Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Primer
  • Published:

Interferometric scattering microscopy

Nature Reviews Methods Primers volume 5, Article number: 23 (2025) Cite this article

Subjects

Abstract

Over the past two decades, interferometric scattering (iSCAT) microscopy has become a powerful label-free imaging method with a range of applications in fundamental science and technology. iSCAT detects the scattering of subwavelength entities through interference with a reference beam of light. Performed in a variety of illumination and detection schemes, iSCAT has exploited both amplitude and phase information to reach single-molecule detection sensitivity; to determine the size, mass and refractive index of nanoparticles; to achieve high spatiotemporal precision in 3D tracking of nanoparticles; to image subcellular nanostructures; and to quantify ultrafast diffusion and transport of energy in solids. In this Primer, we describe the basic principles of iSCAT detection and imaging from theoretical and practical points of view. We discuss various factors that affect the attainable signal-to-noise ratio, which in turn determines crucial performance features such as sensitivity and speed. We survey selected applications in which iSCAT has been instrumental in providing new insights. Finally, we discuss some of the current challenges and potential avenues for advancing the technique further.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: iSCAT microscopy.
Fig. 2: Image background and elimination.
Fig. 3: iSCAT for 3D tracking of individual nanoparticles.
Fig. 4: Sensitive analysis of size of nanoparticles and dynamics of protein assembly.
Fig. 5: Label-free 3D nanoscopic cell imaging.
Fig. 6: Stroboscopic energy flow microscopy in materials.

Similar content being viewed by others

References

  1. Weisenburger, S. & Sandoghdar, V. Light microscopy: an ongoing contemporary revolution. Contemp. Phys. 56, 123–143 (2015).

    Article  ADS  Google Scholar 

  2. Adhikari, S. & Orrit, M. Progress and perspectives in single-molecule optical spectroscopy. J. Chem. Phys. 156, 160903 (2022).

    Article  ADS  Google Scholar 

  3. Mazumder, N. et al. Label-free non-linear multimodal optical microscopy — basics, development, and applications. Front. Phys. https://doi.org/10.3389/fphy.2019.00170 (2019).

    Article  Google Scholar 

  4. Saleh, B. E. A. & Teich, M. C. (eds) Fundamentals of Photonics 3rd edn Vol. 1 and 2 (Wiley, 2019).

  5. Taylor, R. W. & Sandoghdar, V. in Label-Free Super-Resolution Microscopy (ed. Astratov, V.) 25–65 (Springer, 2019).

  6. Zernike, F. Phase contrast, a new method for the microscopic observation of transparent objects. Physica 9, 686–698 (1942).

    Article  ADS  Google Scholar 

  7. Zernike, F. Phase contrast, a new method for the microscopic observation of transparent objects part II. Physica 9, 974–986 (1942).

    Article  ADS  Google Scholar 

  8. Normarski, G. Dispositif oculaire a contraste de phase pour microscope. J. Phys. Radium 11, 9–10 (1950).

    Google Scholar 

  9. Curtis, A. S. G. The mechanism of adhesion of cells to glass: a study by interference reflection microscopy. J. Cell Biol. 20, 199–215 (1964).

    Article  Google Scholar 

  10. Park, Y., Depeursinge, C. & Popescu, G. Quantitative phase imaging in biomedicine. Nat. Photonics 12, 578–589 (2018).

    Article  ADS  Google Scholar 

  11. Javidi, B. et al. Roadmap on digital holography [Invited]. Opt. Express 29, 35078–35118 (2021).

    Article  ADS  Google Scholar 

  12. Lane, N. The unseen world: reflections on Leeuwenhoek (1677) ‘Concerning little animals’. Philos. Trans. R. Soc. B 370, 20140344 (2015).

    Article  Google Scholar 

  13. Cocquyt, T., Zhou, Z., Plomp, J. & van Eijck, L. Neutron tomography of Van Leeuwenhoek’s microscopes. Sci. Adv. 7, eabf2402 (2021).

    Article  ADS  Google Scholar 

  14. Mie, G. Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen. Ann. Phys. 330, 377–445 (1908).

    Article  Google Scholar 

  15. Zimm, B. H. Apparatus and methods for measurement and interpretation of the angular variation of light scattering; preliminary results on polystyrene solutions. J. Chem. Phys. 16, 1099–1116 (1948).

    Article  ADS  Google Scholar 

  16. Thompson, B. J. & Zinky, W. R. Holographic detection of submicron particles. Appl. Opt. 7, 2426–2428 (1968).

    Article  ADS  Google Scholar 

  17. Batchelder, J. S. & Taubenblatt, M. A. Interferometric detection of forward scattered light from small particles. Appl. Phys. Lett. 55, 215–217 (1989).

    Article  ADS  Google Scholar 

  18. Amos, L. A. & Amos, W. B. The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy. J. Cell Sci. 1991, 95–101 (1991).

    Article  Google Scholar 

  19. Kalkbrenner, T., Ramstein, M., Mlynek, J. & Sandoghdar, V. A single gold particle as a probe for apertureless scanning near-field optical microscopy. J. Microsc. 202, 72–76 (2001).

    Article  MathSciNet  Google Scholar 

  20. Klar, T. et al. Surface-plasmon resonances in single metallic nanoparticles. Phys. Rev. Lett. 80, 4249–4252 (1998).

    Article  ADS  Google Scholar 

  21. Boyer, D., Tamarat, P., Maali, A., Lounis, B. & Orrit, M. Photothermal imaging of nanometer-sized metal particles among scatterers. Science 297, 1160–1163 (2002).

