Abstract

The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout rate of 80 megapixels per second. In this study, we coupled the camera to an image intensifier, a 760 nm emission filter and a 50 mm lens, and with a super-bright 627nm LED providing pulsed excitation of a 18 × 18 mm sample area. The resulting macro-imager with compact and rigid optical alignment of its main components was characterised using planar phosphorescent O2 sensors and a resolution plate mask. Several acquisition and image processing algorithms were evaluated to optimise the system resolution and performance for the wide-field PLIM, followed by imaging a variety of phosphorescent samples. The new PLIM system looks promising, particularly for phosphorescence lifetime-based imaging of O2 in various chemical and biological samples.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2019 (4)

J. T. Sharick, J. J. Jeffery, M. R. Karim, C. M. Walsh, K. Esbona, R. S. Cook, and M. C. Skala, “Cellular Metabolic Heterogeneity In Vivo Is Recapitulated in Tumor Organoids,” Neoplasia (N. Y., NY, U. S.) 21(6), 615–626 (2019).
[Crossref]

A. Nomerotski, “Imaging and time stamping of photons with nanosecond resolution in Timepix based optical cameras,” Nucl. Instrum. Methods Phys. Res., Sect. A 937, 26–30 (2019).
[Crossref]

S. Procz, C. A. Avila, J. Fey, G. A. Roque, M. Schuetz, and E. Hamann, “X-ray and gamma imaging with Medipix and Timepix detectors in medical research,” Radiat. Meas. 127, 106104 (2019).
[Crossref]

S. Tsigaridas, M. V. Beuzekom, H. V. D. Graaf, F. Hartjes, K. Heijhoff, N. P. Hessey, P. J. de Jong, and V. Prodanovic, “Timewalk correction for the Timepix3 chip obtained with real particle data,” Nucl. Instrum. Methods Phys. Res., Sect. A 930(April), 185–190 (2019).
[Crossref]

2018 (3)

M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
[Crossref]

Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
[Crossref]

K. Zheng, T. P. Jensen, and D. A. Rusakov, “Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging,” Nat. Protoc. 13(3), 581–597 (2018).
[Crossref]

2017 (9)

C. Dysli, S. Wolf, M. Y. Berezin, L. Sauer, M. Hammer, and M. S. Zinkernagel, “Fluorescence lifetime imaging ophthalmoscopy,” Prog. Retinal Eye Res. 60, 120–143 (2017).
[Crossref]

L. Wei, W. Yan, and D. Ho, “Recent advances in fluorescence lifetime analytical microsystems: Contact optics and CMOS time-resolved electronics,” Sensors 17(12), 2800 (2017).
[Crossref]

L. M. Hirvonen and K. Suhling, “Wide-field TCSPC: Methods and applications,” Meas. Sci. Technol. 28(1), 012003 (2017).
[Crossref]

H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
[Crossref]

W. Zarychta-Wiśniewska, A. Burdzinska, R. Zagozdzon, B. Dybowski, M. Butrym, Z. Gajewski, and L. Paczek, “In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models,” PLoS One 12(9), e0184588 (2017).
[Crossref]

V. Tsytsarev, F. Akkenti, E. Pumbo, Q. Tang, Y. Chen, R. S. Erzurumlu, and D. B. Papkovsky, “Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue,” J. Neurosci. Methods 281, 1–6 (2017).
[Crossref]

A. Nomerotski, I. Chakaberia, M. Fisher-Levine, Z. Janoska, P. Takacs, and T. Tsang, “Characterization of TimepixCam, a fast imager for the time-stamping of optical photons,” J. Instrum. 12(01), C01017 (2017).
[Crossref]

L. M. Hirvonen, M. Fisher-Levine, K. Suhling, and A. Nomerotski, “Photon counting phosphorescence lifetime imaging with TimepixCam,” Rev. Sci. Instrum. 88(1), 013104 (2017).
[Crossref]

