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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

Sample time-lapse average lifetime images of Photofrin® (20 µg/mL) uptake by MLL cells between 1-18 hours. The color legend illustrates the corresponding lifetime values ranging from 1 ns (red) to 10 ns (blue). The pixels corresponding to the photosensitizer in the lifetime images are co-localized with the signal of the steady-state fluorescence images (data not shown). (A) The image shows Photofrin® uptake by the cell membrane at 1 hour of incubation; (B) 2 to 4 hours of incubation. This image was acquired at 2.5 hours of incubation when Photofrin® started to migrate into the cytoplasm; (C) Photofrin® has localized at the peri-nuclear region after 6 hours of incubation; and (D) After 18 hours of incubation, granular spots have appeared in the cells and the redistribution of the drug after prolonged incubation was observed.
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Figure 3: Sample time-lapse average lifetime images of Photofrin® (20 µg/mL) uptake by MLL cells between 1-18 hours. The color legend illustrates the corresponding lifetime values ranging from 1 ns (red) to 10 ns (blue). The pixels corresponding to the photosensitizer in the lifetime images are co-localized with the signal of the steady-state fluorescence images (data not shown). (A) The image shows Photofrin® uptake by the cell membrane at 1 hour of incubation; (B) 2 to 4 hours of incubation. This image was acquired at 2.5 hours of incubation when Photofrin® started to migrate into the cytoplasm; (C) Photofrin® has localized at the peri-nuclear region after 6 hours of incubation; and (D) After 18 hours of incubation, granular spots have appeared in the cells and the redistribution of the drug after prolonged incubation was observed.

Mentions: Steady-state confocal images showed the location and cellular uptake rate of Photofrin® matched the literature, where Photofrin® first binds to the plasma membrane, then moves towards cytoplasm, and finally targets the inner membrane of the mitochondria 20,24,25. These confocal results were also consistent with the FLIM images when cells were stained with 20 µg/mL of Photofrin® as shown in Figure 3.


Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

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

Sample time-lapse average lifetime images of Photofrin® (20 µg/mL) uptake by MLL cells between 1-18 hours. The color legend illustrates the corresponding lifetime values ranging from 1 ns (red) to 10 ns (blue). The pixels corresponding to the photosensitizer in the lifetime images are co-localized with the signal of the steady-state fluorescence images (data not shown). (A) The image shows Photofrin® uptake by the cell membrane at 1 hour of incubation; (B) 2 to 4 hours of incubation. This image was acquired at 2.5 hours of incubation when Photofrin® started to migrate into the cytoplasm; (C) Photofrin® has localized at the peri-nuclear region after 6 hours of incubation; and (D) After 18 hours of incubation, granular spots have appeared in the cells and the redistribution of the drug after prolonged incubation was observed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475212&req=5

Figure 3: Sample time-lapse average lifetime images of Photofrin® (20 µg/mL) uptake by MLL cells between 1-18 hours. The color legend illustrates the corresponding lifetime values ranging from 1 ns (red) to 10 ns (blue). The pixels corresponding to the photosensitizer in the lifetime images are co-localized with the signal of the steady-state fluorescence images (data not shown). (A) The image shows Photofrin® uptake by the cell membrane at 1 hour of incubation; (B) 2 to 4 hours of incubation. This image was acquired at 2.5 hours of incubation when Photofrin® started to migrate into the cytoplasm; (C) Photofrin® has localized at the peri-nuclear region after 6 hours of incubation; and (D) After 18 hours of incubation, granular spots have appeared in the cells and the redistribution of the drug after prolonged incubation was observed.
Mentions: Steady-state confocal images showed the location and cellular uptake rate of Photofrin® matched the literature, where Photofrin® first binds to the plasma membrane, then moves towards cytoplasm, and finally targets the inner membrane of the mitochondria 20,24,25. These confocal results were also consistent with the FLIM images when cells were stained with 20 µg/mL of Photofrin® as shown in Figure 3.

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