Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy

Marina Parker; Samuel A. Mayes; Craig M. Browning; Joshua Deal; Samantha Gunn-Mayes; Naga S. Annamdevula; Thomas C. Rich; Silas J. Leavesley

doi:10.1117/1.JBO.28.2.026502

7 February 2023 Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy

Marina Parker, Samuel A. Mayes, Craig M. Browning, Joshua Deal, Samantha Gunn-Mayes, Naga S. Annamdevula, Thomas C. Rich, Silas J. Leavesley

Author Affiliations +

Journal of Biomedical Optics, Vol. 28, Issue 2, 026502 (February 2023). https://doi.org/10.1117/1.JBO.28.2.026502

Abstract

Significance

Hyperspectral imaging (HSI) technologies offer great potential in fluorescence microscopy for multiplexed imaging, autofluorescence removal, and analysis of autofluorescent molecules. However, there are also associated trade-offs when implementing HSI in fluorescence microscopy systems, such as decreased acquisition speed, resolution, or field-of-view due to the need to acquire spectral information in addition to spatial information. The vast majority of HSI fluorescence microscopy systems provide spectral discrimination by filtering or dispersing the fluorescence emission, which may result in loss of emitted fluorescence signal due to optical filters, dispersive optics, or supporting optics, such as slits and collimators. Technologies that scan the fluorescence excitation spectrum may offer an approach to mitigate some of these trade-offs by decreasing the complexity of the emission light path.

Aim

We describe the development of an optical technique for hyperspectral imaging fluorescence excitation-scanning (HIFEX) on a microscope system.

Approach

The approach is based on the design of an array of wavelength-dependent light emitting diodes (LEDs) and a unique beam combining system that uses a multifurcated mirror. The system was modeled and optimized using optical ray trace simulations, and a prototype was built and coupled to an inverted microscope platform. The prototype system was calibrated, and initial feasibility testing was performed by imaging multilabel slide preparations.

Results

We present results from optical ray trace simulations, prototyping, calibration, and feasibility testing of the system. Results indicate that the system can discriminate between at least six fluorescent labels and autofluorescence and that the approach can provide decreased wavelength switching times, in comparison with mechanically tuned filters.

Conclusions

We anticipate that LED-based HIFEX microscopy may provide improved performance for time-dependent and photosensitive assays.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Citation Download Citation

Marina Parker, Samuel A. Mayes, Craig M. Browning, Joshua Deal, Samantha Gunn-Mayes, Naga S. Annamdevula, Thomas C. Rich, and Silas J. Leavesley "Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy," Journal of Biomedical Optics 28(2), 026502 (7 February 2023). https://doi.org/10.1117/1.JBO.28.2.026502

Received: 3 October 2022; Accepted: 17 January 2023; Published: 7 February 2023

ACCESS THE FULL ARTICLE

JOURNAL ARTICLE
21 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY