Design and fabrication of universal resolution targets for microsphere-assisted microscopy

Vahid Abbasian, Kashif Masud Awan, Arash Darafsheh

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review


Determining the achieved spatial resolution in microsphere-assisted microscopy (MAM) has remained an important and challenging task. While fluorescent-based microscopies use fluorescent nano-beads to determine the point spread function (PSF), such a method is not practical in label-free super-resolution techniques. In some previous studies, objects with certain features have been imaged and those feature sizes have been reported as the resolution; however, determining the resolution in that way is not consistent with the definition of resolution based on point sources. Furthermore, the imaging characteristics in MAM may depend on specimen-specific properties, such as materials and substrate. In this study, we propose a robust and objective technique to address these issues. We fabricate arrays of nano-pillars varying in diameter and periodicity, ranging from 50 to 1000 nm. These pillars consist of a 20 nm Ag layer with a 2 nm Cr adhesion layer, deposited on a silicon substrate. The role of specimen-specific properties can be investigated by considering different materials. Fabrication involves the bilayer PMMA resist lift-off process, ensuring accurate deposition of nanometer-scale features to serve as sub-diffraction-limited targets in optical super-resolution microscopy. A deconvolution method that provides a robust platform for resolution measurement was used. By deconvolving the object profile, obtained from scanning electron microscopy, from the image profile, the PSF of the system is determined. Our proposed technique offers a reliable and standardized approach for quantifying resolution, magnification, and field-of-view in MAM or any other label-free microscopy technique, enabling accurate and objective comparison across different novel microscopy techniques.

Original languageEnglish
Title of host publicationThree-Dimensional and Multidimensional Microscopy
Subtitle of host publicationImage Acquisition and Processing XXXI
EditorsThomas G. Brown, Tony Wilson, Laura Waller
ISBN (Electronic)9781510669550
StatePublished - 2024
EventThree-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXXI 2024 - San Francisco, United States
Duration: Jan 31 2024Feb 1 2024

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceThree-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXXI 2024
Country/TerritoryUnited States
CitySan Francisco


  • Microscopy
  • Microsphere-assisted microscopy
  • Microsphere.
  • Resolution


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