A. Zhao, M. Van Beuzekom, B. Bouwens, D. Byelov, I. Chakaberia, C. Cheng, E. Maddox, A. Nomerotski, P. Svihra, J. Visser, V. Vrba, and T. Weinacht, “Coincidence velocity map imaging using Tpx3Cam, a time stamping optical camera with 1.5 ns timing resolution,” Rev. Sci. Instrum. 88(11), 113104 (2017).
[Crossref]

2016 (1)

M. Fisher-Levine and A. Nomerotski, “TimepixCam: A fast optical imager with time-stamping,” J. Instrum. 11(03), C03016 (2016).
[Crossref]

2015 (2)

L. M. Hirvonen, Z. Petrášek, A. Beeby, and K. Suhling, “Sub-µs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay,” New J. Phys. 17(2), 023032 (2015).
[Crossref]

J. Visser, M. Van Beuzekom, H. Boterenbrood, B. Van Der Heijden, J. I. Muñoz, S. Kulis, B. Munneke, and F. Schreuder, “SPIDR: A read-out system for Medipix3 & Timepix3,” J. Instrum. 10(12), C12028 (2015).
[Crossref]

2014 (4)

H. Lu, J. MacK, Y. Yang, and Z. Shen, “Structural modification strategies for the rational design of red/NIR region BODIPYs,” Chem. Soc. Rev. 43(13), 4778–4823 (2014).
[Crossref]

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
[Crossref]

L. M. Hirvonen, F. Festy, and K. Suhling, “Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution,” Opt. Lett. 39(19), 5602 (2014).
[Crossref]

C. A. Kelly, C. Toncelli, J. P. Kerry, and D. B. Papkovsky, “Discrete O2 sensors produced by a spotting method on polyolefin fabric substrates,” Sens. Actuators, B 203, 935–940 (2014).
[Crossref]

2013 (2)

V. Tsytsarev, H. Arakawa, S. Borisov, E. Pumbo, R. S. Erzurumlu, and D. B. Papkovsky, “In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe,” J. Neurosci. Methods 216(2), 146–151 (2013).
[Crossref]

A. Orte, J. M. Alvarez-Pez, and M. J. Ruedas-Rama, “Fluorescence lifetime imaging microscopy for the detection of intracellular pH with quantum dot nanosensors,” ACS Nano 7(7), 6387–6395 (2013).
[Crossref]

2012 (6)

L. Marcu, “Fluorescence lifetime techniques in medical applications,” Ann. Biomed. Eng. 40(2), 304–331 (2012).
[Crossref]

J. V. Veetil, S. Jin, and K. Ye, “Fluorescence lifetime imaging microscopy of intracellular glucose dynamics,” J. Diabetes Sci. Technol. 6(6), 1276–1285 (2012).
[Crossref]

S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P. M. W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, and C. Dunsby, “Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin,” Dermatol. Res. Pract. 2012, 1–8 (2012).
[Crossref]

R. I. Dmitriev and D. B. Papkovsky, “Optical probes and techniques for O2 measurement in live cells and tissue,” Cell. Mol. Life Sci. 69(12), 2025–2039 (2012).
[Crossref]

W. Becker, “Fluorescence lifetime imaging - techniques and applications,” J. Microsc. 247(2), 119–136 (2012).
[Crossref]

T. Jokic, S. M. Borisov, R. Saf, D. A. Nielsen, M. Kühl, and I. Klimant, “Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes,” Anal. Chem. 84(15), 6723–6730 (2012).
[Crossref]

2011 (1)

R. Beacham, A. Mac Raighne, D. Maneuski, V. O’Shea, S. McVitie, and D. McGrouther, “Medipix2/Timepix detector for time resolved Transmission Electron Microscopy,” J. Instrum. 6(12), C12052 (2011).
[Crossref]

2010 (3)

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
[Crossref]

Z. Petrášek and K. Suhling, “Photon arrival timing with sub-camera exposure time resolution in wide-field time-resolved photon counting imaging,” Opt. Express 18(24), 24888 (2010).
[Crossref]

A. V. Zhdanov, V. I. Ogurtsov, C. T. Taylor, and D. B. Papkovsky, “Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique,” Integr. Biol. 2(9), 443–451 (2010).
[Crossref]

2009 (2)

J. Korczyński and J. Włodarczyk, “Fluorescence lifetime imaging microscopy (FLIM) in biological and medical research,” Postepy Biochem. 55(4), 434–440 (2009).

