Abstract
TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.
Keywords
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Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, Heidelberg
Charbonniere LJ, Hildebrandt N (2008) Lanthanide complexes and quantum dots: a bright wedding for resonance energy transfer. Eur J Inorg Chem 2008:3241–3251
Hosny NA, Lee DA, Knight MM (2012) Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations. J Biomed Opt 17(1):016007-1–016007-12
Fercher A, Borisov SM, Zhdanov AV et al (2011) Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles. ACS Nano 5:5499–5508
Lebedev AY, Cheprakov AV, Sakadzic S et al (2009) Dendritic phosphorescent probes for oxygen imaging in biological systems. Appl Mater Interfaces 1:1292–1304
Sakadžic S, Roussakis E, Yaseen MA et al (2010) Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nat Methods 7:755–759
Gerritsen HC, Sanders R, Draaijer A et al (1997) Fluorescence lifetime imaging of oxygen in cells. J Fluoresc 7:11–16
Papkovsky D, Zhdanov AV, Fercher A et al (2012) Phosphorescent oxygen-sensitive probes. Springer
Papkovsky DB, Dmitriev RI (2013) Biological detection by optical oxygen sensing. Chem Soc Rev 42:8700–8732
Shibata M, Ichioka S, Ando J, Kamiya A (2001) Microvascular and interstitial PO2 measurement in rat skeletal muscle by phosphorescence quenching. J Appl Physiol 91:321–327
Skala MC, Riching KM, Bird DK et al (2007) In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt 12:02401-1–02401-10
Becker W (2005) Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York
Becker W (2015) The bh TCSPC handbook, 6th edn. Becker & Hickl GmbH, Berlin. Available from: www.becker-hickl.com
Becker W (2015) Introduction to multi-dimensional TCSPC. In: Becker W (ed) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York, pp 1–63
Becker W, Shcheslavskiy V, Studier H (2015) TCSPC FLIM with different optical scanning techniques. In: Becker W (ed) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York, pp 65–117
Becker W (2015) Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting. Med Photon 27:41–61
Studier H, Becker W (2014) Megapixel FLIM. Proc SPIE 8948:89481K
Becker W, Su B, Bergmann A et al (2011) Simultaneous fluorescence and phosphorescence lifetime imaging. Proc SPIE 7903:790320
Becker W (2015) Fluorescence lifetime imaging techniques: time-correlated single-photon counting. In: Marcu L, French PMW, Elson DS (eds) Fluorecence lifetime spectroscopy and imaging. Principles and applications in biomedical diagnostics. CRC Press, Taylor & Francis Group, Boca Raton, London, New York, pp 203–232
Shcheslavskiy VI, Neubauer A, Bukowiecki R et al (2016) Combined fluorescence and phosphorescence lifetime imaging. Appl Phys Lett 108:091111-1–091111-5
Becker & Hickl GmbH (2015) DCS-120 confocal scanning FLIM systems. In: User handbook, 6th edn. Available from: www.becker-hickl.com
Sutter instrument, Movable objective microscope. www.sutter.com/microscopes
Toncelli C, Arzhakova OV, Dolgova A et al (2014) Oxygen-sensitive phosphorescent nanomaterials produced from high density polyethylene films by local solvent-crazing. Anal Chem 86(3):1917–1923
Dmitriev RI, Kondrashina AV, Koren K et al (2014) Small molecule phosphorescent probes for O2 imaging in 3D tissue models. Biomater Sci 2:853–866
Dmitriev RI, Zhdanov AV, Nolan YM, Papkovsky DB (2013) Imaging of neurosphere oxygenation with phosphorescent probes. Biomaterials 34:9307–9317
Kalinina S, Shcheslavskiy V, Becker W et al (2016) Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping. J Biophotonics 9(8):800–811
Kurokawa H, Ito H, Inoue M et al (2015) High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime. Sci Rep 5:1–13
Zhdanov AV, Golubeva AV, Okkelman IA et al (2015) Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study. Am J Phys Cell Phys 309:C501–C509
Lukina M, Orlova A, Shirmanova M et al (2017) Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy. Opt Lett 42(4):731–734
Jenkins J, Dmitriev RI, Papkovsky DB (2015) Imaging cell and tissue O2 by TCSPC-PLIM. In: Becker W (ed) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York, pp 225–247
Sanchez WY, Pastore M, Haridass I et al (2015) Fluorescence lifetime imaging of the skin. In: Becker W (ed) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York, pp 457–508
Baggaley E, Botchway SW, Haycock JW et al (2014) Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond. Chem Sci 5:879–886
Baggaley E, Gill MR, Green NH et al (2014) Dinuclear ruthenium(II) complexes as two-photon, time-resolved emission microscopy probes for cellular DNA. Angew Chem Int Ed Eng 53:3367–3371
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Becker, W., Shcheslavskiy, V., Rück, A. (2017). Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation. In: Dmitriev, R. (eds) Multi-Parametric Live Cell Microscopy of 3D Tissue Models. Advances in Experimental Medicine and Biology, vol 1035. Springer, Cham. https://doi.org/10.1007/978-3-319-67358-5_2
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