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Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

Yeh SC, Diamond KR, Patterson MS, Nie Z, Hayward JE, Fang Q - Theranostics (2012)

Bottom Line: Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage.The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters.When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: 1. School of Biomedical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada;

ABSTRACT
Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin(®) at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

No MeSH data available.


Related in: MedlinePlus

Distribution of A2 as a function of different incubation time. A2 was increased from 19% to 35% as cells took up more Photofrin® as the incubation time increased, suggesting contribution of τ2 (mostly from photoproducts of Photofrin® under strong irradiation) increased. However, at the time of 18 hours, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® may have been released and redistributed throughout the cells.
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Figure 6: Distribution of A2 as a function of different incubation time. A2 was increased from 19% to 35% as cells took up more Photofrin® as the incubation time increased, suggesting contribution of τ2 (mostly from photoproducts of Photofrin® under strong irradiation) increased. However, at the time of 18 hours, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® may have been released and redistributed throughout the cells.

Mentions: Figure 6 and Figure 7 show the normalized coefficients, A1 and A2, of the short and long lifetime components. As seen in Figure 6, the significant increase of A2 from 19% to 35% (p < 0.01) was observed when cells are taking up more Photofrin® over time, supporting the assertion that more aggregates and photoproducts of Photofrin® are formed after irradiation. At 18 hours, however, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® might be released and redistributed throughout the cells again. At early incubation times when intracellular concentrations of Photofrin® were low, the autofluorescence, mainly short component of porphyrin species that exhibit 1.7 ns of average fluorescence lifetime in the non-stained control group (with 70 % of τ1 centered at 0.8 ns, and 30 % of τ2 centered at 3.5 ns, data not shown), contributed more to the short lifetime component than Photofrin® itself. Therefore, as the τ1 is very close to the system response (0.2 ns), it may contain contributions from short autofluorescence signals and estimation uncertainties, especially in the case of low signal from Photofrin®. This can be seen in Figure 7, where the short lifetime component contributed the most at the beginning of the incubation, and as the intracellular Photofrin® concentration increased, the relative amplitude A1 decreased from 81% to 65%.


Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

Yeh SC, Diamond KR, Patterson MS, Nie Z, Hayward JE, Fang Q - Theranostics (2012)

Distribution of A2 as a function of different incubation time. A2 was increased from 19% to 35% as cells took up more Photofrin® as the incubation time increased, suggesting contribution of τ2 (mostly from photoproducts of Photofrin® under strong irradiation) increased. However, at the time of 18 hours, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® may have been released and redistributed throughout the cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475212&req=5

Figure 6: Distribution of A2 as a function of different incubation time. A2 was increased from 19% to 35% as cells took up more Photofrin® as the incubation time increased, suggesting contribution of τ2 (mostly from photoproducts of Photofrin® under strong irradiation) increased. However, at the time of 18 hours, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® may have been released and redistributed throughout the cells.
Mentions: Figure 6 and Figure 7 show the normalized coefficients, A1 and A2, of the short and long lifetime components. As seen in Figure 6, the significant increase of A2 from 19% to 35% (p < 0.01) was observed when cells are taking up more Photofrin® over time, supporting the assertion that more aggregates and photoproducts of Photofrin® are formed after irradiation. At 18 hours, however, the images showed depolarization and swelling of mitochondria, therefore the Photofrin® might be released and redistributed throughout the cells again. At early incubation times when intracellular concentrations of Photofrin® were low, the autofluorescence, mainly short component of porphyrin species that exhibit 1.7 ns of average fluorescence lifetime in the non-stained control group (with 70 % of τ1 centered at 0.8 ns, and 30 % of τ2 centered at 3.5 ns, data not shown), contributed more to the short lifetime component than Photofrin® itself. Therefore, as the τ1 is very close to the system response (0.2 ns), it may contain contributions from short autofluorescence signals and estimation uncertainties, especially in the case of low signal from Photofrin®. This can be seen in Figure 7, where the short lifetime component contributed the most at the beginning of the incubation, and as the intracellular Photofrin® concentration increased, the relative amplitude A1 decreased from 81% to 65%.

Bottom Line: Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage.The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters.When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: 1. School of Biomedical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada;

ABSTRACT
Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin(®) at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

No MeSH data available.


Related in: MedlinePlus