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: Practical application to protein-protein interactions using global analysis,” J. R. Soc., Interface 6, suppl. 1(2009).
[Crossref]

2008 (1)

X. Llopart, R. Ballabriga, M. Campbell, L. Tlustos, and W. Wong, “Timepix, a 65 k programmable pixel readout chip for arrival time, energy and/or photon counting measurements: erratum,” Nucl. Instrum. Methods Phys. Res., Sect. A 585(1-2), 106–108 (2008).
[Crossref]

2004 (1)

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt. 9(6), 1230 (2004).
[Crossref]

2003 (1)

H.-J. Lin, P. Herman, and J. R. Lakowicz, “Fluorescence lifetime-resolved pH imaging of living cells,” Cytometry 52A(2), 77–89 (2003).
[Crossref]

1992 (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of calcium using Quin-2,” Cell Calcium 13(3), 131–147 (1992).
[Crossref]

1990 (1)

J. Kavandi, J. Callis, M. Gouterman, G. Khalil, D. Wright, E. Green, D. Burns, and B. McLachlan, “Luminescent barometry in wind tunnels,” Rev. Sci. Instrum. 61(11), 3340–3347 (1990).
[Crossref]

Agronskaia, A. V.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt. 9(6), 1230 (2004).
[Crossref]

Akkenti, F.

V. Tsytsarev, F. Akkenti, E. Pumbo, Q. Tang, Y. Chen, R. S. Erzurumlu, and D. B. Papkovsky, “Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue,” J. Neurosci. Methods 281, 1–6 (2017).
[Crossref]

Al Darwish, R.

R. Al Darwish, L. Marcu, and E. Bezak, “Overview of current applications of the Timepix detector in spectroscopy, radiation and medical physics,” Appl. Spectrosc. Rev.1–19 (2019).

Alvarez-Pez, J. M.

A. Orte, J. M. Alvarez-Pez, and M. J. Ruedas-Rama, “Fluorescence lifetime imaging microscopy for the detection of intracellular pH with quantum dot nanosensors,” ACS Nano 7(7), 6387–6395 (2013).
[Crossref]

Ameer-Beg, S. M.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: Practical application to protein-protein interactions using global analysis,” J. R. Soc., Interface 6, suppl. 1(2009).
[Crossref]

Arakawa, H.

V. Tsytsarev, H. Arakawa, S. Borisov, E. Pumbo, R. S. Erzurumlu, and D. B. Papkovsky, “In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe,” J. Neurosci. Methods 216(2), 146–151 (2013).
[Crossref]

Arginelli, F.

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Boll, R.

M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
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T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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W. Zarychta-Wiśniewska, A. Burdzinska, R. Zagozdzon, B. Dybowski, M. Butrym, Z. Gajewski, and L. Paczek, “In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models,” PLoS One 12(9), e0184588 (2017).
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T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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X. Llopart, R. Ballabriga, M. Campbell, L. Tlustos, and W. Wong, “Timepix, a 65 k programmable pixel readout chip for arrival time, energy and/or photon counting measurements: erratum,” Nucl. Instrum. Methods Phys. Res., Sect. A 585(1-2), 106–108 (2008).
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P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: Practical application to protein-protein interactions using global analysis,” J. R. Soc., Interface 6, suppl. 1(2009).
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A. Nomerotski, I. Chakaberia, M. Fisher-Levine, Z. Janoska, P. Takacs, and T. Tsang, “Characterization of TimepixCam, a fast imager for the time-stamping of optical photons,” J. Instrum. 12(01), C01017 (2017).
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A. Zhao, M. Van Beuzekom, B. Bouwens, D. Byelov, I. Chakaberia, C. Cheng, E. Maddox, A. Nomerotski, P. Svihra, J. Visser, V. Vrba, and T. Weinacht, “Coincidence velocity map imaging using Tpx3Cam, a time stamping optical camera with 1.5 ns timing resolution,” Rev. Sci. Instrum. 88(11), 113104 (2017).
[Crossref]

Chakrabortty, S.

