197 (1998). [CrossRef] [PubMed]
  16. M. Guizar-Sicairos, S. T. Thurman, and J. R. Fienup, “Efficient subpixel image registration algorithms,” Opt. Lett.33(2), 156–158 (2008). [CrossRef] [PubMed]
  17. M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]
  18. R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods7(5), 339–340 (2010). [CrossRef] [PubMed]
  19. L. Nugent-Glandorf and T. T. Perkins, “Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection,” Opt. Lett.29(22), 2611–2613 (2004). [CrossRef] [PubMed]
  20. M. J. Mlodzianoski, J. M. Schreiner, S. P. Callahan, K. Smolková, A. Dlasková, J. Santorová, P. Ježek, and J. Bewersdorf, “Sample drift correction in 3D fluorescence photoactivation localization microscopy,” Opt. Express19(16), 15009–15019 (2011). [CrossRef] [PubMed]
  21. D. Qin, Y. N. Xia, and G. M. Whitesides, “Soft lithography for micro- and nanoscale patterning,” Nat. Protoc.5(3), 491–502 (2010). [CrossRef] [PubMed]
  22. M. Speidel, A. Jonás, and E. L. Florin, “Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging,” Opt. Lett.28(2), 69–71 (2003). [CrossRef] [PubMed]
  23. E. Toprak, H. Balci, B. H. Blehm, and P. R. Selvin, “Three-dimensional particle tracking via bifocal imaging,” Nano Lett.7(7), 2043–2045 (2007). [CrossRef] [PubMed]
  24. S. F. Gibson and F. Lanni, “Diffraction by a Circular Aperture as a model for three-dimensional optical microscopy,” J. Opt. Soc. Am. A6(9), 1357–1367 (1989). [CrossRef] [PubMed]
  25. A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science303(5658), 676–678 (2004). [CrossRef] [PubMed]
  26. E. Toprak, A. Yildiz, M. T. Hoffman, S. S. Rosenfeld, and P. R. Selvin, “Why kinesin is so processive,” Proc. Natl. Acad. Sci. U.S.A.106(31), 12717–12722 (2009). [CrossRef] [PubMed]
  27. S. M. Block, L. S. B. Goldstein, and B. J. Schnapp, “Bead Movement by Single Kinesin Molecules Studied with Optical Tweezers,” Nature348(6299), 348–352 (1990). [CrossRef] [PubMed]
  28. K. M. Zia, M. Zuber, I. A. Bhatti, M. Barikani, and M. A. Sheikh, “Evaluation of biocompatibility and mechanical behavior of polyurethane elastomers based on chitin/1,4-butane diol blends,” Int. J. Biol. Macromol.44(1), 18–22 (2009). [CrossRef] [PubMed]
  29. M. Howarth, K. Takao, Y. Hayashi, and A. Y. Ting, “Targeting quantum dots to surface proteins in living cells with biotin ligase,” Proc. Natl. Acad. Sci. U.S.A.102(21), 7583–7588 (2005). [CrossRef] [PubMed]
  30. M. Howarth, W. H. Liu, S. Puthenveetil, Y. Zheng, L. F. Marshall, M. M. Schmidt, K. D. Wittrup, M. G. Bawendi, and A. Y. Ting, “Monovalent, reduced-size quantum dots for imaging receptors on living cells,” Nat. Methods5(5), 397–399 (2008). [CrossRef] [PubMed]
  31. F. Wang, D. Banerjee, Y. S. Liu, X. Y. Chen, and X. G. Liu, “Upconversion nanoparticles in biological labeling, imaging, and therapy,” Analyst (Lond.)135(8), 1839–1854 (2010). [CrossRef] [PubMed]

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