    Article  ADS  Google Scholar 

  22. Lindfors, K., Kalkbrenner, T., Stoller, P. & Sandoghdar, V. Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy. Phys. Rev. Lett. 93, 037401 (2004).

    Article  ADS  Google Scholar 

  23. Arbouet, A. et al. Direct measurement of the single-metal-cluster optical absorption. Phys. Rev. Lett. 93, 127401 (2004).

    Article  ADS  Google Scholar 

  24. Hsieh, C.-L., Spindler, S., Ehrig, J. & Sandoghdar, V. Tracking single particles on supported lipid membranes: multimobility diffusion and nanoscopic confinement. J. Phys. Chem. B 118, 1545–1554 (2014).

    Article  Google Scholar 

  25. Spindler, S. et al. Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy. J. Phys. Appl. Phys. 49, 274002 (2016).

    Article  Google Scholar 

  26. Huang, Y.-F. et al. Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells. ACS Nano 11, 2575–2585 (2017).

    Article  Google Scholar 

  27. de Wit, G., Albrecht, D., Ewers, H. & Kukura, P. Revealing compartmentalized diffusion in living cells with interferometric scattering microscopy. Biophys. J. 114, 2945–2950 (2018).

    Article  Google Scholar 

  28. Taylor, R. W. et al. Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane. Nat. Photonics 13, 480–487 (2019).

    Article  ADS  Google Scholar 

  29. Vala, M. et al. Nanoscopic structural fluctuations of disassembling microtubules revealed by label‐free super‐resolution microscopy. Small Methods 5, 2000985 (2021).

    Article  Google Scholar 

  30. Vamivakas, A. N. et al. Strong extinction of a far-field laser beam by a single quantum dot. Nano Lett. 7, 2892–2896 (2007).

    Article  ADS  Google Scholar 

  31. Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V. Imaging a single quantum dot when it is dark. Nano Lett. 9, 926–929 (2009).

    Article  ADS  Google Scholar 

  32. Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V. Single-molecule sensitivity in optical absorption at room temperature. J. Phys. Chem. Lett. 1, 3323–3327 (2010).

    Article  Google Scholar 

  33. Celebrano, M., Kukura, P., Renn, A. & Sandoghdar, V. Single-molecule imaging by optical absorption. Nat. Photonics 5, 95–98 (2011).

    Article  ADS  Google Scholar 

  34. Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 5, 4495 (2014).

    Article  ADS  Google Scholar 

  35. Ortega Arroyo, J. et al. Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett. 14, 2065–2070 (2014).

    Article  ADS  Google Scholar 

  36. Young, G. et al. Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018).

    Article  ADS  Google Scholar 

  37. Delor, M., Weaver, H. L., Yu, Q. & Ginsberg, N. S. Imaging material functionality through three-dimensional nanoscale tracking of energy flow. Nat. Mater. 19, 56–62 (2020).

    Article  ADS  Google Scholar 

  38. Su, H. et al. Dark-exciton driven energy funneling into dielectric inhomogeneities in two-dimensional semiconductors. Nano Lett. 22, 2843–2850 (2022).

    Article  ADS  Google Scholar 

  39. Baxter, J. M. et al. Coexistence of incoherent and ultrafast coherent exciton transport in a two-dimensional superatomic semiconductor. J. Phys. Chem. Lett. 14, 10249–10256 (2023).

    Article  Google Scholar 

  40. Gruber, C. G., Frey, L., Guntermann, R., Medina, D. D. & Cortés, E. Early stages of covalent organic framework formation imaged in operando. Nature 630, 872–877 (2024).

    Article  Google Scholar 

  41. Küppers, M., Albrecht, D., Kashkanova, A. D., Lühr, J. & Sandoghdar, V. Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells. Nat. Commun. 14, 1962 (2023).

    Article  ADS  Google Scholar 

  42. Hsiao, Y.-T., Wu, T.-Y., Wu, B.-K., Chu, S.-W. & Hsieh, C.-L. Spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells. Opt. Express 30, 45233–45245 (2022).

    Article  ADS  Google Scholar 

  43. Wrigge, G., Gerhardt, I., Hwang, J., Zumofen, G. & Sandoghdar, V. Efficient coupling of photons to a single molecule and the observation of its resonance fluorescence. Nat. Phys. 4, 60–66 (2008).

    Article  Google Scholar 

  44. Gerhardt, I. et al. Strong extinction of a laser beam by a single molecule. Phys. Rev. Lett. 98, 033601 (2007).

    Article  ADS  Google Scholar 

  45. Loudon, R. The Quantum Theory of Light (Oxford Univ. Press, 2000).

  46. Martin, C. et al. In-line holographic microscopy with model-based analysis. Nat. Rev. Methods Primers 2, 83 (2022).

    Article  Google Scholar 

  47. Thiele, J. C., Pfitzner, E. & Kukura, P. Single-protein optical holography. Nat. Photonics 18, 388–395 (2024).

    Article  ADS  Google Scholar 

  48. Ortega Arroyo, J., Cole, D. & Kukura, P. Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging. Nat. Protoc. 11, 617–633 (2016).

    Article  Google Scholar 

  49. Gemeinhardt, A. & et al. Label-free imaging of single proteins secreted from living cells via iSCAT microscopy. J. Vis. Exp. https://doi.org/10.3791/58486 (2018).