S. Kalinina, P. Schaefer, J. Breymayer, D. Bisinger, S. Chakrabortty, and A. Rueck, “Oxygen sensing PLIM together with FLIM of intrinsic cellular fluorophores for metabolic mapping,” in Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI (International Society for Optics and Photonics, 2018), 10497, p. 104970F.

Charbon, E.

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
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A. Zhao, M. Van Beuzekom, B. Bouwens, D. Byelov, I. Chakaberia, C. Cheng, E. Maddox, A. Nomerotski, P. Svihra, J. Visser, V. Vrba, and T. Weinacht, “Coincidence velocity map imaging using Tpx3Cam, a time stamping optical camera with 1.5 ns timing resolution,” Rev. Sci. Instrum. 88(11), 113104 (2017).
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J. T. Sharick, J. J. Jeffery, M. R. Karim, C. M. Walsh, K. Esbona, R. S. Cook, and M. C. Skala, “Cellular Metabolic Heterogeneity In Vivo Is Recapitulated in Tumor Organoids,” Neoplasia (N. Y., NY, U. S.) 21(6), 615–626 (2019).
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Cui, G.

C. Ianzano, P. Svihra, M. Flament, A. Hardy, G. Cui, A. Nomerotski, and E. Figueroa, “Spatial characterization of photonic polarization entanglement using a Tpx3Cam intensified fast-camera,” 1–22 (2018).

De Gaspari, M.

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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de Jong, P. J.

S. Tsigaridas, M. V. Beuzekom, H. V. D. Graaf, F. Hartjes, K. Heijhoff, N. P. Hessey, P. J. de Jong, and V. Prodanovic, “Timewalk correction for the Timepix3 chip obtained with real particle data,” Nucl. Instrum. Methods Phys. Res., Sect. A 930(April), 185–190 (2019).
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Desch, K.

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
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S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P. M. W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, and C. Dunsby, “Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin,” Dermatol. Res. Pract. 2012, 1–8 (2012).
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W. Zarychta-Wiśniewska, A. Burdzinska, R. Zagozdzon, B. Dybowski, M. Butrym, Z. Gajewski, and L. Paczek, “In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models,” PLoS One 12(9), e0184588 (2017).
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Dymoke-Bradshaw, A. K. L.

H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
[Crossref]

Dysli, C.

C. Dysli, S. Wolf, M. Y. Berezin, L. Sauer, M. Hammer, and M. S. Zinkernagel, “Fluorescence lifetime imaging ophthalmoscopy,” Prog. Retinal Eye Res. 60, 120–143 (2017).
[Crossref]

Erk, B.

M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
[Crossref]

Erzurumlu, R. S.

V. Tsytsarev, F. Akkenti, E. Pumbo, Q. Tang, Y. Chen, R. S. Erzurumlu, and D. B. Papkovsky, “Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue,” J. Neurosci. Methods 281, 1–6 (2017).
[Crossref]

V. Tsytsarev, H. Arakawa, S. Borisov, E. Pumbo, R. S. Erzurumlu, and D. B. Papkovsky, “In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe,” J. Neurosci. Methods 216(2), 146–151 (2013).
[Crossref]

Esbona, K.

J. T. Sharick, J. J. Jeffery, M. R. Karim, C. M. Walsh, K. Esbona, R. S. Cook, and M. C. Skala, “Cellular Metabolic Heterogeneity In Vivo Is Recapitulated in Tumor Organoids,” Neoplasia (N. Y., NY, U. S.) 21(6), 615–626 (2019).
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Festy, F.

