Chip-based label-free incoherent super-resolution optical microscopy

Nikhil Jayakumar; Luis E. Villegas-Hernández; Weisong Zhao; Hong Mao; Firehun T. Dullo; Jean-Claude Tinguely; Krizia Sagini; Alicia Llorente; Balpreet Singh Ahluwalia

doi:10.1038/s41377-025-01914-x

Your Location：

Home >

Browse articles >

Chip-based label-free incoherent super-resolution optical microscopy

Original Articles

- Chip-based label-free incoherent super-resolution optical microscopy
- Light: Science & Applications Vol. 14, Issue 9, Pages: 2772-2783(2025)
- Affiliations：
  
  1.Department of Physics and Technology, UiT The Arctic University of Norway, Tromsø 9037, Norway
  2.Innovation Photonics and Imaging Center, State Key Laboratory of Space Environment Interation with Matters, Key Laboratory of Ultra-Precision Intelligent Instrumentation of Ministry of Industry and Information Technology, School of Instrumentation Science and Engineering, Harbin Institute of Technology, Harbin 150080, China
  3.SINTEF Digital department of Microsystems and Nanotechnology, Gaustadalleen 23C, 0373 Oslo, Norway
  4.Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, 0379 Oslo, Norway
  5.Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, Montebello 0379 Oslo, Norway
  6.Department for Mechanical, Electronics and Chemical Engineering, Oslo Metropolitan University, Oslo, Norway
  7.Department of Clinical Science, Intervention and Technology, Karolinska Institute, Solna, Sweden
- Author bio：
  
  Nikhil Jayakumar (nik.jay.hil@gmail.com)
  Balpreet Singh Ahluwalia (Balpreet.singh.ahluwalia@uit.no)
- Funds：
- DOI：10.1038/s41377-025-01914-x
  CLC：
- Received：16 September 2024，
  
  Revised：2025-05-27，
  
  Accepted：31 May 2025，
  
  Published Online：04 August 2025，
  
  Published：30 September 2025
- Accepted：
Scan QR Code
Jayakumar, N. et al. Chip-based label-free incoherent super-resolution optical microscopy. Light: Science & Applications, 14, 2772-2783 (2025).
DOI：

Jayakumar, N. et al. Chip-based label-free incoherent super-resolution optical microscopy. Light: Science & Applications, 14, 2772-2783 (2025). DOI： 10.1038/s41377-025-01914-x.

摘要

Abstract

The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction

limit. Despite its enormous potential

the necessity to label the sample may adversely influence the delicate biology under investigation. Thus

continued development efforts are needed to surpass the far-field label-free diffraction barrier. The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging. In this article

we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence (PL) of silicon nitride waveguides for near-field illumination of unlabeled samples. The technique is abbreviated EPSLON

Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy. We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules

i.e.

such an illumination permits incoherence between the scattered fields from various locations on the sample plane. Thus

the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time

thereby permitting the application of techniques like structured illumination microscopy (SIM) and intensity-fluctuation-based optical nanoscopy (IFON) in label-free mode to circumvent the diffraction limit. In this proof-of-concept work

we observed a two-point resolution of

$$ \sim $$

180 nm on super-resolved nanobeads and resolution improvements between 1.9× to 2.8× over the diffraction limit

as quantified using Fourier Ring Correlation (FRC)

on various biological samples. We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.

关键词

Keywords

references

Wolf, E. Introduction to the Theory of Coherence and Polarization of Light . (Cambridge University Press, Cambridge, 2007).

Fujimoto, J. G. et al. Optical coherence tomography: an emerging technology for biomedical imaging and optical biopsy. Neoplasia 2 , 9–25 (2000)..

Heintzmann, R. & Cremer, C. G. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proceedings of SPIE 3568, Optical Biopsies and Microscopic Techniques Ⅲ . (SPIE, Stockholm, Sweden, 1999) pp 185–196.

Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198 , 82–87 (2000)..

Dertinger, T. et al. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106 , 22287–22292 (2009)..

Gustafsson, N. et al. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 7 , 12471 (2016)..

Yahiatene, I. et al. Entropy-based super-resolution imaging (ESI): from disorder to fine detail. ACS Photonics 2 , 1049–1056 (2015)..

Cox, S. et al. Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat. Methods 9 , 195–200 (2012)..

Zhao, W. S. et al. Enhanced detection of fluorescence fluctuations for high-throughput super-resolution imaging. Nat. Photonics 17 , 806–813 (2023)..

Agarwal, K. & Macháň, R. Multiple signal classification algorithm for super-resolution fluorescence microscopy. Nat. Commun. 7 , 13752 (2016)..

Ryabukho, V. P. et al. Wiener–Khintchin theorem for spatial coherence of optical wave field. J. Opt. 15 , 025405 (2013)..

Goodman, J. W. Speckle Phenomena in Optics: Theory and Applications . (Roberts and Company Publishers, Englewood, 2007).

Wicker, K. & Heintzmann, R. Resolving a misconception about structured illumination. Nat. Photonics 8 , 342–344 (2014)..