    Article  Google Scholar 

  50. Kukura, P. et al. High-speed nanoscopic tracking of the position and orientation of a single virus. Nat. Methods 6, 923–927 (2009).

    Article  Google Scholar 

  51. Yang, Y. et al. Interferometric plasmonic imaging and detection of single exosomes. Proc. Natl Acad. Sci. USA 115, 10275–10280 (2018).

    Article  ADS  Google Scholar 

  52. Zhang, P. et al. Plasmonic scattering imaging of single proteins and binding kinetics. Nat. Methods 17, 1010–1017 (2020).

    Article  Google Scholar 

  53. Mazaheri, M. et al. iSCAT microscopy and particle tracking with tailored spatial coherence. Optica 11, 1030 (2024).

    Article  Google Scholar 

  54. Daaboul, G. G. et al. Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for ebola and marburg detection. ACS Nano 8, 6047–6055 (2014).

    Article  Google Scholar 

  55. Utterback, J. K. et al. Operando label-free optical imaging of solution-phase ion transport and electrochemistry. ACS Energy Lett. 8, 1785–1792 (2023).

    Article  Google Scholar 

  56. Folie, B. D. et al. Effect of anisotropic confinement on electronic structure and dynamics of band edge excitons in inorganic perovskite nanowires. J. Phys. Chem. A 124, 1867–1876 (2020).

    Article  Google Scholar 

  57. Guzelturk, B. et al. Nonequilibrium thermodynamics of colloidal gold nanocrystals monitored by ultrafast electron diffraction and optical scattering microscopy. ACS Nano 14, 4792–4804 (2020).

    Article  Google Scholar 

  58. Delor, M. et al. Carrier diffusion lengths exceeding 1 μm despite trap-limited transport in halide double perovskites. ACS Energy Lett. 5, 1337–1345 (2020).

    Article  Google Scholar 

  59. Utterback, J. K. et al. Nanoscale disorder generates subdiffusive heat transport in self-assembled nanocrystal films. Nano Lett. 21, 3540–3547 (2021).

    Article  ADS  Google Scholar 

  60. Li, J. et al. Zwitterions in 3D perovskites: organosulfide-halide perovskites. J. Am. Chem. Soc. 144, 22403–22408 (2022).

    Article  Google Scholar 

  61. Weaver, H. L. et al. Detecting, distinguishing, and spatiotemporally tracking photogenerated charge and heat at the nanoscale. ACS Nano 17, 19011–19021 (2023).

    Article  Google Scholar 

  62. Xu, D. et al. Ultrafast imaging of polariton propagation and interactions. Nat. Commun. 14, 3881 (2023).

    Article  ADS  Google Scholar 

  63. Cheng, S.-W. et al. Optical imaging of ultrafast phonon–polariton propagation through an excitonic sensor. Nano Lett. 23, 9936–9942 (2023).

    Article  ADS  Google Scholar 

  64. Tulyagankhodjaev, J. A. et al. Room-temperature wavelike exciton transport in a van der Waals superatomic semiconductor. Science 382, 438–442 (2023).

    Article  ADS  Google Scholar 

  65. Lyu, P.-T. et al. Decrypting material performance by wide-field femtosecond interferometric imaging of energy carrier evolution. J. Am. Chem. Soc. 144, 13928–13937 (2022).

    Article  Google Scholar 

  66. Schnedermann, C. et al. Ultrafast tracking of exciton and charge carrier transport in optoelectronic materials on the nanometer scale. J. Phys. Chem. Lett. 10, 6727–6733 (2019).

    Article  Google Scholar 

  67. Schnedermann, C. et al. Sub-10 fs time-resolved vibronic optical microscopy. J. Phys. Chem. Lett. 7, 4854–4859 (2016).

    Article  Google Scholar 

  68. Sung, J. et al. Long-range ballistic propagation of carriers in methylammonium lead iodide perovskite thin films. Nat. Phys. 16, 171–176 (2020).

    Article  Google Scholar 

  69. Dastjerdi, H. M. et al. Optimized analysis for sensitive detection and analysis of single proteins via interferometric scattering microscopy. J. Phys. Appl. Phys. 55, 054002 (2022).

    Article  ADS  Google Scholar 

  70. Bohren, C. F. & Huffman, D. R. Absorption and Scattering of Light by Small Particles 1st edn (Wiley-VCH, 1983).

  71. Kalkbrenner, T., Håkanson, U. & Sandoghdar, V. Tomographic plasmon spectroscopy of a single gold nanoparticle. Nano Lett. 4, 2309–2314 (2004).

    Article  ADS  Google Scholar 

  72. Jacobsen, V., Klotzsch, E. & Sandoghdar, V. Interferometric detection and tracking of nanoparticles. In Nano Biophotonics: Science and Technology. Proc. 3rd International Nanophotonics Symposium (eds Masuhara, H., Kawata, S. & Tokunaga, F.) 143–159 (Elsevier, 2007).

  73. Taylor, R. W. & Sandoghdar, V. Interferometric scattering microscopy: seeing single nanoparticles and molecules via Rayleigh scattering. Nano Lett. 19, 4827–4835 (2019).

    Article  ADS  Google Scholar 

  74. Špačková, B. et al. Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles. Nat. Methods 19, 751–758 (2022).