Fey, J.

S. Procz, C. A. Avila, J. Fey, G. A. Roque, M. Schuetz, and E. Hamann, “X-ray and gamma imaging with Medipix and Timepix detectors in medical research,” Radiat. Meas. 127, 106104 (2019).
[Crossref]

Figueroa, E.

C. Ianzano, P. Svihra, M. Flament, A. Hardy, G. Cui, A. Nomerotski, and E. Figueroa, “Spatial characterization of photonic polarization entanglement using a Tpx3Cam intensified fast-camera,” 1–22 (2018).

Fishburn, M.

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
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M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
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L. M. Hirvonen, M. Fisher-Levine, K. Suhling, and A. Nomerotski, “Photon counting phosphorescence lifetime imaging with TimepixCam,” Rev. Sci. Instrum. 88(1), 013104 (2017).
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A. Nomerotski, I. Chakaberia, M. Fisher-Levine, Z. Janoska, P. Takacs, and T. Tsang, “Characterization of TimepixCam, a fast imager for the time-stamping of optical photons,” J. Instrum. 12(01), C01017 (2017).
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M. Fisher-Levine and A. Nomerotski, “TimepixCam: A fast optical imager with time-stamping,” J. Instrum. 11(03), C03016 (2016).
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Flament, M.

C. Ianzano, P. Svihra, M. Flament, A. Hardy, G. Cui, A. Nomerotski, and E. Figueroa, “Spatial characterization of photonic polarization entanglement using a Tpx3Cam intensified fast-camera,” 1–22 (2018).

French, P. M. W.

H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
[Crossref]

S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P. M. W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, and C. Dunsby, “Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin,” Dermatol. Res. Pract. 2012, 1–8 (2012).
[Crossref]

Fu, Y.

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
[Crossref]

Gajewski, Z.

W. Zarychta-Wiśniewska, A. Burdzinska, R. Zagozdzon, B. Dybowski, M. Butrym, Z. Gajewski, and L. Paczek, “In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models,” PLoS One 12(9), e0184588 (2017).
[Crossref]

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H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
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M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
[Crossref]

Gilbey, J.

P. R. Barber, S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic, “Multiphoton time-domain fluorescence lifetime imaging microscopy: Practical application to protein-protein interactions using global analysis,” J. R. Soc., Interface 6, suppl. 1(2009).
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Görlitz, F.

H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
[Crossref]

Gouterman, M.

J. Kavandi, J. Callis, M. Gouterman, G. Khalil, D. Wright, E. Green, D. Burns, and B. McLachlan, “Luminescent barometry in wind tunnels,” Rev. Sci. Instrum. 61(11), 3340–3347 (1990).
[Crossref]

Graaf, H. V. D.

S. Tsigaridas, M. V. Beuzekom, H. V. D. Graaf, F. Hartjes, K. Heijhoff, N. P. Hessey, P. J. de Jong, and V. Prodanovic, “Timewalk correction for the Timepix3 chip obtained with real particle data,” Nucl. Instrum. Methods Phys. Res., Sect. A 930(April), 185–190 (2019).
[Crossref]

Green, E.

J. Kavandi, J. Callis, M. Gouterman, G. Khalil, D. Wright, E. Green, D. Burns, and B. McLachlan, “Luminescent barometry in wind tunnels,” Rev. Sci. Instrum. 61(11), 3340–3347 (1990).
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Gromov, V.

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
[Crossref]

Guanti, M.

S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P. M. W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, and C. Dunsby, “Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin,” Dermatol. Res. Pract. 2012, 1–8 (2012).
[Crossref]

Guardoli, D.

S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P. M. W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, and C. Dunsby, “Multiphoton laser microscopy and fluorescence lifetime imaging for the evaluation of the skin,” Dermatol. Res. Pract. 2012, 1–8 (2012).
[Crossref]

Hamann, E.

