Chip-based wide field-of-view nanoscopy

Journal name:
Nature Photonics
Volume:
11,
Pages:
322–328
Year published:
DOI:
doi:10.1038/nphoton.2017.55
Received
Accepted
Published online

Abstract

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

At a glance

Figures

  1. Implementation of chip-based nanoscopy.
    Figure 1: Implementation of chip-based nanoscopy.

    a, Channel-like waveguide geometries are realized by etching the slab waveguide either partially or completely down to the SiO2 substrate. In either case, the light is mainly guided by channels of 25–500 µm breadth. b, Five channels can easily be seen in the photograph of a strip waveguide chip, marked by arrows. Scale bar, 1 cm. c, Light guided inside the waveguide is the source of the evanescent field illuminating samples on top of the surface. d, The optical set-up consists of a simple upright microscope for fluorescence detection and an illumination unit to provide coupling to the input facet of the waveguides, either through an objective lens or via a lensed fibre.

  2. Demonstration of chip-based dSTORM.
    Figure 2: Demonstration of chip-based dSTORM.

    a, Diffraction-limited and dSTORM imaging of immunostained tubulin in liver sinusoidal endothelial cells (LSECs). b, Measuring a lateral profile of 540 nm width along a straight microtubule (magenta marking in the inset of the dSTORM image in a) reveals its hollow structure. c, The resolution capability is further investigated by imaging DNA-origami nanorulers of (50 ± 5) nm specified length, which can be clearly resolved with waveguide-based dSTORM, similar to objective-based TIRF dSTORM. d, Analysing their line profiles, a mean nanoruler length of 49 nm is found in both cases, confirming that the chip-based implementation shows comparable performance to a conventional inverted dSTORM set-up. e, Waveguide chip-based illumination also allows for using a low-magnification/low-NA (×20/NA 0.45) objective lens for dSTORM imaging over a FOV of 0.5 mm × 0.5 mm. f, A detail from the white box in e. g, The profile over adjacent tubulin filaments reveals their separation by 138 nm.

  3. Demonstration of chip-based ESI.
    Figure 3: Demonstration of chip-based ESI.

    a, Spatial fluorescence intensity fluctuations are induced by changing the mode pattern of the waveguide during image acquisition. Accordingly, these measurements show a diffraction-limited image of the labelled structure multiplied by the mode pattern. A stack of ∼200 frames is used as input data for the fluctuation analysis reconstruction algorithm, resulting in one super-resolved image. b, Imaging tubulin in an LSEC. The comparison of the diffraction-limited image, the corresponding ESI reconstruction and the dSTORM image shows the gradually increasing resolution. Scale bar, 5 µm. c, Magnification of the same region (indicated by the rectangle in b) for the three different imaging modalities. Scale bar, 0.5 µm. The dSTORM image of the same structure verifies the applicability of chip-based ESI. d, The line profiles reveal a resolution of 106 nm for chip-based ESI (green line) by imaging adjacent microtubules, as simultaneously observed in the dSTORM image (blue dashed line).

  4. Imaging the same sample under varied acquisition conditions reveals the specific strengths of the different approaches.
    Figure 4: Imaging the same sample under varied acquisition conditions reveals the specific strengths of the different approaches.

    As TIRF excitation is provided over the entire width of the waveguide, arbitrary objective lenses can be used for detection. a,d, Using a ×20 magnification objective lens allows for chip-based TIRF imaging over a FOV of 0.46 mm width. b,c,eh, Both fluctuation-based ESI (b,e) and localization-based dSTORM (c,f) result in an optical resolution enhancement obtained using the NA 0.4 lens and thus resolve actin bundles at 334 nm distance (h). Hence, both techniques provide a tool for scanning large FOVs at high resolution to identify cells of interest, which can be re-imaged using a high-magnification/high-NA lens in a subsequent step (g), accomplishing superior resolution. The choice between wide-FOV ESI or dSTORM can be made by either prioritizing short acquisition times (choosing ESI) or best resolution (choosing dSTORM). The images in dg show a detail of the region marked by the box in a. Scale bars, 20 µm (a); 2 µm (d).

  5. Multi-colour chip-based dSTORM reveals the interplay between actin (magenta) and the membrane (green) in LSECs.
    Figure 5: Multi-colour chip-based dSTORM reveals the interplay between actin (magenta) and the membrane (green) in LSECs.

    a, Groups of fenestrations form sieve-plate superstructures that are surrounded by thicker actin bundles. Inset: actin is present between neighbouring fenestrations, where it colocalizes with the plasma membrane. Scale bars, 5 µm (main image); 1 µm (inset). b, Line profiles taken at different positions in the liver cell, as shown in a, reveal diameters of ∼200 nm and smaller for the chosen fenestrations, which can only be resolved optically by super-resolution microscopy. The profiles underline the visual impressions of colocalization.

