A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos

Jakub Nedbal; Francesco Mattioli Della Rocca; Iveta Ivanova; Andrew Allan; Jeremy Graham; Richard Walker; Robert K. Henderson; klaus suhling

doi:10.1038/s41598-024-56122-1

A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos

Jakub Nedbal, Francesco Mattioli Della Rocca, Iveta Ivanova, Andrew Allan, Jeremy Graham, Richard Walker, Robert K. Henderson, klaus suhling

School of Engineering

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192x128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with +38ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

Original language	English
Article number	7247
Number of pages	17
Journal	Scientific Reports
Volume	14
Issue number	1
Early online date	27 Mar 2024
DOIs	https://doi.org/10.1038/s41598-024-56122-1
Publication status	Published - Dec 2024

Keywords / Materials (for Non-textual outputs)

Algorithms
Microscopy, Fluorescence/methods
Single photon avalanche diode (SPAD)
FLIM video
Giant unilamellar vesicle (GUV)
Fluorescence lifetime imaging (FLIM)
Algae
Selective-plane illumination microscopy
Nonlinearity correction

Access to Document

10.1038/s41598-024-56122-1

https://www.nature.com/articles/s41598-024-56122-1Licence: Creative Commons: Attribution (CC-BY)

Development of wide-field TCSPC fluorescence microscopy for cell membrane studies
Henderson, R.
BBSRC
1/01/18 → 31/12/20
Project: Research

Cite this

Nedbal, J., Mattioli Della Rocca, F., Ivanova, I., Allan, A., Graham, J., Walker, R., Henderson, R. K., & suhling, K. (2024). A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos. Scientific Reports, 14(1), Article 7247. https://doi.org/10.1038/s41598-024-56122-1

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title = "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos",

abstract = "A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192x128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with +38ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.",

keywords = "Algorithms, Microscopy, Fluorescence/methods, Single photon avalanche diode (SPAD), FLIM video, Giant unilamellar vesicle (GUV), Fluorescence lifetime imaging (FLIM), Algae, Selective-plane illumination microscopy, Nonlinearity correction",

author = "Jakub Nedbal and {Mattioli Della Rocca}, Francesco and Iveta Ivanova and Andrew Allan and Jeremy Graham and Richard Walker and Henderson, {Robert K.} and klaus suhling",

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month = dec,

doi = "10.1038/s41598-024-56122-1",

language = "English",

volume = "14",

journal = "Scientific Reports",

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AU - Ivanova, Iveta

AU - Allan, Andrew

AU - Graham, Jeremy

AU - Walker, Richard

AU - Henderson, Robert K.

AU - suhling, klaus

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AB - A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192x128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with +38ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

KW - Algorithms

KW - Microscopy, Fluorescence/methods

KW - Single photon avalanche diode (SPAD)

KW - FLIM video

KW - Giant unilamellar vesicle (GUV)

KW - Fluorescence lifetime imaging (FLIM)

KW - Algae

KW - Selective-plane illumination microscopy

KW - Nonlinearity correction

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JO - Scientific Reports

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A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

Access to Document

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Projects

Development of wide-field TCSPC fluorescence microscopy for cell membrane studies

Cite this