S. Procz, C. A. Avila, J. Fey, G. A. Roque, M. Schuetz, and E. Hamann, “X-ray and gamma imaging with Medipix and Timepix detectors in medical research,” Radiat. Meas. 127, 106104 (2019).
[Crossref]

Hammer, M.

C. Dysli, S. Wolf, M. Y. Berezin, L. Sauer, M. Hammer, and M. S. Zinkernagel, “Fluorescence lifetime imaging ophthalmoscopy,” Prog. Retinal Eye Res. 60, 120–143 (2017).
[Crossref]

Hardy, A.

C. Ianzano, P. Svihra, M. Flament, A. Hardy, G. Cui, A. Nomerotski, and E. Figueroa, “Spatial characterization of photonic polarization entanglement using a Tpx3Cam intensified fast-camera,” 1–22 (2018).

Hares, J. D.

H. Sparks, F. Görlitz, D. J. Kelly, S. C. Warren, P. A. Kellett, E. Garcia, A. K. L. Dymoke-Bradshaw, J. D. Hares, M. A. A. Neil, C. Dunsby, and P. M. W. French, “Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging,” Rev. Sci. Instrum. 88(1), 013707 (2017).
[Crossref]

Hartjes, F.

S. Tsigaridas, M. V. Beuzekom, H. V. D. Graaf, F. Hartjes, K. Heijhoff, N. P. Hessey, P. J. de Jong, and V. Prodanovic, “Timewalk correction for the Timepix3 chip obtained with real particle data,” Nucl. Instrum. Methods Phys. Res., Sect. A 930(April), 185–190 (2019).
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T. Jokic, S. M. Borisov, R. Saf, D. A. Nielsen, M. Kühl, and I. Klimant, “Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes,” Anal. Chem. 84(15), 6723–6730 (2012).
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L. M. Hirvonen, M. Fisher-Levine, K. Suhling, and A. Nomerotski, “Photon counting phosphorescence lifetime imaging with TimepixCam,” Rev. Sci. Instrum. 88(1), 013104 (2017).
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M. Fisher-Levine and A. Nomerotski, “TimepixCam: A fast optical imager with time-stamping,” J. Instrum. 11(03), C03016 (2016).
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C. Ianzano, P. Svihra, M. Flament, A. Hardy, G. Cui, A. Nomerotski, and E. Figueroa, “Spatial characterization of photonic polarization entanglement using a Tpx3Cam intensified fast-camera,” 1–22 (2018).

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J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of calcium using Quin-2,” Cell Calcium 13(3), 131–147 (1992).
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R. Beacham, A. Mac Raighne, D. Maneuski, V. O’Shea, S. McVitie, and D. McGrouther, “Medipix2/Timepix detector for time resolved Transmission Electron Microscopy,” J. Instrum. 6(12), C12052 (2011).
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C. A. Kelly, C. Toncelli, J. P. Kerry, and D. B. Papkovsky, “Discrete O2 sensors produced by a spotting method on polyolefin fabric substrates,” Sens. Actuators, B 203, 935–940 (2014).
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V. Tsytsarev, H. Arakawa, S. Borisov, E. Pumbo, R. S. Erzurumlu, and D. B. Papkovsky, “In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe,” J. Neurosci. Methods 216(2), 146–151 (2013).
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A. V. Zhdanov, V. I. Ogurtsov, C. T. Taylor, and D. B. Papkovsky, “Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique,” Integr. Biol. 2(9), 443–451 (2010).
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J. Jenkins, R. I. Dmitriev, and D. B. Papkovsky, “Imaging cell and tissue O2 by TCSPC-PLIM,” in Advanced Time-Correlated Single Photon Counting Applications (Springer, 2015), pp. 225–247.

