Simultaneous multi‐spectral, single‐photon fluorescence imaging using a plasmonic colour filter array

• Published in: Journal of biophotonics Vol. 14; no. 7; pp. e2746 - n/a
• Main Authors: Connolly, Peter W. R., Valli, Jessica, Shah, Yash D., Altmann, Yoann, Grant, James, Accarino, Claudio, Rickman, Colin, Cumming, David R. S., Buller, Gerald S.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY‐VCH Verlag GmbH & Co. KGaA 01.07.2021; Wiley Subscription Services, Inc
• Subjects: Algorithms; Aluminum; Arrays; Avalanche diodes; Biological properties; Biological samples; CFA; Color; colour filter array; FLIM; Fluorescence; Glass substrates; Gratings (spectra); image processing; Image reconstruction; imaging; metasurface; microscopy; Photon avalanches; Photons; Pixels; plasmonic; Plasmonics; single‐photon avalanche diode; SPAD; Spectra; Thin films; Transmission efficiency; image processing; colour filter array; fluorescence; plasmonic; single-photon avalanche diode; SPAD; FLIM; microscopy; imaging; CFA; metasurface
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.202000505

We present the first realisation of simultaneous multi‐spectral fluorescence imaging using a single‐photon avalanche diode (SPAD) array, where the spectral unmixing is facilitated by a plasmonic metasurface mosaic colour filter array (CFA). A 64 × 64 pixel format silicon SPAD array is used to record widefield fluorescence and brightfield data from four biological samples. A plasmonic metasurface composed of an arrangement of circular and elliptical nanoholes etched into an aluminium thin film deposited on a glass substrate provides the high transmission efficiency CFA, enabling a bespoke spectral unmixing algorithm to reconstruct high fidelity, full colour images from as few as ∼3 photons per pixel. This approach points the way toward real‐time, single‐photon sensitive multi‐spectral fluorescence imaging. Furthermore, this is possible without additional bulky components such as a filter wheel, prism or diffraction grating, nor the need for multiple sample exposures or multiple detectors. This paper demonstrates multispectral fluorescence imaging with the samples undergoing excitation illumination from multiple wavelengths simultaneously. The multi‐spectral fluorescence passes through a mosaic plasmonic colour filter array affixed to a SPAD array. This allows for a quick spectral unmixing process and allows high quality multispectral detection and image reconstruction using shorter exposures, significantly quicker than equivalent automated filter‐wheel based systems, and without the need for image splitting prisms or gratings.