    Article  Google Scholar 

  75. Ignatovich, F. V. & Novotny, L. Real-time and background-free detection of nanoscale particles. Phys. Rev. Lett. 96, 013901 (2006).

    Article  ADS  Google Scholar 

  76. Yurdakul, C. et al. High-throughput, high-resolution interferometric light microscopy of biological nanoparticles. ACS Nano 14, 2002–2013 (2020).

    Article  Google Scholar 

  77. Cole, D., Young, G., Weigel, A., Sebesta, A. & Kukura, P. Label-free single-molecule imaging with numerical-aperture-shaped interferometric scattering microscopy. ACS Photonics 4, 211–216 (2017).

    Article  Google Scholar 

  78. Liebel, M., Hugall, J. T. & van Hulst, N. F. Ultrasensitive label-free nanosensing and high-speed tracking of single proteins. Nano Lett. 17, 1277–1281 (2017).

    Article  ADS  Google Scholar 

  79. Avci, O., Campana, M. I., Yurdakul, C. & Ünlü, M. S. Pupil function engineering for enhanced nanoparticle visibility in wide-field interferometric microscopy. Optica 4, 247–254 (2017).

    Article  ADS  Google Scholar 

  80. Brokmann, X., Coolen, L., Hermier, J.-P. & Dahan, M. Emission properties of single CdSe/ZnS quantum dots close to a dielectric interface. Chem. Phys. 318, 91–98 (2005).

    Article  Google Scholar 

  81. Lee, K. G. et al. A planar dielectric antenna for directional single-photon emission and near-unity collection efficiency. Nat. Photonics 5, 166–169 (2011).

    Article  ADS  Google Scholar 

  82. Cheng, C.-Y., Liao, Y.-H. & Hsieh, C.-L. High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy. Nanoscale 11, 568–577 (2019).

    Article  Google Scholar 

  83. Li, N. et al. Photonic resonator interferometric scattering microscopy. Nat. Commun. 12, 1744 (2021).

    Article  ADS  Google Scholar 

  84. Gentner, C. et al. Enhanced quantitative wavefront imaging for nano-object characterization. ACS Nano 18, 19247–19256 (2024).

    Article  Google Scholar 

  85. Hogenboom, D. O., DiMarzio, C. A., Gaudette, T. J., Devaney, A. J. & Lindberg, S. C. Three-dimensional images generated by quadrature interferometry. Opt. Lett. 23, 783–785 (1998).

    Article  ADS  Google Scholar 

  86. Diezmann, L., von, Lee, M. Y., Lew, M. D. & Moerner, W. E. Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy. Optica 2, 985–993 (2015).

    Article  ADS  Google Scholar 

  87. McGorty, R., Schnitzbauer, J., Zhang, W. & Huang, B. Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy. Opt. Lett. 39, 275–278 (2014).

    Article  ADS  Google Scholar 

  88. Mahmoodabadi, R. G. et al. Point spread function in interferometric scattering microscopy (iSCAT). Part I: aberrations in defocusing and axial localization. Opt. Express 28, 25969–25988 (2020).

    Article  ADS  Google Scholar 

  89. Kasaian, K., Mazaheri, M. & Sandoghdar, V. Long-range three-dimensional tracking of nanoparticles using interferometric scattering microscopy. ACS Nano 18, 30463–30472 (2024).

    Article  Google Scholar 

  90. Brooks, N. J., Liu, C.-C., Chen, Y.-H. & Hsieh, C.-L. Point spread function engineering for spiral phase interferometric scattering microscopy enables robust 3D single-particle tracking and characterization. ACS Photonics 11, 5239–5250 (2024).

    Article  Google Scholar 

  91. Hitzelhammer, F. et al. Unified simulation platform for interference microscopy. ACS Photonics 11, 2745–2756 (2024).

    Article  Google Scholar 

  92. Dastjerdi, H. M., Mahmoodabadi, R. G., Bär, M., Sandoghdar, V. & Köstler, H. PiSCAT: a Python package for interferometric scattering microscopy. J. Open Source Softw. 7, 4024 (2022).

    Article  ADS  Google Scholar 

  93. Wu, H.-M., Lin, Y.-H., Yen, T.-C. & Hsieh, C.-L. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking. Sci. Rep. 6, 20542 (2016).

    Article  ADS  Google Scholar 

  94. Lin, S., He, Y., Feng, D., Piliarik, M. & Chen, X.-W. Optical fingerprint of flat substrate surface and marker-free lateral displacement detection with angstrom-level precision. Phys. Rev. Lett. 129, 213201 (2022).

    Article  ADS  Google Scholar 

  95. Holanová, K., Vala, M. & Piliarik, M. Optical imaging and localization of prospective scattering labels smaller than a single protein. Opt. Laser Technol. 109, 323–327 (2019).

    Article  ADS  Google Scholar 

  96. Jacobsen, V., Stoller, P., Brunner, C., Vogel, V. & Sandoghdar, V. Interferometric optical detection and tracking of very small gold nanoparticles at a water-glass interface. Opt. Express 14, 405–414 (2006).

    Article  ADS  Google Scholar 

  97. Li, Q. -Y., Lyu, P. -T., Kang, B., Chen, H. -Y. & Xu, J. -J. Label-free optical imaging of ion channel activity on living cells. Preprint at https://arxiv.org/abs/2312.16461 (2023).

  98. Namink, K., Meng, X., Koper, M. T. M. & Kukura, P. & Faez, S. Electric-double-layer-modulation microscopy. Phys. Rev. Appl. 13, 044065 (2020).

