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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 τ2 (slow component) as a function of different incubation times. The four different locations (cell membrane, cytoplasm, mitochondria, and redistribution) were determined based on the preliminary results from confocal images. Image segmentation was performed at 4 hours and 4.5 hours of incubation when Photofrin® localized at the mitochondria and cytoplasm. Significant short τ2 was observed (4.3 ± 0.5 ns) in the cell membrane group (p < 0.05), while Photofrin® exhibited the mean lifetime of 7.1 ± 0.3 ns for the remaining intracellular locations. It was observed that the standard deviation was reduced when Photofrin® localized at the mitochondria.
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Figure 4: Distribution of τ2 (slow component) as a function of different incubation times. The four different locations (cell membrane, cytoplasm, mitochondria, and redistribution) were determined based on the preliminary results from confocal images. Image segmentation was performed at 4 hours and 4.5 hours of incubation when Photofrin® localized at the mitochondria and cytoplasm. Significant short τ2 was observed (4.3 ± 0.5 ns) in the cell membrane group (p < 0.05), while Photofrin® exhibited the mean lifetime of 7.1 ± 0.3 ns for the remaining intracellular locations. It was observed that the standard deviation was reduced when Photofrin® localized at the mitochondria.

Mentions: The FLIM datasets acquired at different incubation times were then classified into four categories based on the intracellular distribution: namely membrane, cytoplasm, mitochondria, and redistribution groups. Various sources of fluorescence may contribute to the total exponential decay signal, such as photoproducts, different molecular constituents of Photofrin®, and autofluorescence. Rather than use the average fluorescence lifetime to monitor the Photofrin® uptake process, the fluorescence lifetimes and amplitudes were plotted separately. Figure 4 shows the slow lifetime component of Photofrin® when it is localized at different intracellular components over various incubation periods. The values of the longer lifetime component (τ2) are consistent with previous spectral-resolved FLIM studies, where the fluorescence signals of photoproducts and aggregated Photofrin® (emission range at 651 nm - 687 nm) increased after irradiation and exhibited long lifetime components of approximately 8 ns 13,16. In Figure 4, τ2 did not change significantly from 2 hours to 18 hours of incubation. The individual standard deviation of τ2 , however, was reduced from around 1 ns to 0.4 ns after 4.5 hours of incubation, when Photofrin® was localized in the mitochondrial regions. The overlap of cytoplasmic and mitochondria groups at 4 hours and 4.5 hours of incubation was analyzed based on the segmentation technique. At the transition stage, the values of fluorescence lifetime and standard deviation between two groups were similar. It should be noted that Photofrin® exhibited the slow decay time (τ2) of 4.3 ± 0.5 ns when it was bound to the cell membrane. The significant lifetime change compared to all other incubation periods (p < 0.01 except for redistribution group where p < 0.05) suggested that the differences between the cell membrane microenvironment and other regions may result in more quenching of the fluorescence.


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 τ2 (slow component) as a function of different incubation times. The four different locations (cell membrane, cytoplasm, mitochondria, and redistribution) were determined based on the preliminary results from confocal images. Image segmentation was performed at 4 hours and 4.5 hours of incubation when Photofrin® localized at the mitochondria and cytoplasm. Significant short τ2 was observed (4.3 ± 0.5 ns) in the cell membrane group (p < 0.05), while Photofrin® exhibited the mean lifetime of 7.1 ± 0.3 ns for the remaining intracellular locations. It was observed that the standard deviation was reduced when Photofrin® localized at the mitochondria.
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Related In: Results  -  Collection

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Figure 4: Distribution of τ2 (slow component) as a function of different incubation times. The four different locations (cell membrane, cytoplasm, mitochondria, and redistribution) were determined based on the preliminary results from confocal images. Image segmentation was performed at 4 hours and 4.5 hours of incubation when Photofrin® localized at the mitochondria and cytoplasm. Significant short τ2 was observed (4.3 ± 0.5 ns) in the cell membrane group (p < 0.05), while Photofrin® exhibited the mean lifetime of 7.1 ± 0.3 ns for the remaining intracellular locations. It was observed that the standard deviation was reduced when Photofrin® localized at the mitochondria.
Mentions: The FLIM datasets acquired at different incubation times were then classified into four categories based on the intracellular distribution: namely membrane, cytoplasm, mitochondria, and redistribution groups. Various sources of fluorescence may contribute to the total exponential decay signal, such as photoproducts, different molecular constituents of Photofrin®, and autofluorescence. Rather than use the average fluorescence lifetime to monitor the Photofrin® uptake process, the fluorescence lifetimes and amplitudes were plotted separately. Figure 4 shows the slow lifetime component of Photofrin® when it is localized at different intracellular components over various incubation periods. The values of the longer lifetime component (τ2) are consistent with previous spectral-resolved FLIM studies, where the fluorescence signals of photoproducts and aggregated Photofrin® (emission range at 651 nm - 687 nm) increased after irradiation and exhibited long lifetime components of approximately 8 ns 13,16. In Figure 4, τ2 did not change significantly from 2 hours to 18 hours of incubation. The individual standard deviation of τ2 , however, was reduced from around 1 ns to 0.4 ns after 4.5 hours of incubation, when Photofrin® was localized in the mitochondrial regions. The overlap of cytoplasmic and mitochondria groups at 4 hours and 4.5 hours of incubation was analyzed based on the segmentation technique. At the transition stage, the values of fluorescence lifetime and standard deviation between two groups were similar. It should be noted that Photofrin® exhibited the slow decay time (τ2) of 4.3 ± 0.5 ns when it was bound to the cell membrane. The significant lifetime change compared to all other incubation periods (p < 0.01 except for redistribution group where p < 0.05) suggested that the differences between the cell membrane microenvironment and other regions may result in more quenching of the fluorescence.

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