References

  1. Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190, 165175 (2010).
  2. Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 8287 (2000).
  3. Heintzmann, R. & Cremer, C. G. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc. SPIE 3568, 185196 (1999).
  4. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780782 (1994).
  5. Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935939 (2006).
  6. Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 2228722292 (2009).
  7. Yahiatène, I., Hennig, S., Müller, M. & Huser, T. Entropy-based super-resolution imaging (ESI): from disorder to fine detail. ACS Photon. 2, 10491056 (2015).
  8. Rust, M. J., Bates, M. & Zhuang, X. Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution. Nat. Methods 3, 793795 (2006).
  9. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 61726176 (2008).
  10. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 16421645 (2006).
  11. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 42584272 (2006).
  12. Hoyer, P., Staudt, T., Engelhardt, J. & Hell, S. W. Quantum dot blueing and blinking enables fluorescent microscopy. Nano Lett. 11, 245250 (2010).
  13. Xu, J. Q., Tehrani, K. F. & Kner, P. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy. ACS Nano 9, 29172925 (2015).
  14. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159161 (2007).
  15. Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417423 (2011).
  16. Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).
  17. Grandin, H. M., Stadler, B., Textor, M. & Voros, J. Waveguide excitation fluorescence microscopy: a new tool for sensing and imaging the biointerface. Biosens. Bioelectron. 21, 14761482 (2006).
  18. Agnarsson, B., Ingthorsson, S., Gudjonsson, T. & Leosson, K. Evanescent-wave fluorescence microscopy using symmetric planar waveguides. Opt. Express 17, 50755082 (2009).
  19. Agnarsson, B., Jonsdottir, A. B., Arnfinnsdottir, N. B. & Leosson, K. On-chip modulation of evanescent illumination and live-cell imaging with polymer waveguides. Opt. Express 19, 2292922935 (2011).
  20. Shen, H. et al. TIRF microscopy with ultra-short penetration depth. Opt. Express 22, 1072810734 (2014).
  21. Agnarsson, B. et al. Evanescent light-scattering microscopy for label-free interfacial imaging: from single sub-100 nm vesicles to live cells. ACS Nano 9, 1184911862 (2015).
  22. Ramachandran, S., Cohen, D. A., Quist, A. P. & Lal, R. High performance, LED powered, waveguide based total internal reflection microscopy. Sci. Rep. 3, 2133 (2013).
  23. Dhakal, A. et al. Evanescent excitation and collection of spontaneous Raman spectra using silicon nitride nanophotonic waveguides. Opt. Lett. 39, 40254028 (2014).
  24. Fedyanin, D. Y. & Stebunov, Y. V. All-nanophotonic NEMS biosensor on a chip. Sci. Rep. 5, 10968 (2015).
  25. Yurtsever, G. et al. Photonic integrated Mach–Zehnder interferometer with an on-chip reference arm for optical coherence tomography. Biomed. Opt. Express 5, 10501061 (2014).
  26. Sørensen, K. K., Simon-Santamaria, J., McCuskey, R. S. & Smedsrød, B. Liver sinusoidal endothelial cells. Compr. Physiol. 5, 17511574 (2015).
  27. Weber, K., Rathke, P. C. & Osborn, M. Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy. Proc. Natl Acad. Sci. USA 75, 18201824 (1978).
  28. Olivier, N., Keller, D., Gonczy, P. & Manley, S. Resolution doubling in 3D-STORM imaging through improved buffers. PLoS ONE 8, e69004 (2013).
  29. Vaughan, J. C., Jia, S. & Zhuang, X. Ultra-bright photoactivatable fluorophores created by reductive caging. Nat. Methods 9, 11811184 (2012).
  30. Endesfelder, U. & Heilemann, M. Art and artifacts in single-molecule localization microscopy: beyond attractive images. Nat. Methods 11, 235238 (2014).
  31. Bourg, N. et al. Direct optical nanoscopy with axially localized detection. Nat. Photon. 9, 587593 (2015).
  32. Endesfelder, U., Malkusch, S., Fricke, F. & Heilemann, M. A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment. Histochem. Cell Biol. 141, 629638 (2014).
  33. Nieuwenhuizen, R. P. et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557562 (2013).
  34. Banterle, N., Bui, K. H., Lemke, E. A. & Beck, M. Fourier ring correlation as a resolution criterion for super-resolution microscopy. J. Struct. Biol. 183, 363367 (2013).
  35. Douglass, K. M., Sieben, C., Archetti, A., Lambert, A. & Manley, S. Super-resolution imaging of multiple cells by optimised flat-field epi-illumination. Nat. Photon. 10, 705708 (2016).
  36. Smedsrød, B. & Pertoft, H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J. Leukoc. Biol. 38, 213230 (1985).
  37. Ahluwalia, B. S. et al. Fabrication of submicrometer high refractive index tantalum pentoxide waveguides for optical propulsion of microparticles. IEEE Photon. Technol. Lett. 21, 14081410 (2009).
  38. Ventalon, C. & Mertz, J. Quasi-confocal fluorescence sectioning with dynamic speckle illumination. Opt. Lett. 30, 33503352 (2005).
  39. Kim, M., Park, C., Rodriguez, C., Park, Y. & Cho, Y. H. Superresolution imaging with optical fluctuation using speckle patterns illumination. Sci. Rep. 5, 16525 (2015).
  40. Wolter, S. et al. rapidSTORM: accurate, fast open-source software for localization microscopy. Nat. Methods 9, 10401041 (2012).
  41. Smith, C. S., Joseph, N., Rieger, B. & Lidke, K. A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Methods 7, 373375 (2010).
  42. Cogger, V. C., Roessner, U., Warren, A., Fraser, R. & Le Couteur, D. G. A sieve-raft hypothesis for the regulation of endothelial fenestrations. Comput. Struct. Biotechnol. J. 8, e201308003 (2013).
  43. Mönkemöller, V. et al. Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy. Phys. Chem. Chem. Phys. 16, 1257612581 (2014).
  44. Mönkemöller, V., Øie, C., Hübner, W., Huser, T. & McCourt, P. Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations. Sci Rep. 5, 16279 (2015).
  45. Braet, F. & Wisse, E. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review. Comp. Hepatol. 1, 1 (2002).
  46. Wang, X. et al. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies. Lab Chip 11, 36563662 (2011).
  47. Helle, Ø. I., Ahluwalia, B. S. & Hellesø, O. G. Optical transport, lifting and trapping of micro-particles by planar waveguides. Opt. Express 23, 66016612 (2015).
  48. Dullo, F. T. & Hellesø, O. G. On-chip phase measurement for microparticles trapped on a waveguide. Lab Chip 15, 39183924 (2015).
  49. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484488 (2011).
  50. Diekmann, R. et al. Nanoscopy of bacterial cells immobilized by holographic optical tweezers. Nat. Commun. 7, 13711 (2016).
  51. Prieto, F. et al. An integrated optical interferometric nanodevice based on silicon technology for biosensor applications. Nanotechnology 14, 907912 (2003).
  52. van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 9911009 (2011).
  53. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676682 (2012).
  54. Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 23892390 (2014).

