1932

Abstract

The phasor approach to fluorescence lifetime imaging has become a common method to analyze complicated fluorescence signals from biological samples. The appeal of the phasor representation of complex fluorescence decays in biological systems is that a visual representation of the decay of entire cells or tissues can be used to easily interpret fundamental biological states related to metabolism and oxidative stress. Phenotyping based on autofluorescence provides new avenues for disease characterization and diagnostics. The phasor approach is a transformation of complex fluorescence decays that does not use fits to model decays and therefore has the same information content as the original data. The phasor plot is unique for a given system, is highly reproducible, and provides a robust method to evaluate the existence of molecular interactions such as Förster resonance energy transfer or the response of ion indicators. Recent advances permitquantification of multiple components from phasor plots in fluorescence lifetime imaging microscopy, which is not presently possible using data fitting methods, especially in biological systems.

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2021-05-06
2024-05-19
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Literature Cited

  1. 1. 
    Adamczyk B. 2017. Complex numbers and phasors. Foundations of Electromagnetic Compatibility: With Practical Applications109–40 Hoboken, NJ: Wiley. , 1st ed..
    [Google Scholar]
  2. 2. 
    Aguilar-Arnal L, Ranjit S, Stringari C, Orozco-Solis R, Gratton E, Sassone-Corsi P 2016. Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species. PNAS 113:4512715–20
    [Google Scholar]
  3. 3. 
    Alfonso-García A, Smith TD, Datta R, Luu TU, Gratton E et al. 2016. Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy. J. Biomed. Opt. 21:446005
    [Google Scholar]
  4. 4. 
    Bagatolli LA 2012. LAURDAN fluorescence properties in membranes: a journey from the fluorometer to the microscope. Fluorescent Methods to Study Biological Membranes G Mely, Y Duportail 3–36 Berlin: Springer
    [Google Scholar]
  5. 5. 
    Bismuto E, Jameson DM, Gratton E. 1987. Dipolar relaxations in glycerol: a dynamic fluorescence study of 4-2′-dimethylamino6′-naphthoylcyclohexanecarboxylic acid (DANCA). J. Am. Chem. Soc. 109:82354–57
    [Google Scholar]
  6. 6. 
    Castro-Castillo V, Gajardo J, Sandoval-Altamirano C, Gunther G, Gratton E et al. 2019. CAPRYDAA, an anthracene dye analog to LAURDAN: a comparative study using cuvette and microscopy. J. Mater. Chem. B 8:188–99
    [Google Scholar]
  7. 7. 
    Cerrada A, Haller T, Cruz A, Pérez-Gil J. 2015. Pneumocytes assemble lung surfactant as highly packed/dehydrated states with optimal surface activity. Biophys. J. 109:112295–306
    [Google Scholar]
  8. 8. 
    Chance B, Nioka S, Warren W, Yurtsever G 2005. Mitochondrial NADH as the bellwether of tissue O2 delivery. Adv. Exp. Med. Biol. 566:231–42
    [Google Scholar]
  9. 9. 
    Chen YC, Clegg RM. 2011. Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency. J. Microsc. 244:121–37
    [Google Scholar]
  10. 10. 
    Cutrale F, Salih A, Gratton E. 2013. Spectral phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR. Methods Appl. Fluoresc. 1:035001
    [Google Scholar]
  11. 11. 
    Cutrale F, Trivedi V, Trinh LA, Chiu C-L, Choi JM et al. 2016. Hyper-spectral phasor analysis enables multiplexed 5D in vivo imaging. Nat. Methods 14:2149–52
    [Google Scholar]
  12. 12. 
    Datta R, Alfonso-García A, Cinco R, Gratton E. 2015. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress. Sci. Rep. 5:9848
    [Google Scholar]
  13. 13. 
    Datta R, Heylman C, George SC, Gratton E. 2016. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes. Biomed. Opt. Express 7:51690–701
    [Google Scholar]
  14. 14. 
    De Oliveira HM, Nunes FD. 2014. About the phasor pathways in analogical amplitude modulations. Int. J. Res. Eng. Sci. 2:111–18
    [Google Scholar]
  15. 15. 
    Digman MA, Caiolfa VR, Zamai M, Gratton E. 2008. The phasor approach to fluorescence lifetime imaging analysis. Biophys. J. 94:2L14–16
    [Google Scholar]
  16. 16. 
    Dvornikov A, Gratton E. 2018. Hyperspectral imaging in highly scattering media by the spectral phasor approach using two filters. Biomed. Opt. Express 9:84833–40
    [Google Scholar]
  17. 17. 
    Dvornikov A, Malacrida L, Gratton E. 2019. The DIVER microscope for imaging in scattering media. Methods Protoc 2:253
    [Google Scholar]
  18. 18. 
