Abstract

We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells. We applied spectral phasors to isolate intracellular RNA species with similar spectral properties. We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level. Most notable were transcripts in the nucleoli which were spectrally similar to RNA clusters in the cytoplasm. We propose that these species represent ribosomal RNA and clustered ribonucleoprotein complexes. Further, we observed within this cluster Cajal bodies in the proximity of the nucleolus. In addition, transcripts in the cytoplasm undertook a filamentous morphology composed of multiple puncti structures which individually localized along and close to mitochondria but were distinct from mitochondria.

© 2012 OSA

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References

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  1. R. R. Cowden and S. K. Curtis, “Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids,” Histochemistry77(4), 535–542 (1983).
    [CrossRef] [PubMed]
  2. Z. Darzynkiewicz and S. P. Carter, “Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride),” Cancer Res.48(5), 1295–1299 (1988).
    [PubMed]
  3. Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
    [PubMed]
  4. F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
    [CrossRef] [PubMed]
  5. G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
    [CrossRef] [PubMed]
  6. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
    [CrossRef] [PubMed]
  7. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
    [CrossRef] [PubMed]
  8. E. B. van Munster and T. W. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005).
    [PubMed]
  9. B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
    [CrossRef]
  10. F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012).
    [CrossRef] [PubMed]
  11. T. Kiss, “Biogenesis of small nuclear RNPs,” J. Cell Sci.117(25), 5949–5951 (2004).
    [CrossRef] [PubMed]
  12. P. J. Santangelo, N. Nitin, and G. Bao, “Live cell imaging of messenger RNA co-localization with mitochondria,” in Proceedings of the 2005 Summer Bioengineering Conference (2005), pp. 701–702.
  13. P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
    [CrossRef] [PubMed]
  14. P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
    [CrossRef] [PubMed]

2012 (2)

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012).
[CrossRef] [PubMed]

2011 (1)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

2009 (1)

G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
[CrossRef] [PubMed]

2008 (1)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

2005 (2)

E. B. van Munster and T. W. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005).
[PubMed]

P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
[CrossRef] [PubMed]

2004 (2)

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

T. Kiss, “Biogenesis of small nuclear RNPs,” J. Cell Sci.117(25), 5949–5951 (2004).
[CrossRef] [PubMed]

1988 (2)

Z. Darzynkiewicz and S. P. Carter, “Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride),” Cancer Res.48(5), 1295–1299 (1988).
[PubMed]

F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
[CrossRef] [PubMed]

1986 (1)

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

1983 (1)

R. R. Cowden and S. K. Curtis, “Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids,” Histochemistry77(4), 535–542 (1983).
[CrossRef] [PubMed]

Andrews, L. M.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

Bader, A. N.

Bao, G.

G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
[CrossRef] [PubMed]

P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
[CrossRef] [PubMed]

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

Caiolfa, V. R.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Carter, S. P.

Z. Darzynkiewicz and S. P. Carter, “Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride),” Cancer Res.48(5), 1295–1299 (1988).
[PubMed]

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Cinquin, A.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

Cinquin, O.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

Cowden, R. R.

R. R. Cowden and S. K. Curtis, “Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids,” Histochemistry77(4), 535–542 (1983).
[CrossRef] [PubMed]

Crissman, H. A.

F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
[CrossRef] [PubMed]

Curtis, S. K.

R. R. Cowden and S. K. Curtis, “Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids,” Histochemistry77(4), 535–542 (1983).
[CrossRef] [PubMed]

Darzynkiewicz, Z.

Z. Darzynkiewicz and S. P. Carter, “Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride),” Cancer Res.48(5), 1295–1299 (1988).
[PubMed]

F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
[CrossRef] [PubMed]

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Digman, M. A.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Donovan, P. J.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

Fereidouni, F.

Gadella, T. W.

E. B. van Munster and T. W. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005).
[PubMed]

Gerritsen, H. C.

Gratton, E.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Jones, M. R.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

Kapuscinski, J.

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Kiss, T.

T. Kiss, “Biogenesis of small nuclear RNPs,” J. Cell Sci.117(25), 5949–5951 (2004).
[CrossRef] [PubMed]

Markham, J.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

Melamed, M. R.

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Nitin, N.

P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
[CrossRef] [PubMed]

Nix, B.

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

Rhee, W. J.

G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
[CrossRef] [PubMed]

Santangelo, P. J.

P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
[CrossRef] [PubMed]

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

Schmid, F. A.

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Stringari, C.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

Traganos, F.

F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
[CrossRef] [PubMed]

Tsourkas, A.

G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
[CrossRef] [PubMed]

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

van Munster, E. B.

E. B. van Munster and T. W. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005).
[PubMed]

Wright, B. K.

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

Zamai, M.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Adv. Biochem. Eng. Biotechnol. (1)

E. B. van Munster and T. W. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng. Biotechnol.95, 143–175 (2005).
[PubMed]

Annu. Rev. Biomed. Eng. (1)

G. Bao, W. J. Rhee, and A. Tsourkas, “Fluorescent probes for live-cell RNA detection,” Annu. Rev. Biomed. Eng.11(1), 25–47 (2009).
[CrossRef] [PubMed]

Biophys. J. (2)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008).
[CrossRef] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J.103(1), L7–L9 (2012).
[CrossRef]

Cancer Res. (2)

Z. Darzynkiewicz and S. P. Carter, “Photosensitizing effects of the tricyclic heteroaromatic cationic dyes pyronin Y and toluidine blue O (tolonium chloride),” Cancer Res.48(5), 1295–1299 (1988).
[PubMed]

Z. Darzynkiewicz, J. Kapuscinski, S. P. Carter, F. A. Schmid, and M. R. Melamed, “Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA,” Cancer Res.46(11), 5760–5766 (1986).
[PubMed]

Exp. Cell Res. (1)

F. Traganos, H. A. Crissman, and Z. Darzynkiewicz, “Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells,” Exp. Cell Res.179(2), 535–544 (1988).
[CrossRef] [PubMed]

Histochemistry (1)

R. R. Cowden and S. K. Curtis, “Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids,” Histochemistry77(4), 535–542 (1983).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

P. J. Santangelo, N. Nitin, and G. Bao, “Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons,” J. Biomed. Opt.10(4), 044025 (2005).
[CrossRef] [PubMed]

J. Cell Sci. (1)

T. Kiss, “Biogenesis of small nuclear RNPs,” J. Cell Sci.117(25), 5949–5951 (2004).
[CrossRef] [PubMed]

Nucleic Acids Res. (1)

P. J. Santangelo, B. Nix, A. Tsourkas, and G. Bao, “Dual FRET molecular beacons for mRNA detection in living cells,” Nucleic Acids Res.32(6), e57 (2004).
[CrossRef] [PubMed]

Opt. Express (1)

Proc. Natl. Acad. Sci. U.S.A. (1)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13582–13587 (2011).
[CrossRef] [PubMed]

Other (1)

P. J. Santangelo, N. Nitin, and G. Bao, “Live cell imaging of messenger RNA co-localization with mitochondria,” in Proceedings of the 2005 Summer Bioengineering Conference (2005), pp. 701–702.

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Figures (5)

Fig. 1
Fig. 1

Z-stack intensity images and corresponding image averaged emission profiles of PY labeled species in a live NIH3T3 cell. The color coded arrows link each emission spectrum with the corresponding plane of focus. Small spectral shifts were observed for each plane.

Fig. 2
Fig. 2

Fluorescence intensity (A) and spectral phasor z-stack images (B) of PY labeled species in a live NIH3T3 cell with cluster selection through the use of three cursors (radius = 3-5 nm) in the distribution plot (C: centre and magnified right). Cluster selection provided three main species. One selection (yellow painted) mainly in the nucleoli and sporadically in the cytoplasm may be associated with rRNA and mRNP complexes. The second selection (purple) was positioned throughout the cytoplasm. Species in the nucleus (blue color) appeared positioned around the nuclear periphery or surrounding the nucleoli and represented a linear combination of PY transcripts and background. Cluster distributions were also globally assessed for PY in DMEM and DMEM/background. Background displayed minimal migration from the centre of the distribution plot with DMEM inducing deviation from 0 as pictured (C: left). PY in solution displayed a compact cluster at an average wavelength of 557.7 ± 1.5nm width 37.1 ± 0.7nm positioned slightly below 0 line of reference (C: left).

Fig. 3
Fig. 3

Fluorescence intensity (A) and spectral phasor z-stack images (B) of PY labeled species in a live NIH3T3 cell with cluster selection through the use of three cursors with a maximum radii of 4nm within the distribution plot (C: left and magnified: right). Cluster selection again provided three main species.

Fig. 4
Fig. 4

Spectral phasor images (A) and spectral phasor plot (B) of PY labeled species in a live NIH3T3 cell. The two images display the same plane of focus but two different zooms. Cluster selection occurred through the use of a single cursor with a radius of 3nm. The PY selected for with the yellow cluster (lower images) displayed similar properties to the other cells however there were small detected clusters within the nucleus which bear similar characteristics to Cajal bodies (number present, size and morphology).

Fig. 5
Fig. 5

Fluorescence microscopy image (A) of live NIH3T3 cells dual labeled with mito-eGFP (green) and PY (red) 512 × 512 image size. The time lapse images (B) show the spatial position of the transcripts (red) with the mitochondrial matrix (green). Note the mobilization of the transcripts which appear to move along single mitochondria (black arrows) as a function of time (6 second intervals).

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