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L. M. Hirvonen, Z. Petrášek, A. Beeby, and K. Suhling, “Sub-µs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay,” New J. Phys. 17(2), 023032 (2015).
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Z. Petrášek and K. Suhling, “Photon arrival timing with sub-camera exposure time resolution in wide-field time-resolved photon counting imaging,” Opt. Express 18(24), 24888 (2010).
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Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
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T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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S. Procz, C. A. Avila, J. Fey, G. A. Roque, M. Schuetz, and E. Hamann, “X-ray and gamma imaging with Medipix and Timepix detectors in medical research,” Radiat. Meas. 127, 106104 (2019).
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Prodanovic, V.

S. Tsigaridas, M. V. Beuzekom, H. V. D. Graaf, F. Hartjes, K. Heijhoff, N. P. Hessey, P. J. de Jong, and V. Prodanovic, “Timewalk correction for the Timepix3 chip obtained with real particle data,” Nucl. Instrum. Methods Phys. Res., Sect. A 930(April), 185–190 (2019).
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Pumbo, E.

V. Tsytsarev, F. Akkenti, E. Pumbo, Q. Tang, Y. Chen, R. S. Erzurumlu, and D. B. Papkovsky, “Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue,” J. Neurosci. Methods 281, 1–6 (2017).
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V. Tsytsarev, H. Arakawa, S. Borisov, E. Pumbo, R. S. Erzurumlu, and D. B. Papkovsky, “In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe,” J. Neurosci. Methods 216(2), 146–151 (2013).
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Qu, J.

Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
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Richardson, J.

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
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Rolles, D.

M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
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Rompotis, D.

M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
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Roque, G. A.

S. Procz, C. A. Avila, J. Fey, G. A. Roque, M. Schuetz, and E. Hamann, “X-ray and gamma imaging with Medipix and Timepix detectors in medical research,” Radiat. Meas. 127, 106104 (2019).
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Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
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Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
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C. Dysli, S. Wolf, M. Y. Berezin, L. Sauer, M. Hammer, and M. S. Zinkernagel, “Fluorescence lifetime imaging ophthalmoscopy,” Prog. Retinal Eye Res. 60, 120–143 (2017).
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T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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ACS Nano (1)

A. Orte, J. M. Alvarez-Pez, and M. J. Ruedas-Rama, “Fluorescence lifetime imaging microscopy for the detection of intracellular pH with quantum dot nanosensors,” ACS Nano 7(7), 6387–6395 (2013).
[Crossref]

Anal. Chem. (1)

T. Jokic, S. M. Borisov, R. Saf, D. A. Nielsen, M. Kühl, and I. Klimant, “Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes,” Anal. Chem. 84(15), 6723–6730 (2012).
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Cell Calcium (1)

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Cell. Mol. Life Sci. (1)

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Chem. Soc. Rev. (1)

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[Crossref]

Detect. Imaging Devices Infrared, Focal Plane, Single Phot. (1)

M. Gersbach, R. Trimananda, Y. Maruyama, M. Fishburn, D. Stoppa, J. Richardson, R. Walker, R. K. Henderson, and E. Charbon, “High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor,” Detect. Imaging Devices Infrared, Focal Plane, Single Phot. 7780, 77801H (2010).
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Integr. Biol. (1)

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[Crossref]

J. Biomed. Opt. (1)

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt. 9(6), 1230 (2004).
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J. Diabetes Sci. Technol. (1)

J. V. Veetil, S. Jin, and K. Ye, “Fluorescence lifetime imaging microscopy of intracellular glucose dynamics,” J. Diabetes Sci. Technol. 6(6), 1276–1285 (2012).
[Crossref]

J. Innovative Opt. Health Sci. (1)

Z. Wang, Y. Zheng, D. Zhao, Z. Zhao, L. Liu, A. Pliss, F. Zhu, J. Liu, J. Qu, and P. Luan, “Applications of fluorescence lifetime imaging in clinical medicine,” J. Innovative Opt. Health Sci. 11(01), 1830001 (2018).
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J. Instrum. (5)