    Eichorst JP, Wen Teng K, Clegg RM 2014. Polar plot representation of time-resolved fluorescence. Fluorescence Spectroscopy and Microscopy: Methods and Protocols Y Engelborghs, AJWG Visser 97–112 Totowa, NJ: Humana Press
    [Google Scholar]
  19. 19. 
    Fereidouni F, Bader AN, Colonna A, Gerritsen HC. 2014. Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin. J. Biophoton. 7:8589–96
    [Google Scholar]
  20. 20. 
    Fereidouni F, Bader AN, Gerritsen HC. 2012. Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. Opt. Express 20:1212729–41
    [Google Scholar]
  21. 21. 
    Gaviola E. 1926. Die Abklingungszeiten der Fluoreszenz von Farbstofflösungen. Ann. Phys. 386:26681–710
    [Google Scholar]
  22. 22. 
    Golfetto O, Hinde E, Gratton E. 2013. Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes. Biophys. J. 104:1238–47
    [Google Scholar]
  23. 23. 
    Golfetto O, Hinde E, Gratton E. 2015. The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells. Methods Mol. Biol. 1232:273–90
    [Google Scholar]
  24. 24. 
    Hinde E, Digman MA, Welch C, Hahn KM, Gratton E. 2012. Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy. Microsc. Res. Tech. 75:3271–81
    [Google Scholar]
  25. 25. 
    James NG, Ross JA, Stefl M, Jameson DM. 2011. Applications of phasor plots to in vitro protein studies. Anal. Biochem. 410:170–76
    [Google Scholar]
  26. 26. 
    Jameson DM. 2014. Introduction to Fluorescence Boca Raton, FL: CRC Press. , 1st ed..
  27. 27. 
    Jameson DM, Gratton E, Hall RD. 1984. The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry. Appl. Spectrosc. Rev. 20:155–106
    [Google Scholar]
  28. 28. 
    Kim SM, Nguyen TT, Ravi A, Kubiniok P, Finicle BT et al. 2018. PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells. Cancer Discov 8:7866–83
    [Google Scholar]
  29. 29. 
    Kolb DA, Weber G. 1975. Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide oxalate. Biochemistry 10:204471–76
    [Google Scholar]
  30. 30. 
    Lajevardipour A, Chon JWM, Chattopadhyay A, Clayton AHA. 2016. Imaging cellular dynamics with spectral relaxation imaging microscopy: distinct spectral dynamics in Golgi membranes of living cells. Sci. Rep. 6:37038
    [Google Scholar]
  31. 31. 
    Ma N, Digman MA, Malacrida L, Gratton E. 2016. Measurements of absolute concentrations of NADH in cells using the phasor FLIM method. Biomed. Opt. Express 7:72441–52
    [Google Scholar]
  32. 32. 
    Malacrida L, Astrada S, Briva A, Bollati-Fogolín M, Gratton E, Bagatolli LA. 2016. Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures. Biochim. Biophys. Acta Biomembr. 1858:112625–35
    [Google Scholar]
  33. 33. 
    Malacrida L, Gratton E. 2018. LAURDAN fluorescence and phasor plots reveal the effects of a H2O2 bolus in NIH-3T3 fibroblast membranes dynamics and hydration. Free Radic. . Biol. Med. 128:144–56
    [Google Scholar]
  34. 34. 
    Malacrida L, Gratton E, Jameson DM. 2015. Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches. Methods Appl. Fluoresc. 3:4047001
    [Google Scholar]
  35. 35. 
    Malacrida L, Jameson DM, Gratton E. 2017. A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes. Sci. Rep. 7:9215
    [Google Scholar]
  36. 36. 
    Martelo L, Fedorov A, Berberan-Santos MN. 2015. Fluorescence phasor plots using time domain data: effect of the instrument response function. J. Phys. Chem. B 119:3210267–74
    [Google Scholar]
  37. 37. 
    Martelo L, Fedorov A, Berberan-Santos MN. 2015. Phasor representation of monomer-excimer kinetics: general results and application to pyrene. J. Phys. Chem. B 119:4815023–29
    [Google Scholar]
  38. 38. 
    Martelo L, Fedorov A, Berberan-Santos MN. 2017. Monomer-excimer mixed fluorescence decays in the phasor space. J. Lumin. 192:64–70
    [Google Scholar]
  39. 39. 
    Osseiran S, Roider EM, Wang H, Suita Y, Murphy M et al. 2017. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy. J. Biomed. Opt. 22:12125004
    [Google Scholar]
  40. 40. 
    Ranjit S, Datta R, Dvornikov A, Gratton E. 2019. Multicomponent analysis of phasor plot in a single pixel to calculate changes of metabolic trajectory in biological systems. J. Phys. Chem. A 123:459865–7
    [Google Scholar]
  41. 41. 
    Ranjit S, Dvornikov A, Dobrinskikh E, Wang X, Luo Y et al. 2017. Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy. Biomed. Opt. Express 8:7371–78
    [Google Scholar]
  42. 42. 
    Ranjit S, Dvornikov A, Levi M, Furgeson S, Gratton E. 2016. Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images. Biomed. Opt. Express 7:93519–30
    [Google Scholar]
  43. 43. 
    Ranjit S, Dvornikov A, Stakic M, Hong S-H, Levi M et al. 2015. Imaging fibrosis and separating collagens using second harmonic generation and phasor approach to fluorescence lifetime imaging. Sci. Rep. 5:13378
    [Google Scholar]
  44. 44. 
    Ranjit S, Lanzano L, Gratton E. 2014. Mapping diffusion in a living cell via the phasor approach. Biophys. J. 107:122775–85
    [Google Scholar]
  45. 45. 
    Ranjit S, Malacrida L, Gratton E. 2018. Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card. Microsc. Res. Tech. 81:9980–89
    [Google Scholar]
  46. 46. 
    Ranjit S, Malacrida L, Jameson DM, Gratton E. 2018. Fit-free analysis of fluorescence lifetime imaging data using the phasor approach. Nat. Protoc. 13:91979–2004
    [Google Scholar]
  47. 47. 
    Reinhart GD, Marzola P, Jameson DM, Gratton E. 1991. A method for on-line background subtraction in frequency domain fluorometry. J. Fluoresc. 1:3153–62
    [Google Scholar]
  48. 48. 
    Sameni S, Malacrida L, Tan Z, Digman MA. 2018. Alteration in fluidity of cell plasma membrane in Huntington disease revealed by spectral phasor analysis. Sci. Rep. 8:734
    [Google Scholar]
  49. 49. 
    Sarmento MJ, Oneto M, Pelicci S, Pesce L, Scipioni L et al. 2018. Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures. Nat. Commun. 9:13415
    [Google Scholar]
  50. 50. 
    Scipioni L, Gratton E, Diaspro A, Lanzanò L. 2016. Phasor analysis of local ICS detects heterogeneity in size and number of intracellular vesicles. Biophys. J. 111:3619–29
    [Google Scholar]
  51. 51. 
    Sediqi H, Wray A, Jones C, Jones M. 2018. Application of spectral phasor analysis to sodium microenvironments in myoblast progenitor cells. PLOS ONE 13:10e0204611
    [Google Scholar]
  52. 52. 
    Sena F, Sotelo-Silveira M, Astrada S, Botella MA, Malacrida L, Borsani O. 2017. Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1–5 drought hypersensitive mutant. Plant Physiol. Biochem. 119:224–31
    [Google Scholar]
  53. 53. 
    Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW et al. 2007. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. PNAS 104:4919494–99
    [Google Scholar]
  54. 54. 
    Spencer RD, Weber G. 1969. Measurements of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer. Ann. N. Y. Acad. Sci. 158:1361–76
    [Google Scholar]
  55. 55. 
    Štefl M, James NG, Ross JA, Jameson DM. 2011. Applications of phasors to in vitro time-resolved fluorescence measurements. Anal. Biochem. 410:62–69
    [Google Scholar]
  56. 56. 
    Stringari C, Abdeladim L, Malkinson G, Mahou P, Solinas X et al. 2017. Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing. Sci. Rep. 7:3792
    [Google Scholar]
  57. 57. 
    Stringari C, Nourse JL, Flanagan LA, Gratton E. 2012. Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential. PLOS ONE 7:11e48014
    [Google Scholar]
  58. 58. 
    Valeur B, Berberan-Santos MN. 2012. Molecular Fluorescence: Principles and Applications Hoboken, NJ: Wiley. , 2nd ed..
  59. 59. 
    Vallmitjana A, Dvornikov A, Torrado B, Jameson DM, Ranjit S, Gratton E. 2020. Resolution of 4 components in the same pixel in FLIM images using the phasor approach. Methods Appl. Fluoresc. 8:3035001
    [Google Scholar]
  60. 60. 
    Vallmitjana A, Torrado B, Dvornikov A, Ranjit S, Gratton E. 2020. Blind resolution of lifetime components in individual pixels of fluorescence lifetime images using the phasor approach. J. Phys. Chem. B 124:4510126–37
    [Google Scholar]
  61. 61. 
    Weber G. 1981. Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements. J. Phys. Chem. 85:8949–53
    [Google Scholar]
  62. 62. 
    Winkler U, Hirrlinger J. 2015. Crosstalk of signaling and metabolism mediated by the NAD(+)/NADH redox state in brain cells. Neurochem. Res. 40:122394–401
    [Google Scholar]
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