Near-isotropic super-resolution microscopy with axial interference speckle illumination

Hajun Yoo, Kwanhwi Ko, Sukhyeon Ka, Gwiyeong Moon, Hyunwoong Lee, Seongmin Im, Peng Xi and Donghyun Kim ()
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Hajun Yoo: Yonsei University
Kwanhwi Ko: Yonsei University
Sukhyeon Ka: Yonsei University
Gwiyeong Moon: Yonsei University
Hyunwoong Lee: Yonsei University
Seongmin Im: Yonsei University
Peng Xi: College of Future TechnologyPeking University
Donghyun Kim: Yonsei University

Nature Communications, 2025, vol. 16, issue 1, 1-11

Abstract: Abstract Super-resolution microscopy has pushed the limits of biological imaging. However, achieving isotropic resolution across all spatial dimensions remains a challenge and often requires a complex and highly sensitive optical setup. Herein, we introduce axial interference speckle illumination-engineered structured illumination microscopy (AXIS-SIM), a minimal-modification approach that utilizes constructive interference from a simple back-reflecting mirror to enhance the axial resolution without additional phase control or complex beam shaping. AXIS-SIM provides superior optical sectioning and improves axial resolution beyond the typical axial resolution of conventional 3D-structured illumination microscopy (~300 nm), achieving lateral and axial resolutions of 108.5 and 140.1 nm, respectively. Furthermore, its robustness against alignment errors and sample-induced aberrations enables high-throughput 3D super-resolution imaging of diverse biological specimens. We demonstrate its potential by visualizing the 3D morphology of cell membranes, resolving the nanoscale distribution of lysosomes and microtubules and tracking lysosomal movements with enhanced axial clarity.

Date: 2025
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DOI: 10.1038/s41467-025-64366-2

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