Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. One of the
important modes cell death by photodynamic therapy (PDT) is apoptosis. The translocation of Bax from the cytosol into
the mitochondrial outer membrane is a central event during apoptosis. In this study, we detected the kinetics of Bax
activation in the cells with different photosensitizers treatments. Using laser scanning confocal microscopy, we found
that Photofrin were localized primarily in mitochondria and N-aspartyl chlorine e6 (NPe6) preferentially localized to
lysosomes. The results showed Bax can be activated and translocated from the cytosol to mitochondria by Photofrin-PDT
treatment but not by NPe6-PDT treatment in ASTC-a-1 cells. The activation of Bax started 15 minutes after Photofrin-
PDT treatment and the process of activation completed within 45 minutes. These results indicated that the initiation and
process of Bax activation are different corresponding to different sublocalization photosensitizers treatments. Our data
suggests that different sublocalization photosensitizers in PDT treatments photodamaged different organelles and will
trigger various apoptotic pathways in PDT treatment.
Apoptosis is an important cellular event that plays a key role in pathogeny and therapy of many diseases. The
mechanisms of the initiation and regulation of PDT-induced apoptosis are complex. Some PDT-associated apoptosis
pathways involved plasma membrane death receptors, mitochondria, lysosomes and endoplasmic reticulum (ER). In
order to determine the apoptosis pathway induced by Photofrin-PDT, we used fluorescence resonance energy transfer
(FRET) technique and probe SCAT3 to monitor the dynamics of caspase-3 activation after PDT treatment and also
measured caspase-8 activity. With laser scanning confocal microscopy, we found that Photofrin were localized primarily
in mitochondria, the primary targets of Photofrin-PDT. Formation of mitochondrial reactive oxygen species (ROS) was
detected within minutes after PDT treatment. This was followed by mitochondrial membrane potential (ΔΨm),
cytochrome c release, caspase-9 activity, caspase-3 activity and apoptosis. After PDT treatment, caspase-3 was activated
rapidly while caspase-8 remained inactivated. Our results indicated that PDT-induced apoptosis was initiated from
mitochondria pathway and independent of caspase-8 activation. The activation of caspase-3 by PDT started 20 minutes
after treatment and completed in about 15 minutes. PDT-induced apoptosis is directly initiated from mitochondria
pathway and not involved in the death receptors-dependent pathway. Our results demonstrated that FRET could be an
effective tool to determine PDT-induced apoptosis and other cell death mechanism.
Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. In this
study, a recombinant caspase-3 substrate was used as a fluorescence resonance energy transfer (FRET) probe to detect
the activation of caspase-3, and to monitor apoptosis in human lung adenocarcinoma (ASTC-a- 1) cells. With laser
scanning confocal microscopy, we found that Photofrin were localized primarily in mitochondria, the primary targets of
Photofrin-PDT. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the onset and
completion of caspase-3 activation induced by PDT is more rapidly than that by tumor necrosis factor-α (TNF-α). The
activation of caspase-3 by PDT started 20 minutes after treatment and completed in about 15 minutes. In comparison,
the onset of caspase-3 activation by TNF-a was delayed by 3 hours and the completion of caspase-3 activation required a
significantly longer time (approximately 90 minutes). These results indicated that the initiation and process of caspase-3
activation are different corresponding to different treatment methods. Our data suggest that caspase-3 activation
mediated by the cell surface death receptors is slower than that of the mitochondrial pathway and the mitochondria is an
efficient target to induce apoptosis.
Apoptosis is one of the important modes in PDT-induced cell death. Activation of caspase-3 is considered to be the final step in many apoptosis pathways. In this study, we used SCAT3, a fluorescence resonance energy transfer (FRET) probe containing caspase-3 substrate, to study the dynamics of caspase-3 activation in living ASTC-a-1 cells expressing stably SCAT3. The FRET analysis results indicated that caspase-3 activation in response to tumor necrosis factor-α or PDT resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The SCAT3 was cleaved immediately after PDT treatment, but that for TNF-a treatment was delayed two hours. Our experimental results suggested that the different apoptotic pathways induced by TNF-α or PDT caused different cleavage kinetics of SCAT3. This study shows that FRET technique based on GFPs could be used to study the mechanism of PDT-induced apoptosis in living cells.
Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.
It is reported that apoptosis of cancer cells in photodynamic therapy (PDT) is caused by 1O2 generated in photosensitization. In order to study the mechanism of this kind of 1O2-induced apoptosis, it is necessary to establish a special technique to dynamically detect intracellular production and localization of 1O2. FCLA, as a chemiluminescence probe to detect singlet oxygen (1O2) and superoxide (O2-.), has been used successfully in photodynamic and sonodynamic diagnosis in tissue level, recently. This paper reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. Human lung cancer cell line (ASTC-a-1) was used in the experiment. The result of this research showed that both HpD and FCLA could permeate through cellular membrane and localize to prinuclear area, when HpD or FCLA was incubated with cells. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane was different from that of FCLA. Intracellular FCLA concentration reached a peak after incubation for only 30 - 45 minutes, but amount of HpD in cells approached the equilibrium after incubation for near 22 h. In the experiment, we did not observe the evidence of FCLA or HpD penetrating into nucleolus. This study suggests that it is possibly to use a specific chemiluminescence probe to dynamcially detect the production and localization of 1O2 or 02-. in cell.
Photodynamic therapy (PDT) utilizes photon energy to activate a pre-administered photosensitizer drug in tissue to achieve a localized tumor control. PDT cell killing mechanism is directly related to the reactive oxygen species (ROS) produced during the photochemical reactions. Conventional PDT dosimetry evaluates distributions of the photosensitizer drug, photon propagation and absorption, and availability of molecular oxygen in the target tissue. Yet, the ultimate bullet for the damaging effect is ROS. An evaluation of ROS production during PDT should provide a more direct marker for PDT. Fluoresceinyl Cypridina Luciferin Analog (FCLA) is a chemiluminescence probe that specifically interacts with ROS (singlet oxygen and/or superoxide). The work is a preliminary investigation of the feasibility using FCLA as a means to evaluate ROS production in PDT.
With the increasing demand of optical storage capacity, multi-level (ML) optical storage based on multi-level-amplitude modulation is receiving more and more attention, for it transmits more information ove a fixed bandwidth channel by using the available SNR more efficiently. We proposed an ML optical storage technology in azo LCP film based on polarization angle division. In this paper, the write strategy is developed by a ML write-compensation system that corrects for non-linearities of the data signal. The dynamic transmittance range is extended with the full monotonic range of angle and the varaition of each amplitude level relative to our dynamic range is improved. The experiments of imroved linear and nonlinear 16-level storage in azo LCP are demonstrated. The effects of thickness non-uniformity on linear and nonlinear 16-level storage are discussed.
The self-injection locking single frequency Yb-doped fiber ring laser is reported. The system shows compact, stable and tunable. In the primary experiment, the Self-injection locking single frequency fiber ring laser with wavelength 1.05325)mum, power exceeding 3.5mW,line-width less than 36 MHz was manufactured. The laser shows stability, low threshold and high power. No mode-hopping was observed within 2 hours. The single polarization output can be obtained when use single polarization fiber.
By using composite self-focus rod, LD-pumped frequency doubling Nd:YVO4 laser and 3D fine regulation stand, a new Fizeau-type interferometer is developed. It can apply to measuring the micro-vibration and deformation of micro- surface at the same time. The theoretical analysis and the experimental results for the interferometer of self-focus sensor are given.
KEYWORDS: Near field scanning optical microscopy, Fiber lasers, Near field, Signal detection, Laser beam diagnostics, Cladding, Optical microscopes, Optical imaging, Near field optics, Waveguides
Passive and active fiber probes, used for the scanning near- field optical microscope, show different image properties. For the passive fiber probe, a better optical image contrast has been obtained with a parabolic taper than with a linear taper. The reflected fields in different fiber tapers have been discussed. For the active fiber probe, an ASE probe made of Er3+-doped fiber has shown a higher efficiency of several times and a lower image noise than the usual passive probe.
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