    Article  ADS  Google Scholar 

  99. Spindler, S., Sibold, J., Gholami Mahmoodabadi, R., Steinem, C. & Sandoghdar, V. High-speed microscopy of diffusion in pore-spanning lipid membranes. Nano Lett. 18, 5262–5271 (2018).

    Article  ADS  Google Scholar 

  100. Robert, H. M. L. et al. Fast photothermal spatial light modulation for quantitative phase imaging at the nanoscale. Nat. Commun. 12, 2921 (2021).

    Article  ADS  Google Scholar 

  101. Foley, E. D. B., Kushwah, M. S., Young, G. & Kukura, P. Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers. Nat. Methods 18, 1247–1252 (2021).

    Article  Google Scholar 

  102. Witkowska, A., Spindler, S., Mahmoodabadi, R. G., Sandoghdar, V. & Jahn, R. Differential diffusional properties in loose and tight docking prior to membrane fusion. Biophys. J. 119, 2431–2439 (2020).

    Article  ADS  Google Scholar 

  103. Dahmardeh, M., Mirzaalian Dastjerdi, H., Mazal, H., Köstler, H. & Sandoghdar, V. Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa. Nat. Methods 20, 442–447 (2023).

    Article  Google Scholar 

  104. Simon, F., Weiss, L. E. & van Teeffelen, S. A guide to single-particle tracking. Nat. Rev. Methods Primers 4, 66 (2024).

    Article  Google Scholar 

  105. Kashkanova, A. D. et al. Precision single-particle localization using radial variance transform. Opt. Express 29, 11070–11083 (2021).

    Article  ADS  Google Scholar 

  106. Krishnan, M., Mojarad, N., Kukura, P. & Sandoghdar, V. Geometry-induced electrostatic trapping of nanometric objects in a fluid. Nature 467, 692–695 (2010).

    Article  ADS  Google Scholar 

  107. Taylor, R. W. et al. High-precision protein-tracking with interferometric scattering microscopy. Front. Cell Dev. Biol. 8, 590158 (2020).

    Article  Google Scholar 

  108. Shechtman, Y. Recent advances in point spread function engineering and related computational microscopy approaches: from one viewpoint. Biophys. Rev. 12, 1303–1309 (2020).

    Article  Google Scholar 

  109. de Wit, X. M. et al. Precise characterization of nanometer-scale systems using interferometric scattering microscopy and Bayesian analysis. Appl. Opt. 62, 7205–7215 (2023).

    Article  ADS  Google Scholar 

  110. Dong, J., Maestre, D., Conrad-Billroth, C. & Juffmann, T. Fundamental bounds on the precision of iSCAT, COBRI and dark-field microscopy for 3D localization and mass photometry. J. Phys. Appl. Phys. 54, 394002 (2021).

    Article  Google Scholar 

  111. Lin, Y.-H., Chang, W.-L. & Hsieh, C.-L. Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds. Opt. Express 22, 9159–9170 (2014).

    Article  ADS  Google Scholar 

  112. Isojima, H., Iino, R., Niitani, Y., Noji, H. & Tomishige, M. Direct observation of intermediate states during the stepping motion of kinesin-1. Nat. Chem. Biol. 12, 290–297 (2016).

    Article  Google Scholar 

  113. Kashkanova, A. D., Blessing, M., Gemeinhardt, A., Soulat, D. & Sandoghdar, V. Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions. Nat. Methods 19, 586–593 (2022).

    Article  Google Scholar 

  114. Ortiz-Orruño, U., Quidant, R., van Hulst, N. F., Liebel, M. & Ortega Arroyo, J. Simultaneous sizing and refractive index analysis of heterogeneous nanoparticle suspensions. ACS Nano 17, 221–229 (2023).

    Article  Google Scholar 

  115. Lee, I.-B. et al. Interferometric scattering microscopy with polarization-selective dual detection scheme: capturing the orientational information of anisotropic nanometric objects. ACS Photonics 5, 797–804 (2018).

    Article  Google Scholar 

  116. Stoller, P., Jacobsen, V. & Sandoghdar, V. Measurement of the complex dielectric constant of a single gold nanoparticle. Opt. Lett. 31, 2474–2476 (2006).

    Article  ADS  Google Scholar 

  117. Daaboul, G. G. et al. High-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification. Nano Lett. 10, 4727–4731 (2010).

    Article  ADS  Google Scholar 

  118. McDonald, M. P. et al. Visualizing single-cell secretion dynamics with single-protein sensitivity. Nano Lett. 18, 513–519 (2018).

    Article  ADS  Google Scholar 

  119. Malay, A. D. et al. An ultra-stable gold-coordinated protein cage displaying reversible assembly. Nature 569, 438–442 (2019).

    Article  ADS  Google Scholar 

  120. Soltermann, F. et al. Quantifying protein–protein interactions by molecular counting with mass photometry. Angew. Chem. Int. Ed. 59, 10774–10779 (2020).

    Article  Google Scholar 

  121. Sendker, F. L. et al. Emergence of fractal geometries in the evolution of a metabolic enzyme. Nature 628, 894–900 (2024).

    Article  ADS  Google Scholar 

  122. Olerinyova, A. et al. Mass photometry of membrane proteins. Chem 7, 224–236 (2021).

    Article  Google Scholar 

  123. Heermann, T., Steiert, F., Ramm, B., Hundt, N. & Schwille, P. Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle. Nat. Methods 18, 1239–1246 (2021).

