A custom two-in-one HIST and line-scanning confocal excitation module
- 1University of North Carolina at Chapel Hill
Abstract
Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.
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BibTeX
@misc{arthur2026custom,
title = {{A custom two-in-one HIST and line-scanning confocal excitation module}},
author = {C. Arthur and D. E. Milkie and A. J. Ulmer and J. P. Ellis and H. K. Kim and F. Song and A. Chavez and W. R. Legant},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751507},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751507v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A custom two-in-one HIST and line-scanning confocal excitation module
AU - C. Arthur
AU - D. E. Milkie
AU - A. J. Ulmer
AU - J. P. Ellis
AU - H. K. Kim
AU - F. Song
AU - A. Chavez
AU - W. R. Legant
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751507
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751507v1
AB - Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.
ER -