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

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Engineering

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Technical Summary

FLIM is a key technique to image the interaction of proteins, and is independent of fluorophore concentration, which is hard to control in cells. Time-correlated single photon counting (TCSPC) FLIM has the highest sensitivity of all FLIM approaches, but while scanning TCSPC FLIM is routinely implemented, some microscopy methods are performed without beam scanning, employing wide-field camera-based detection instead, e.g. time-lapse and TIRF microscopy. There is a technology gap for wide-field TCSPC FLIM, and this proposal will fill that gap: advanced 192x256 pixel SPAD array cameras with on-pixel time-to-digital converters - the most advanced TCSPC imaging detectors to date - will be adapted for wide-field TCSPC-based FLIM and TIRF microscopy, and bespoke data management solutions for this application implemented. They provide the high level of sensitivity, specificity and speed required - without beam scanning - to elucidate the control of receptor self-association at the membrane of living cells and how this is regulated by inflammatory insults.

Regulation of epithelial cell junction integrity is vital to many processes including embryonic development, tissue homeostasis, wound healing and inflammation. One transmembrane receptor playing a role in these processes is the coxsackie virus adenovirus receptor (CAR). The crystal structure of the CAR D1 domain has shown that D1 is able to form homodimers in solution, but how CAR dimerisation is regulated in intact cells remains unknown. Understanding how, where and when CAR dimerises is essential to dissecting its role in controlling epithelial cell adhesion, but hampered by the limitations of currently available techniques such as standard biochemical or immunofluorescence analysis which compromises the cell and does not provide spatial or temporal information. Current TIRF microscopes lack the versatility, time-resolution and photon throughput capabilities to address this issue.

Planned Impact

In addition to the academic beneficiaries, commercial private sector beneficiaries may include STMicroelectronics who
have developed the first high volume products based on SPAD sensors ("flightsense") for time-of-flight ranging. A recent
spinoff company (PhotonForce) from the University of Edinburgh is commercialising SPAD image sensors in
scientific/medical applications and is a likely licensee of IP generated in the project. The SPAD arrays can be used for
range finding in mobile phones to switch off the display when the device is held to the ear, thus contributing to saving
energy - an important mass market development (see http://www.st.com/content/st_com/en/about/media-center/pressitem.
html/stmicroelectronics-proximity-sensor-solves-smartphone-hang-ups.html). Our development and refinement of
photon arrival timing techniques in this proposal may be able to further optimise this approach. Moreover, our novel
fluorescence and photon arrival time detection technology will lead to SPAD array cameras optimised specifically for timeresolved
fluorescence microscopy, which could be manufactured by PhotonForce. The proposed project would thus
facilitate their entry into the life sciences fluorescence microscopy market. We will also organise a workshop for the
academic community and industry in the final year of the project. Moreover, we will invite the industrial collaborators
STMicroelectronics and PhotonForce to join the project review meetings every 4 months either in person or via skype. This
would allow them to develop their applications alongside the main thrust of the project ensuring that beneficiaries are well
represented even at the genesis of the project. In the longer term, when the SPAD array technology developed in this
proposal is taken up by the biophotonics research community.
Beyond the field of fluorescence microscopy and inflammation, general photon time-of-flight measurement techniques such
as photon-counting light detection and ranging (lidar) and photon counting optical tomography would benefit significantly
from TCSPC detection. In lidar, single photon sensitivity and large number of pixels would allow rapid detection of reflected
laser pulses, speeding up the process of mapping an archaeological site, for example, or industrial processes such as lidarbased
non-contact inspection of car bodies or aircraft wings for fractures. In photon counting optical tomography, image
acquisition could be sped up by orders of magnitude, as currently only 10s of detectors are used to map the specimen, now
10s of 1000s could, in principle, be used.
The single photon sensitive SPAD array cameras with picosecond resolution will allow us to observe the moment a cell
responds to a chemical stimulus, and the location of that stimulus, at the level of single proteins. This will help us to
understand how inflammation occurs, on a molecular basis. The technology we will develop will dramatically improve our
understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the life
sciences - an area of great interest for the pharmaceutical industry. Their aim is to establish the efficacy of a new drug early
in its development and on a molecular basis, reducing the reliance on lengthy clinical trials. The pharmaceutical industry
saves money by adopting this approach, and patients benefit from an earlier availability of a new drug.
The public will also benefit from outreach activities, for example SPAD cameras were demonstrated at the Royal Society
Summer Science Exhibition 2014, and the PI frequently talks at events such as the Pint of Science festival and the Crick's
Science Museum Lates event "The Future of Biomedical Discovery", attracting 7000 visitors. He also oversaw the design
and creation of the fluorescence exhibit in the Physics stand at the Big Bang fair in London's ExCel exhibition centre in
2011, an event attracting more than 29,00
 
Title Figure 6A: Diffusing C. vulgaris cells, partly treated with DCMU, and imaged with fluorescence lifetime contrast. 
Description Video of fluorescence lifetime images acquired at one frame per second and showing diffusing C. vulgaris cells. The cells are live and dark acclimed and part treated with DCMU herbicide. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_6A_Diffusing_i_C_vulgaris_i_cells_partly_treated_with...
 
Title Figure 7A: Immobilized C. vulgaris cells, untreated, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live control C. vulgaris cells without DCMU treatment in a 3D fly-through. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is intrinsic without any drug effect.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7A_Immobilized_i_C_vulgaris_i_cells_untreated_and_ima...
 
Title Figure 7A: Immobilized C. vulgaris cells, untreated, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live control C. vulgaris cells without DCMU treatment in a rotating projection around the Y axis. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is intrinsic without any drug effect.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7A_Immobilized_i_C_vulgaris_i_cells_untreated_and_ima...
 
Title Figure 7B: Immobilized C. vulgaris cells, treated treated with DCMU, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized C. vulgaris cells treated with DCMU in a 3D fly-through. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7B_Immobilized_i_C_vulgaris_i_cells_treated_treated_w...
 
Title Figure 7B: Immobilized C. vulgaris cells, treated treated with DCMU, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized C. vulgaris cells treated with DCMU in a rotating projection around the Y axis. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7B_Immobilized_i_C_vulgaris_i_cells_treated_treated_w...
 
Title Figure 7C: Mixture of Immobilized C. vulgaris cells either treated with DCMU or not, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live C. vulgaris cells in a 3D axial stack fly-through. The cells were live and dark acclimed during the imaging. Part of the cells were untreated and part were treated with DCMU to form a mixture of both cell types. The fluorescence lifetime is differs between the cells in the field of view, depending on their exposure to DCMU.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7C_Mixture_of_Immobilized_i_C_vulgaris_i_cells_either...
 
Title Figure 7C: Mixture of Immobilized C. vulgaris cells either treated with DCMU or not, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the X axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live C. vulgaris cells in a 3D projection rotating around the X axis. The cells were live and dark acclimed during the imaging. Part of the cells were untreated and part were treated with DCMU to form a mixture of both cell types. The fluorescence lifetime is differs between the cells in the field of view, depending on their exposure to DCMU.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7C_Mixture_of_Immobilized_i_C_vulgaris_i_cells_either...
 
Title Figure 7D: Immobilized D. quadricauda cells, untreated, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live D. quadricauda cells in 3D axial fly-through. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is intrinsic without any drug effect.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7D_Immobilized_i_D_quadricauda_i_cells_untreated_and_...
 
Title Figure 7D: Immobilized D. quadricauda cells, untreated, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live D. quadricauda cells in a 3D projection rotating around the Y axis. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is intrinsic without any drug effect.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7D_Immobilized_i_D_quadricauda_i_cells_untreated_and_...
 
Title Figure 7E: Immobilized D. quadricauda cells, treated with DCMU, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live D. quadricauda cells treated with DCMU in a 3D axial fly-through. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7E_Immobilized_i_D_quadricauda_i_cells_treated_with_D...
 
Title Figure 7E: Immobilized D. quadricauda cells, treated with DCMU, and imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live D. quadricauda cells treated with DCMU in a 3D projection rotating around the Y axis. The cells were live and dark acclimed during the imaging. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_7E_Immobilized_i_D_quadricauda_i_cells_treated_with_D...
 
Title Figure 8A: Immobilized DPPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DPPC/cholesterol mixture and labelled with di-4-ANEPPDHQ dye in a 3D axial projection.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8A_Immobilized_DPPC_GUVs_imaged_with_fluorescence_lif...
 
Title Figure 8A: Immobilized DPPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DPPC/cholesterol mixture and labelled with di-4-ANEPPDHQ dye in a 3D projection rotating around the Y axis.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8A_Immobilized_DPPC_GUVs_imaged_with_fluorescence_lif...
 
Title Figure 8A: Immobilized DPPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DPPC/cholesterol mixture and labelled with di-4-ANEPPDHQ dye in a 3D projection rotating around the Y axis.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8A_Immobilized_DPPC_GUVs_imaged_with_fluorescence_lif...
 
Title Figure 8B: Immobilized DOPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DOPC and labelled with di-4-ANEPPDHQ dye in a 3D stack fly-through.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8B_Immobilized_DOPC_GUVs_imaged_with_fluorescence_lif...
 
Title Figure 8B: Immobilized DOPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D axial stack fly-through. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DOPC and labelled with di-4-ANEPPDHQ dye in a 3D stack fly-through.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8B_Immobilized_DOPC_GUVs_imaged_with_fluorescence_lif...
 
Title Figure 8B: Immobilized DOPC GUVs imaged with fluorescence lifetime contrast and selective plane illumination. 3D projection rotating around the Y axis. 
Description Video of fluorescence lifetime images acquired with 1 um axial spacing and showing immobilized live GUVs made of DOPC and labelled with di-4-ANEPPDHQ dye in a 3D projection rotating around the Y axis.This video is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Art Film/Video/Animation 
Year Produced 2024 
URL https://kcl.figshare.com/articles/media/Figure_8B_Immobilized_DOPC_GUVs_imaged_with_fluorescence_lif...
 
Description The 192x128 SPAD camera has been installed in a microscope in King's College and is generating images. King's College have now made a chracterisation and
benchmarking of the camera relative to other microscopy cameras.
Exploitation Route The microscope and camera system will be employed for research for many years to come. The sensor has been commercialized by Horiba as the Flimera camera and widely distributed to the research community. A second version of the original sensor has been submitted for manufacture to increase the number of available devices and increase the reliability and yield.
Sectors Healthcare

Pharmaceuticals and Medical Biotechnology

 
Description In this project we supported King's College London in the use of a 192 x 128 SPAD camera sensor. The same device has been commercialised by Horiba as Flimera and a second version of the device has been submitted for manufacture and licensed by the University to Horiba.
First Year Of Impact 2019
Sector Electronics,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Developing single-photon super-resolution microscopy
Amount £320,654 (GBP)
Funding ID EP/Y023137/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2024 
End 03/2027
 
Title Figure 5: Raw data, MATLAB scripts, and processed microalgae FLIM image stills 
Description Image still data in transmission and fluorescence lifetime contrast have been acquired with microalgaeSamples consisted of C. vulgaris and D. quadricauda cells, either treated with DCMU or left untreated. The cells were live and dark acclimed and part treated with DCMU herbicide. The fluorescence lifetime of those cells is increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching.The images were created using the accompanying makeFLIM.m script for MATLAB. The calibration data from the SPADlinarization package (https://doi.org/10.18742/21325584) will be needed to run this script.This dataset is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Figure_5_Raw_data_MATLAB_scripts_and_processed_microalgae_...
 
Title Figure 6: Raw data, MATLAB scripts, and processed time series video of diffusing C. vulgaris cells with FLIM contrast 
Description Image time series data with fluorescence lifetime contrast have been acquired with microalgae C. vulgaris. A mixture of cells treated with DCMU and untreated control cells was used The fluorescence lifetime of the DCMU-treated cells was increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching. The continuous stream of photon timestamps (11-Mar-2021_19_36_17_1000us_00001.mat) was segmented into individual 1-second integration time frames using the accompanying divideStream.m script for MATLAB.The video of the time series frames, analyzed in TRI2, was created by another script createVideo.m. The calibration data from the SPADlinarization package (https://doi.org/10.18742/21325584) will be needed to run this script.This dataset is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Figure_6_Raw_data_MATLAB_scripts_and_processed_time_series...
 
Title Figure 7: Raw data, MATLAB scripts, and processed videos of lightsheet image stacks of algae cells 
Description 3D volume stacks of images of algae cells embedded in agarose. C. vulgaris and D. quadricauda cells were either treated with DCMU, left untreated as controls and a mixture of treated and untreated C. vulgaris cells. The fluorescence lifetime of the DCMU-treated cells was increased due DCMU blocking electron acceptor oxidation and thus reaction center quenching. There are three folders in the package:Dquadricauda_lightsheetCvulgaris_lightsheetCvulgaris_mixture_lightsheetEach folder contains a MATLAB script makeFLIM.m, which produces videos and graphs from the SPAD output data analyzed in TRI2. The calibration data from the SPADlinearization package (https://doi.org/10.18742/21325584) will be needed to run this script.This dataset is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Figure_7_Raw_data_MATLAB_scripts_and_processed_videos_of_l...
 
Title Figure 8: Raw data, MATLAB scripts, and processed videos of lightsheet image stacks of GUVs 
Description 3D volume stacks of images of GUVs embedded in agarose. GUVs made of DPPC or DOPC were labelled with di-4-ANEPPDHQ. The fluorescence lifetime of the di-4-ANEPPDHQ changes with the membrane lipid packing state and thus the bilayer constituent.The MATLAB script makeFLIM.m produces videos and graphs from the SPAD output data analyzed in TRI2. The calibration data from the SPADlinearization package (https://doi.org/10.18742/21325584) will be needed to run this script.This dataset is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Figure_8_Raw_data_MATLAB_scripts_and_processed_videos_of_l...
 
Title Instrument Response Function Measurement for Macroscopic Painting Imaging 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the FLIM macroscopic imaging system. This dataset is an input to the MATLAB SPADlinearization.m script and is associated with the data in the article "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained from the macroscopic FLIM setup used without an emission filters and with a piece of office paper as the scattering sample. The dataset is used to create calibration structures for the two optical systems (macroscopic and microscopic). The calibration structures can be reproduced by running the SPADlinearization.m script. Both the resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Macr...
 
Title Instrument Response Function Measurement for Microscopic Painting Cross-Section Analysis 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the microscope. This dataset is an input to the MATLAB SPADlinearization.m script. It was used in the preparation of the data in manuscript "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained with a quenched fluorescein sample on a TCSPC microscope. The dataset is used to create calibration structures for the microscopic optical system. The calibration structures can be reproduced by running the SPADlinearization.m script on the raw data. The resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Cros...
 
Title Instrument Response Function Measurement for Painting Cross Section Analysis 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the microscope. This dataset is an input to the MATLAB SPADlinearization.m script. It was used in the preparation of the data in manuscript "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained with a quenched fluorescein sample on a TCSPC microscope. The dataset is used to create calibration structures for the microscopic optical system. The calibration structures can be reproduced by running the SPADlinearization.m script on the raw data. The resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Cros...
 
Title Instrument Response Function Measurement for Painting Cross Section Analysis 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the microscope. This dataset is an input to the MATLAB SPADlinearization.m script. It was used in the preparation of the data in manuscript "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained with a quenched fluorescein sample on a TCSPC microscope. The dataset is used to create calibration structures for the microscopic optical system. The calibration structures can be reproduced by running the SPADlinearization.m script on the raw data. The resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Cros...
 
Title Instrument Response Function Measurement for Painting Macroscopic Imaging 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the FLIM macroscopic imaging system. This dataset is an input to the MATLAB SPADlinearization.m script and is associated with the data in the article "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained from the macroscopic FLIM setup used without an emission filters and with a piece of office paper as the scattering sample. The dataset is used to create calibration structures for the two optical systems (macroscopic and microscopic). The calibration structures can be reproduced by running the SPADlinearization.m script. Both the resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Macr...
 
Title Instrument Response Function Measurement for Painting Macroscopic Imaging 
Description This is the dataset used for calibrating the SPAD camera timing skew and to generate an instrument response function for the FLIM macroscopic imaging system. This dataset is an input to the MATLAB SPADlinearization.m script and is associated with the data in the article "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". The measurement was obtained from the macroscopic FLIM setup used without an emission filters and with a piece of office paper as the scattering sample. The dataset is used to create calibration structures for the two optical systems (macroscopic and microscopic). The calibration structures can be reproduced by running the SPADlinearization.m script. Both the resulting calibration structure and the scripts are available in another dataset in this collection called "SPAD Linearization Code". 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://kcl.figshare.com/articles/dataset/Instrument_Response_Function_Measurement_for_Painting_Macr...
 
Title Figure 3: Explanation of TDC linearization through simulation 
Description The dataset contains a Mathworks MATLAB code rebinningSIM.m, which creates graphs that demonstrate the function of the Monte-Carlo algorithm for rebinning time-correlated single photon data from a SPAD array to correct for TDC linearization and timing offset. Run the code to generate a new set of images, change parameters of the code to alter these images. Each run of the code results in different output as it uses random number generator to simulate the data. This source code is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos"This software is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Technology Software 
Year Produced 2024 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/Figure_3_Explanation_of_TDC_linearization_through_simulat...
 
Title Figure 3: Explanation of TDC linearization through simulation 
Description The dataset contains a Mathworks MATLAB code rebinningSIM.m, which creates graphs that demonstrate the function of the Monte-Carlo algorithm for rebinning time-correlated single photon data from a SPAD array to correct for TDC linearization and timing offset. Run the code to generate a new set of images, change parameters of the code to alter these images. Each run of the code results in different output as it uses random number generator to simulate the data. This source code is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos"This software is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Technology Software 
Year Produced 2024 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/Figure_3_Explanation_of_TDC_linearization_through_simulat...
 
Title Figures 1, 2 & 4: Demonstration of linearization algorithm function and performance 
Description Graphs software package contains a script and input data to synthesize graphs found in Figures 1, 2 & 4 of the publication. It is a Mathworks MATLAB script. Running graphs.m will produce graphs contained in the publication.Graphs in Fig 2B and 2C are produced by the acquireHistogram.m script found in the SPADlinarization package (https://doi.org/10.18742/21325584)This software is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Technology Software 
Year Produced 2024 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/Figures_1_2_4_Demonstration_of_linearization_algorithm_fu...
 
Title SPAD Linearization Code 
Description This software package contains code for Mathworks MATLAB with some code written in C, but compiled for MATLAB for Linux and Windows. The code is used to process data from a wide-field SPAD array sensor with embedded time-to-digital converters. It has two functions: (1) Creating correction datasets from calibration measurements and (2) using these calibration measurements to correct data before their fluorescence lifetime analysis. The dataset contains tow calibration sets for a particular SPAD sensor chip. One dataset is for macroscopic imaging, the other one for microscopic imaging. They can be used to analyze the existing data, but calibration measurements for a different chip would need to be made for any data acquired on a different sensor. More information about the algorithm and its use is available from: Nedbal, J., Della Rocca, F.M., Walker, R., Henderson, R.K., Suhling, K.: Correction of time-resolved spad array measurements for accurate single-photon time-resolved biological imaging. In: Advanced Photon Counting Techniques XV, vol. 11721, pp. 65-78 (2021). SPIE. The code and the data are current as of the publication date of "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". Newer versions of the code may be found at: https://github.com/jnedbal/SPADcorrection 
Type Of Technology Software 
Year Produced 2023 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/SPAD_Linearization_Code/20411565
 
Title SPAD Linearization Code 
Description This software package contains code for Mathworks MATLAB with some code written in C, but compiled for MATLAB for Linux and Windows. The code is used to process data from a wide-field SPAD array sensor with embedded time-to-digital converters. It has two functions: (1) Creating correction datasets from calibration measurements and (2) using these calibration measurements to correct data before their fluorescence lifetime analysis. The dataset contains two calibration sets for a particular SPAD sensor chip. One dataset is for macroscopic imaging, the other one for microscopic imaging. They can be used to analyze the existing data, but calibration measurements for a different chip would need to be made for any data acquired on a different sensor. More information about the algorithm and its use is available from: Nedbal, J., Della Rocca, F.M., Walker, R., Henderson, R.K., Suhling, K.: Correction of time-resolved SPAD array measurements for accurate single-photon time-resolved biological imaging. In: Advanced Photon Counting Techniques XV, vol. 11721, pp. 65-78 (2021). SPIE. The code and the data are current as of the publication date of "Visualising Varnish Removal for Conservation of Paintings by Fluorescence Lifetime Imaging (FLIM)". Newer versions of the code may be found at: https://github.com/jnedbal/SPADcorrection 
Type Of Technology Software 
Year Produced 2023 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/SPAD_Linearization_Code/20411565/1
 
Title SPAD linearization code for processing calibration and measurement data from the SPAD camera 
Description The SPADlinearization software contains a Mathworks MATLAB script SPADcorrection.m. This is used to create calibration data from measurements of dark count map (dark sensor), instrument response function (pulsed light illuminated sensor), and code density map (constant light illuminated sensor) acquired on the microscope with the QuantiCam camera. The script produces calibration data. There are two calibration datasets in the filebinCorrection78MHz_NKT_chip12_Allura.full.matbinCorrection78MHz_NKT_chip12_FITC.full.matDiffering in the calibration solution (Allura Red or quenched fluorescein) and the fluorescence wavelength measured (667 nm and 535 nm, respectively). These calibration measurements are only suitable for the single sensor and optical setup used in the published experiments and is not generally applicable. The calibrations need to be done for each optical setup and sensor separately.processFLIM.m is a script used to process fluroescence decay measurement data from the QuantiCam SPAD camera. This script was used to process all measurement data presented in the publication.QuantiCAM_calibration_and_measurement_guide.pdf is a protocol explaining how calibration data is acquired and processed and how fluorescence lifetime measurements are processed.Most recent version of the source code is available at:https://www.github.com/jnedbal/SPADcorrection/This software is a supplement to the article "A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos" 
Type Of Technology Software 
Year Produced 2024 
Open Source License? Yes  
URL https://kcl.figshare.com/articles/software/SPAD_linearization_code_for_processing_calibration_and_me...
 
Company Name Singular Photonics 
Description Singular Photonics manufactures semiconductors for applications in 4D imaging. 
Year Established 2024 
Impact Early days, recently launched with £400k investment from Scottish Enterprise.
Website https://www.singularphotonics.com/