    Article  Google Scholar 

  124. Lee, S.-H. et al. Characterizing and tracking single colloidal particles with video holographic microscopy. Opt. Express 15, 18275–18282 (2007).

    Article  ADS  Google Scholar 

  125. Kashkanova, A. D. et al. Label-free discrimination of extracellular vesicles from large lipoproteins. J. Extracell. Vesicles 12, 12348 (2023).

    Article  Google Scholar 

  126. Andrecka, J., Spillane, K. M., Ortega-Arroyo, J. & Kukura, P. Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy. ACS Nano 7, 10662–10670 (2013).

    Article  Google Scholar 

  127. de Wit, G., Danial, J. S. H., Kukura, P. & Wallace, M. I. Dynamic label-free imaging of lipid nanodomains. Proc. Natl Acad. Sci. USA 112, 12299–12303 (2015).

    Article  ADS  Google Scholar 

  128. Goldfain, A. M., Garmann, R. F., Jin, Y., Lahini, Y. & Manoharan, V. N. Dynamic measurements of the position, orientation, and dna content of individual unlabeled bacteriophages. J. Phys. Chem. B 120, 6130–6138 (2016).

    Article  Google Scholar 

  129. Garmann, R. F., Goldfain, A. M. & Manoharan, V. N. Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1909223116 (2019).

  130. Bujak, Ł. et al. Fast leaps between millisecond confinements govern ase1 diffusion along microtubules. Small Methods 5, 2100370 (2021).

    Article  Google Scholar 

  131. Mickolajczyk, K. J., Geyer, E. A., Kim, T., Rice, L. M. & Hancock, W. O. Direct observation of individual tubulin dimers binding to growing microtubules. Proc. Natl Acad. Sci. USA 116, 7314–7322 (2019).

    Article  ADS  Google Scholar 

  132. Nguyen, T. L. et al. Quantitative phase imaging: recent advances and expanding potential in biomedicine. ACS Nano 16, 11516–11544 (2022).

    Article  Google Scholar 

  133. Park, J.-S., Lee, I.-B., Moon, H.-M., Hong, S.-C. & Cho, M. Long-term cargo tracking reveals intricate trafficking through active cytoskeletal networks in the crowded cellular environment. Nat. Commun. 14, 7160 (2023).

    Article  ADS  Google Scholar 

  134. Park, J.-S. et al. Fluorescence-combined interferometric scattering imaging reveals nanoscale dynamic events of single nascent adhesions in living cells. J. Phys. Chem. Lett. 11, 10233–10241 (2020).

    Article  Google Scholar 

  135. Cheng, C.-Y. & Hsieh, C.-L. Background estimation and correction for high-precision localization microscopy. ACS Photonics 4, 1730–1739 (2017).

    Article  Google Scholar 

  136. Hsiao, Y.-T., Tsai, C.-N., Chen, T.-H. & Hsieh, C.-L. Label-free dynamic imaging of chromatin in live cell nuclei by high-speed scattering-based interference microscopy. ACS Nano 16, 2774–2788 (2022).

    Article  Google Scholar 

  137. Limozin, L. & Sengupta, K. Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion. ChemPhysChem 10, 2752–2768 (2009).

    Article  Google Scholar 

  138. Sülzle, J. et al. Label-free imaging of DNA interactions with 2D materials. ACS Photonics 11, 737–744 (2024).

    Article  Google Scholar 

  139. Ginsberg, N. S. & Tisdale, W. A. Spatially resolved photogenerated exciton and charge transport in emerging semiconductors. Annu. Rev. Phys. Chem. 71, 1–30 (2020).

    Article  ADS  Google Scholar 

  140. Kennedy, C. L., Hill, A. H., Massaro, E. S. & Grumstrup, E. M. Ultrafast excited-state transport and decay dynamics in cesium lead mixed halide perovskites. ACS Energy Lett. 2, 1501–1506 (2017).

    Article  Google Scholar 

  141. Zhu, T., Snaider, J. M., Yuan, L. & Huang, L. Ultrafast dynamic microscopy of carrier and exciton transport. Annu. Rev. Phys. Chem. 70, 219–244 (2019).

    Article  ADS  Google Scholar 

  142. Akselrod, G. M. et al. Subdiffusive exciton transport in quantum dot solids. Nano Lett. 14, 3556–3562 (2014).

    Article  ADS  Google Scholar 

  143. Penwell, S. B., Ginsberg, L. D. S., Noriega, R. & Ginsberg, N. S. Resolving ultrafast exciton migration in organic solids at the nanoscale. Nat. Mater. 16, 1136–1141 (2017).

    Article  ADS  Google Scholar 

  144. Kulig, M. et al. Exciton diffusion and halo effects in monolayer semiconductors. Phys. Rev. Lett. 120, 207401 (2018).

    Article  ADS  Google Scholar 

  145. Balasubrahmaniyam, M. et al. From enhanced diffusion to ultrafast ballistic motion of hybrid light–matter excitations. Nat. Mater. 22, 338–344 (2023).

    Article  ADS  Google Scholar 

  146. Li, Y. et al. Light-assisted diazonium functionalization of graphene and spatial heterogeneities in reactivity. J. Phys. Chem. Lett. 10, 4788–4793 (2019).