Kistner, J. et al. Photoluminescence from silicon nitride—no quantum effect. J. Appl. Phys. 110 , 023520 (2011)..

Coucheron, D. A. et al. Study of waveguide background at visible wavelengths for on-chip nanoscopy. Opt. Express 29 , 20735–20746 (2021)..

Diekmann, R. et al. Chip-based wide field-of-view nanoscopy. Nat. Photonics 11 , 322–328 (2017)..

Jayakumar, N. et al. On-chip TIRF nanoscopy by applying Haar wavelet kernel analysis on intensity fluctuations induced by chip illumination. Opt. Express 28 , 35454–35468 (2020)..

Villegas- Hernández, L. E. et al. Chip-based multimodal super-resolution microscopy for histological investigations of cryopreserved tissue sections. Light 11 , 43 (2022)..

Kim, M. et al. Superresolution imaging with optical fluctuation using speckle patterns illumination. Sci. Rep. 5 , 16525 (2015)..

Choi, Y. et al. Wide-field super-resolution optical fluctuation imaging through dynamic near-field speckle illumination. Nano Lett. 22 , 2194–2201 (2022)..

Zernike, F. How I discovered phase contrast. Science 121 , 345–349 (1955)..

Munro, P. R. T. & Török, P. Vectorial, high numerical aperture study of Nomarski's differential interference contrast microscope. Opt. Express 13 , 6833–6847 (2005)..

Hoffman, R. The modulation contrast microscope: principles and performance. J. Microsc. 110 , 205–222 (1977)..

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

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

Baek, Y. & Park, Y. Intensity-based holographic imaging via space-domain Kramers–Kronig relations. Nat. Photonics 15 , 354–360 (2021)..

Zheng, G. A., Horstmeyer, R. & Yang, C. Wide-field, high-resolution Fourier ptychographic microscopy. Nat. Photonics 7 , 739–745 (2013)..

Ruh, D. et al. Superior contrast and resolution by image formation in rotating coherent scattering (ROCS) microscopy. Optica 5 , 1371–1381 (2018)..

Redding, B., Choma, M. A. & Cao, H. Speckle-free laser imaging using random laser illumination. Nat. Photonics 6 , 355–359 (2012)..

Ojaghi, A. et al. Ultraviolet hyperspectral interferometric microscopy. Sci. Rep. 8 , 9913 (2018)..

Liu, X. W. et al. Fluorescent nanowire ring illumination for wide-field far-field subdiffraction imaging. Phys. Rev. Lett. 118 , 076101 (2017)..

Jayakumar, N. et al. Multi-moded high-index contrast optical waveguide for super-contrast high-resolution label-free microscopy. Nanophotonics 11 , 3421–3436 (2022)..

Ilovitsh, T. et al. Cellular imaging using temporally flickering nanoparticles. Sci. Rep. 5 , 8244 (2015)..

Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21 , 72–84 (2019)..

Betzig, E. & Chichester, R. J. Single molecules observed by near-field scanning optical microscopy. Science 262 , 1422–1425 (1993)..

Pendry, J. B. Negative refraction makes a perfect lens. Phys. Rev. Lett. 85 , 3966–3969 (2000)..

Wang, Z. B. et al. Optical virtual imaging at 50 nm lateral resolution with a white-light nanoscope. Nat. Commun. 2 , 218 (2011)..

Darafsheh, A. et al. Advantages of microsphere-assisted super-resolution imaging technique over solid immersion lens and confocal microscopies. Appl. Phys. Lett. 104 , 061117 (2014)..

Rogers, E. T. F. et al. A super-oscillatory lens optical microscope for subwavelength imaging. Nat. Mater. 11 , 432–435 (2012)..

Barsi, C. & Fleischer, J. W. Nonlinear abbe theory. Nat. Photonics 7 , 639–643 (2013)..

Zalevsky, Z & Mendlovic, D. Optical Superresolution . (Springer, New York, 2004).

Gur, A. et al. Linear optics based nanoscopy. Opt. Express 18 , 22222–22231 (2010)..

Lin, C. P. et al. UV photonic integrated circuits for far-field structured illumination autofluorescence microscopy. Nat. Commun. 13 , 4360 (2022)..

Dong, B. Q. et al. Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast. Proc. Natl Acad. Sci. USA 113 , 9716–9721 (2016)..

Astratov, V. Label-Free Super-Resolution Microscopy . (Springer, Cham, 2019).

Cotte, Y. et al. Marker-free phase nanoscopy. Nat. Photonics 7 , 113–117 (2013)..

Maire, G. et al. Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy. Opt. Lett. 43 , 2173–2176 (2018)..

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

Ströhl, F. et al. Super-condenser enables label free nanoscopy. Opt. Express 27 , 25280–25292 (2019)..

Tang, M. W. et al. High-refractive-index chip with periodically fine-tuning gratings for tunable virtual-wavevector spatial frequency shift universal super-resolution imaging. Adv. Sci. 9 , 2103835 (2022)..