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Author information

  1. These authors contributed equally to this work.

    • Robin Diekmann &
    • Øystein I. Helle

Affiliations

  1. Department of Physics, Bielefeld University, 33615 Bielefeld, Germany

    • Robin Diekmann,
    • Thomas R. Huser &
    • Mark Schüttpelz
  2. Department of Physics and Technology, UiT-The Arctic University of Norway, 9037 Tromsø, Norway

    • Øystein I. Helle,
    • Cristina I. Øie &
    • Balpreet S. Ahluwalia
  3. Department of Medical Biology, UiT-The Arctic University of Norway, 9037 Tromsø, Norway

    • Peter McCourt
  4. Department of Internal Medicine and NSF Center for Biophotonics, University of California, Davis, Sacramento, California 95817, USA

    • Thomas R. Huser

Contributions

B.S.A. and M.S. conceived the project. All authors designed the research. C.I.Ø. isolated the cells and stained and prepared the biological samples. R.D. and Ø.I.H. built the set-up, prepared the non-biological samples, performed the experiments, performed the simulations, reconstructed the images, analysed the data and created the figures. R.D., Ø.I.H., M.S. and B.S.A. mainly wrote the paper. All authors reviewed the manuscript.

Competing financial interests

M.S. and B.S.A. have applied for patent GB1606268.9 for chip-based optical nanoscopy. The other authors declare no competing financial interests.

Corresponding authors

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