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J Microsc. 2012 May;246(2):113-23. doi: 10.1111/j.1365-2818.2012.03596.x. Epub 2012 Feb 22.

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy.

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  • 1Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77005, USA.


We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores' emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging.

© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

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