M. Fisher-Levine and A. Nomerotski, “TimepixCam: A fast optical imager with time-stamping,” J. Instrum. 11(03), C03016 (2016).
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A. Nomerotski, I. Chakaberia, M. Fisher-Levine, Z. Janoska, P. Takacs, and T. Tsang, “Characterization of TimepixCam, a fast imager for the time-stamping of optical photons,” J. Instrum. 12(01), C01017 (2017).
[Crossref]

T. Poikela, J. Plosila, T. Westerlund, M. Campbell, M. De Gaspari, X. Llopart, V. Gromov, R. Kluit, M. Van Beuzekom, F. Zappon, V. Zivkovic, C. Brezina, K. Desch, Y. Fu, and A. Kruth, “Timepix3: A 65 K channel hybrid pixel readout chip with simultaneous ToA/ToT and sparse readout,” J. Instrum. 9(05), C05013 (2014).
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M. Fisher-Levine, R. Boll, F. Ziaee, C. Bomme, B. Erk, D. Rompotis, T. Marchenko, A. Nomerotski, and D. Rolles, “Time-resolved ion imaging at free-electron lasers using TimepixCam,” J. Synchrotron Radiat. 25(2), 336–345 (2018).
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Figures (8)

Fig. 1.
Fig. 1. (Left) Experimental setup of the PLIM macro-imager based on Tpx3Cam. (Right) Components of the intensified Tpx3Cam.
Fig. 2.
Fig. 2. Pulsing scheme of the imager. The LED is pulsed every 100 µs with 50 ns duration of the pulse. Tpx3Cam is provided with a synchronous pulse 10 µs before the LED, which is time-stamped and included into the data stream. The camera records the photon hits for 100 µs until the next LED pulse.
Fig. 3.
Fig. 3. Samples used to characterize the imager: (a) USAF resolution test target; (b) Planar PtBP-RL100 based O2 sensor film and (c) its absorption spectrum; (d) PtBP-RL100 sensor spots on PVDF membrane, (e) Sensor film with PtBP and aza-Bodipy dyes in RL100, and (f) its absorption spectrum. Spectra were measured on an 8453UV-Visible spectrophotometer (Agilent).
Fig. 4.
Fig. 4. Single photon events detected with the Tpx3Cam setup. (a) TOT, i.e. intensity, (b) arrival time, (c-e) Re-scaled arrival time for individual photon events showing the “timewalk” effect.
Fig. 5.
Fig. 5. Images of 1951 USAF resolution test target overlaid on top of a phosphorescent O2 sensor foil. (a) Sum image showing the PLIM imager field of view. (b-e) images of the area indicated in (a) centroided with different methods: (b) sum, (c) brightest pixel (BP), (d) earliest timecode, (e) centre-of-mass with ⅕ pixel accuracy (CoM 5). (f) Cross-sections of the bars in the area indicated in (e), where the sub-pixel centroiding can still resolve lines with ∼40 µm spacing, that cannot be resolved with the other centroiding methods.
Fig. 6.
Fig. 6. Instrument Resolution Function (IRF) position in the measurement time interval is shown for all hit pixels and for the centroided pixels. In both cases the distributions are shown before and after the TOT correction. Gaussian fit of the centroided and TOT corrected distribution corresponds to the IRF FWHM of 30.6 ns. Background was described with a constant in the fit.
Fig. 7.
Fig. 7. Two-dimensional distributions of time, in microseconds, versus TOT, in nanoseconds, for four cases: (a) all hit pixels; (b) all hit pixels with TOT correction applied; (c) centroided pixels; (d) centroided pixels with TOT correction applied.
Fig. 8.
Fig. 8. PLIM images obtained with Timepix3 imager: (a) PtBP-RL100 based O2 sensor spotted on porous PVDF membrane; (b) Lifetime gradient caused by varying O2 concentration in a tube with respiring HCT116 cells precipitated at the bottom; (c) Phosphorescent decays for areas marked in (b) with bi-exponential fits and residuals of fits.

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