Pang, C. L. et al. On-chip super-resolution imaging with fluorescent polymer films. Adv. Funct. Mater. 29 , 1900126 (2019)..

Lee, Y. U. et al. Hyperbolic material enhanced scattering nanoscopy for label-free super-resolution imaging. Nat. Commun. 13 , 6631 (2022)..

Goodman, J. W. Introduction to Fourier Optics . 2nd edn. (Roberts & Company, Greenwood Village, 2005).

Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102 , 13081–13086 (2005)..

Berezin, M. Y. & Achilefu, S. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110 , 2641–2684 (2010)..

Chowdhury, S., Dhalla, A. H. & Izatt, J. Structured oblique illumination microscopy for enhanced resolution imaging of non-fluorescent, coherently scattering samples. Biomed. Opt. Express 3 , 1841–1854 (2012)..

Wolf, E. A macroscopic theory of interference and diffraction of light from finite sources Ⅱ. Fields with a spectral range of arbitrary width. Proc. R. Soc. A 230 , 246–265 (1955)..

Prieto, F. et al. An integrated optical interferometric nanodevice based on silicon technology for biosensor applications. Nanotechnology 14 , 907–912 (2003)..

Tinguely, J. C., Helle, Ø. I. & Ahluwalia, B. S. Silicon nitride waveguide platform for fluorescence microscopy of living cells. Opt. Express 25 , 27678–27690 (2017)..

Dhakal, A. et al. Silicon nitride background in nanophotonic waveguide enhanced Raman spectroscopy. Materials 10 , 140 (2017)..

Aydinli, A., Serpengüzel, A. & Vardar, D. Visible photoluminescence from low temperature deposited hydrogenated amorphous silicon nitride. Solid State Commun. 98 , 273–277 (1996)..

Vlasukova, L. A. et al. Optical properties of silicon nitride films formed by plasma-chemical vapor deposition. J. Appl. Spectrosc. 80 , 89–92 (2013)..

Giorgis, F., Vinegoni, C. & Pavesi, L. Optical absorption and photoluminescence properties of a −Si 1 − x N x : H films deposited by plasma-enhanced CVD. Phys. Rev. B 61 , 4693–4698 (2000)..

Smith, J. et al. Single photon emission and single spin coherence of a nitrogen vacancy center encapsulated in silicon nitride. Appl. Phys. Lett. 116 , 134001 (2020)..

Helle, Ø. I. et al. Structured illumination microscopy using a photonic chip. Nat. Photonics 14 , 431–438 (2020)..

Engdahl, A. K. et al. Large field-of-view super-resolution optical microscopy based on planar polymer waveguides. ACS Photonics 8 , 1944–1950 (2021)..

Opstad, I. S. et al. Fluorescence fluctuations-based super-resolution microscopy techniques: an experimental comparative study. Preprint at arXiv https://doi.org/10.48550/arXiv.2008.09195 https://doi.org/10.48550/arXiv.2008.09195 (2020)..

Patrakka, J. et al. The number of podocyte slit diaphragms is decreased in minimal change nephrotic syndrome. Pediatr. Res. 52 , 349–355 (2002)..

Siegerist, F. et al. Super-resolution microscopy: a technique to revolutionize research and diagnosis of glomerulopathies. Glomerular Dis. 3 , 19–28 (2022)..

Zhao, W. S. et al. Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation. Light 12 , 298 (2023)..

Donczo, B. & Guttman, A. Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: the Holy Grail for molecular diagnostics. J. Pharm. Biomed. Anal. 155 , 125–134 (2018)..

Akilesh, S. Normal kidney function and structure. in: Pathobiology of Human Disease (eds McManus, L. M. & Mitchell, R. N. ) (Amsterdam: Academic Press, 2014) pp 2716–2733.

Duan, S. Y. et al. The thickness of glomerular basement membrane predicts complete remission in primary membranous nephropathy. Ren. Fail. 45 , 2179335 (2023)..

Chang, B. J. et al. Subdiffraction scattered light imaging of gold nanoparticles using structured illumination. Opt. Lett. 36 , 4773–4775 (2011)..

Jayakumar, N. & Ahluwalia, B. S. From superior contrast to super resolution label free optical microscopy. npj Imaging 3 , 1 (2025)..

Sage, D. et al. DeconvolutionLab2: an open-source software for deconvolution microscopy. Methods 115 , 28–41 (2017)..

Chazot, C. A. C. et al. Luminescent surfaces with tailored angular emission for compact dark-field imaging devices. Nat. Photonics 14 , 310–315 (2020)..

Kuai, Y. et al. Planar photonic chips with tailored angular transmission for high-contrast-imaging devices. Nat. Commun. 12 , 6835 (2021)..

Kandel, M. E. et al. Phase imaging with computational specificity (PICS) for measuring dry mass changes in sub-cellular compartments. Nat. Commun. 11 , 6256 (2020)..

Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19 , 780–782 (1994)..

Views

Downloads

CSCD

Alert me when the article has been cited

Submit

Tools

Publicity Resources

No data

Related Author

No data

Related Institution

No data

⁰