    Article  Google Scholar 

  147. Li, W., Li, Y. & Xu, K. Azidated graphene: direct azidation from monolayers, click chemistry, and bulk production from graphite. Nano Lett. 20, 534–539 (2020).

    Article  ADS  Google Scholar 

  148. Li, W., Li, Y. & Xu, K. Facile, electrochemical chlorination of graphene from an aqueous NaCl solution. Nano Lett. 21, 1150–1155 (2021).

    Article  ADS  Google Scholar 

  149. Merryweather, A. J., Schnedermann, C., Jacquet, Q., Grey, C. P. & Rao, A. Operando optical tracking of single-particle ion dynamics in batteries. Nature 594, 522–528 (2021).

    Article  ADS  Google Scholar 

  150. Xu, C. et al. Operando visualization of kinetically induced lithium heterogeneities in single-particle layered Ni-rich cathodes. Joule 6, 2535–2546 (2022).

    Article  Google Scholar 

  151. Merryweather, A. J. et al. Operando monitoring of single-particle kinetic state-of-charge heterogeneities and cracking in high-rate Li-ion anodes. Nat. Mater. 21, 1306–1313 (2022).

    Article  ADS  Google Scholar 

  152. Shan, X., Patel, U., Wang, S., Iglesias, R. & Tao, N. Imaging local electrochemical current via surface plasmon resonance. Science 327, 1363–1366 (2010).

    Article  ADS  Google Scholar 

  153. Godeffroy, L., Ciocci, P., Lemineur, J.-F. & Kanoufi, F. Watching operando nanoscale electrochemical deposition by optical microscopy. Curr. Opin. Electrochem. 36, 101165 (2022).

    Article  Google Scholar 

  154. Ciocci, P., Lemineur, J.-F., Noël, J.-M., Combellas, C. & Kanoufi, F. Differentiating electrochemically active regions of indium tin oxide electrodes for hydrogen evolution and reductive decomposition reactions. An situ optical microscopy approach. Electrochim. Acta 386, 138498 (2021).

    Article  Google Scholar 

  155. Lemineur, J.-F., Noël, J.-M., Combellas, C. & Kanoufi, F. Optical monitoring of the electrochemical nucleation and growth of silver nanoparticles on electrode: from single to ensemble nanoparticles inspection. J. Electroanal. Chem. 872, 114043 (2020).

    Article  Google Scholar 

  156. Lemineur, J.-F. et al. Imaging and quantifying the formation of single nanobubbles at single platinum nanoparticles during the hydrogen evolution reaction. ACS Nano 15, 2643–2653 (2021).

    Article  Google Scholar 

  157. Vala, M., Palounek, D., Robert, H. M. L. & Piliarik, M. Quantitative detection of optical anisotropy of single microtubules by polarization-sensitive interferometric scattering microscopy. J. Phys. Appl. Phys. 54, 204001 (2021).

    Article  ADS  Google Scholar 

  158. Jiao, N., Lin, S., Feng, D., He, Y. & Chen, X.-W. Defocus-integration interferometric scattering microscopy for speckle suppression and enhancing nanoparticle detection on a substrate. Opt. Lett. 49, 2841–2844 (2024).

    Article  ADS  Google Scholar 

  159. Pang, G., Shen, C., Cao, L. & Hengel, A. V. D. Deep learning for anomaly detection: a review. ACM Comput. Surv. 54, 1–38 (2021).

    Article  Google Scholar 

  160. Jeppesen, D. K., Zhang, Q., Franklin, J. L. & Coffey, R. J. Extracellular vesicles and nanoparticles: emerging complexities. Trends Cell Biol. 33, 667–681 (2023).

    Article  Google Scholar 

  161. Morales-Inostroza, L. et al. An optofluidic antenna for enhancing the sensitivity of single-emitter measurements. Nat. Commun. 15, 2545 (2024).

    Article  ADS  Google Scholar 

  162. Wen, C., Bertosin, E., Shi, X., Dekker, C. & Schmid, S. Orientation-locked DNA origami for stable trapping of small proteins in the nanopore electro-osmotic trap. Nano Lett. 23, 788–794 (2023).

    Article  ADS  Google Scholar 

  163. Svirelis, J. et al. Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates. Nat. Commun. 14, 5131 (2023).

    Article  ADS  Google Scholar 

  164. Wieszczycka, K. et al. Surface functionalization — the way for advanced applications of smart materials. Coord. Chem. Rev. 436, 213846 (2021).

    Article  Google Scholar 

  165. Goswami, N. et al. Label-free SARS-CoV-2 detection and classification using phase imaging with computational specificity. Light. Sci. Appl. 10, 176 (2021).

    Article  ADS  Google Scholar 

  166. Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    Article  ADS  Google Scholar 

  167. Gu, M. Advanced Optical Imaging Theory. Springer Series in Optical Sciences Vol. 75 (Springer, 2000).

  168. Xia, Q. et al. Single virus fingerprinting by widefield interferometric defocus-enhanced mid-infrared photothermal microscopy. Nat. Commun. 14, 6655 (2023).

    Article  ADS  Google Scholar 

  169. Yurdakul, C., Zong, H., Bai, Y., Cheng, J.-X. & Ünlü, M. S. Bond-selective interferometric scattering microscopy. J. Phys. Appl. Phys. 54, 364002 (2021).

    Article  ADS  Google Scholar 

  170. LIGO Scientific Collaboration and Virgo Collaborationet al. Observation of gravitational waves from a binary black hole merger. Phys. Rev. Lett. 116, 061102 (2016).

    Article  ADS  MathSciNet  Google Scholar 

  171. Zumofen, G., Mojarad, N. M., Sandoghdar, V. & Agio, M. Perfect reflection of light by an oscillating dipole. Phys. Rev. Lett. 101, 180404 (2008).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

The authors thank their former and current team members who have contributed to the development of iSCAT over the past 20 years. The authors also thank R. Taylor and M. Miller for their comments on the manuscript. M.P. acknowledges funding by the Czech Science Foundation (project no. 23-07703S) and Operational Programme Johannes Amos Comenius financed by European Structural and Investment Funds and the Czech Ministry of Education, Youth and Sports (project no. SENDISO — CZ.02.01.01/00/22_008/0004596). N.S.G. acknowledges the support of the STROBE Center for Realtime Imaging, a National Science Foundation Science and Technology Center (grant DMR 1548924), the Center for Computational Study of Excited State Phenomena in Energy Materials (grant no. C2SEPEM) under the US Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division (contract no. DE-AC02-05CH11231), as part of the Computational Materials Sciences Program, and a David and Lucile Packard Fellowship. P.K. is funded by the European Research Council Consolidator (grant PHOTOMASS 819593) and the Engineering and Physical Research Council Leadership Fellowship EP/T03419X/1. V.S. thanks the Max Planck Society for continuous financial support.

Author information

Authors and Affiliations

  1. Departments of Chemistry and Physics, University of California Berkeley, Berkeley, CA, USA

    Naomi S. Ginsberg

  2. Materials Science Division, Chemical Science Division and Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Naomi S. Ginsberg

  3. STROBE, National Science Foundation Science and Technology Center, University of California Berkeley, Berkeley, CA, USA

    Naomi S. Ginsberg

  4. Kavli Energy NanoSciences Institute at Berkeley, Berkeley, CA, USA

    Naomi S. Ginsberg

  5. Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan

    Chia-Lung Hsieh

  6. The Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, UK

    Philipp Kukura

  7. Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, UK

    Philipp Kukura

  8. Institute of Photonics and Electronics of the Czech Academy of Sciences, Prague, Czech Republic

    Marek Piliarik

  9. Max Planck Institute for the Science of Light, Erlangen, Germany

    Vahid Sandoghdar

  10. Max-Planck-Zentrum für Physik und Medizin, Erlangen, Germany

    Vahid Sandoghdar

  11. Department of Physics, Friedrich-Alexander University Erlangen–Nürnberg, Erlangen, Germany

    Vahid Sandoghdar

Authors
  1. Naomi S. Ginsberg
    View author publications

    Search author on:PubMed Google Scholar

  2. Chia-Lung Hsieh
    View author publications

    Search author on:PubMed Google Scholar

  3. Philipp Kukura
    View author publications

    Search author on:PubMed Google Scholar

  4. Marek Piliarik
    View author publications

    Search author on:PubMed Google Scholar

  5. Vahid Sandoghdar
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Introduction (V.S., N.S.G. and P.K.); Experimentation (V.S., N.S.G., P.K. and C.-L.H.); Results (V.S., N.S.G., C.-L.H., P.K. and M.P.); Applications (V.S., N.S.G., C.-L.H., P.K. and M.P.); Reproducibility and data deposition (M.P., V.S. and C.-L.H.); Limitations and optimizations (V.S.); Outlook (V.S. and N.S.G.); overview of the Primer (V.S.).

Corresponding author

Correspondence to Vahid Sandoghdar.

Ethics declarations

Competing interests

P.K. is founder, shareholder and non-executive director of Refeyn Ltd. V.S. is a member of the Scientific Advisory Board of Bruker Switzerland AG. N.S.G., C.-L.H. and M.P. declare no competing interests.

Peer review

Peer review information

Nature Reviews Methods Primers thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Accumulation

Summing multiple measurements to improve the signal-to-noise ratio.

Airy function

A mathematical representation of the diffraction pattern created by a circular aperture.

Beam scanning mode

An imaging configuration whereby a focused beam is moved sequentially across the field of view.

Bit depth

The number of bits used in the digitalization process to encode the signal in each pixel, determining intensity resolution.

Extinction

A phenomenon in which energy is lost from a travelling beam owing to its interaction with an object.

Fresnel coefficients

Multiplicative coefficients that describe the ratio of the amplitude of an electric field of light upon reflection from and transmission through interfaces of two different materials.

Gouy phase

The phase shift experienced by a beam of light when confined in the transverse direction, as is the case for a Gaussian beam. The phase shift is strongest in the region of the focal plane.

Interference

A phenomenon in which two or more coherent waves are superposed, resulting in a wave of added amplitudes. Depending on the relative phases of each wave, the total amplitude at any given moment or location might be larger or smaller than the individual components.

Measurement integration time

The time interval over which a signal is accumulated.

Pixel well depth

The maximum number of photoelectrons that a detector pixel can accumulate before becoming saturated.

Point spread function

(PSF). A 3D intensity distribution describing the response of an imaging system to a point-like source.

Wide-field mode

An imaging configuration that captures a large area without scanning the illumination beam.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ginsberg, N.S., Hsieh, CL., Kukura, P. et al. Interferometric scattering microscopy. Nat Rev Methods Primers 5, 23 (2025). https://doi.org/10.1038/s43586-025-00391-1

Download citation

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1038/s43586-025-00391-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing