This PDF file contains the front matter associated with SPIE Proceedings Volume 8224, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Photodynamic therapy (PDT) for cancer is known to induce an immune response against the tumor, in addition to its well-known direct cell-killing and vascular destructive effects. PDT is becoming increasingly used as a therapy for localized infections. However there has not to date been a convincing report of an immune response being generated against a microbial pathogen after PDT in an animal model. We have studied PDT as a therapy for bacterial arthritis caused by Staphylococcus aureus infection in the mouse knee. We had previously found that PDT of an infection caused by injection of MRSA (5X10⁷ CFU) into the mouse knee followed 3 days later by 1 μg of Photofrin and 635- nm diode laser illumination with a range of fluences within 5 minutes, gave a biphasic dose response. The greatest reduction of MRSA CFU was seen with a fluence of 20 J/cm2, whereas lower antibacterial efficacy was observed with fluences that were either lower or higher. We then tested the hypothesis that the host immune response mediated by neutrophils was responsible for most of the beneficial antibacterial effect. We used bioluminescence imaging of luciferase expressing bacteria to follow the progress of the infection in real time. We found similar results using intra-articular methylene blue and red light, and more importantly, that carrying out PDT of the noninfected joint and subsequently injecting bacteria after PDT led to a significant protection from infection. Taken together with substantial data from studies using blocking antibodies we believe that the pre-conditioning PDT regimen recruits and stimulates neutrophils into the infected joint which can then destroy bacteria that are subsequently injected and prevent infection.

Topical application of a potent immunological modulator, imiquimod, followed by laser irradiation has been used for the treatment of late-stage melanoma patients. This novel approach, laser-assisted laser immunotherapy (LIT), targets the root course of melanoma, a highly metastatic cancer. We started a phase I clinical trial in 2006 with promising initial outcomes. The laser-imiquimod combination showed significant palliative effects for these patients with multiple treatment cycles. For the returning patients, we found that the recurrent tumors were less aggressive than usually seen in untreated patients. The current protocol uses a light-absorbing dye for selective laser photothermal interaction with a non-invasive treatment mode. It has limitations for patient treatment, particularly for large, deeper tumors, and for patients with dark pigmented skins. This study provides some information on the treated patients (both stage IV and stage IV) during the past several years. We also discuss the future directions of LIT, particularly in the area of photothermal treatment mode with a new approach of interstitial irradiation. The current results in melanoma treatment using LIT indicate that the combination of photothermal therapy and immunological stimulation may hold the key for the treatment of late-stage, metastatic cancers, not only for cutaneous cancers such as melanoma and breast cancer, but also for deep and internal tumors using different operations modes such as interstitial laser irradiation.

Laser immunotherapy (LIT) was developed to treat metastatic cancers using a combination of laser irradiation and immunological stimulation. The original design of LIT employs a non-invasive, selective laser photothermal interaction, using an in situ light-absorbing dye. However, this non-invasive treatment mode faces challenges in treating deep, large tumors. Furthermore, it has difficulties in the cases of highly pigmented skin overlying target tumors. To overcome these limitations, interstitial laser immunotherapy (ILIT) was proposed. In ILIT, a cylindrical, side-fire fiber diffuser is placed inside the target tumor to induce thermal damage. To enhance the interstitial irradiation induced photothermal interaction, an immunological modifier, glycated chitosan (GC), is injected into the tumor after the laser treatment. In this study, a cylindrical diffuser with an active length of 1 cm was used to treat tumors of 1 to 1.5 cm in size. Different laser powers (1 to 3 watts) and different irradiation durations (10 to 30 minutes) were used to test the thermal effects of ILIT. Different doses of the GC (1.0%, 0.1 to 0.6 ml per rat) were used to determine the immunological effects of ILIT. Our results show that the animal survival depends on both laser dose and GC dose. A dose of 0.2 ml per tumor appeared to result in the highest survival rate under interstitial laser irradiation with 2.5 watts and 20 minutes. While the results in this study are not conclusive, they indicate that interstitial laser irradiation can be combined with immunotherapy to treat metastatic cancers. Furthermore, our results suggest that an optimal combination of laser dose and GC dose could be obtained for future clinical protocols using interstitial laser immunotherapy.

Selective photothermal interaction using dye-assisted non-invasive laser irradiation has limitations when treating deeper tumors or when the overlying skin is heavily pigmented. We developed an interstitial laser irradiation method to induce the desired photothermal effects. An 805-nm near-infrared laser with a cylindrical diffuser was used to treat rat mammary tumors by placing the active tip of the fiber inside the target tumors. Three different power settings (1.0 to 1.5 watts) were applied to treat animal tumors with an irradiation duration of 10 minutes. The temperature distributions of the treated tumors were measured by a 7.1-Tesla magnetic resonance imager using proton resonance frequency (PRF) method. Three-dimensional temperature profiles were reconstructed and assessed using PRF. This is the first time a 7.1-Tesla magnetic resonance imager has been used to monitor interstitial laser irradiation via PRF. This study provides a basic understanding of the photothermal interaction needed to control the thermal damage inside tumor using interstitial laser irradiation. It also shows that PRF can be used effectively in monitoring photothermal interaction. Our long-term goal is to develop a PRF-guided laser therapy for cancer treatment.

Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. Hepatocellular carcinoma may metastasize to lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor: the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed "in vivo flow cytometer" combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

The objectives of this project are to intrinsically and quantitatively investigate the advantages of a phase-contrast (PC) tomosynthesis prototype in removing the superimposed structure noise and the effects on image qualities by an off-center shift of the object along the tube-sweep direction. Experimentally, phantoms are assembled with standard resolution patterns. Phase-contrast tomosynthesis images are acquired and reconstructed. In order to study the effects of the limited angular projections on the in-plane resolutions, all the images were reconstructed in the 2×2 binning mode only, as we will study the phase contrast effects elsewhere. The in-plane slices reconstructed from each of the experiments examined and the in-plane limiting resolutions are determined. For comparison, the resolution patterns and phantoms are also imaged by single projections. Under single x-ray projection, with only one resolution pattern, the limiting resolution of the system is 8 lp/mm; with 2 resolution patterns superimposed, the image of the resolution patterns is blurred for distinguishing line pairs. The PC tomosynthesis in-plane images show that the limiting resolution of the system is 7 lp/mm. For objects with a shift along the horizontal axis (the tube-sweep direction) by 0.8 inch, the spatial resolution is degraded to 4 lp/mm and blur occurs. As is expected, the PC tomosynthesis prototype studied in this project reveals superimposed fine structures of the object; the effect induced by the object's off-center shift is determined in a quantified way. The in-plane resolution of this system can be further improved by optimizing the system alignment, and the reconstruction algorithms.

The purpose of this paper is to report a new automatic scanning scheme for high throughput microscopic systems aiming to facilitate disease diagnosis in genetic laboratories. To minimize the impact of the random vibration and mechanical drifting of the scanning stage in microscopic image acquisition, auto-focusing operations are usually applied repeatedly during the scanning process. Such methods ensure the acquisition of well focused images for clinical diagnosis, but are time consuming. The technique investigated in this preliminary study applies the auto-focusing operations at a limited number of locations on the slide. For the rest of the imaging field, the focusing position is quickly adjusted through linear interpolation. In this initial validation study, blood pathological slides containing both metaphase and interphase cells are scanned. For a selected area of 6.9mm×6.9mm, a number of 2×2, 3×2, 3×3, and 4×4 positions are evenly sampled for auto-focusing operations. Respectively, 25, 29, 40, and 41 clinically meaningful cells are identified for each sampling scheme. For the specific case investigated, the results demonstrate that the 4 position auto-focusing scheme could obtain the adequate number of clinically meaningful cells for the diagnosis. The schemes with more auto-focusing operations provide an option for high reliability diagnosis when clinically necessary. More comprehensive research is planned, and that may lead to optimal design of trade-off for developing the scanning scheme of the high throughput microscopic systems.

The ability to modulate the myofibroblast phenotype will have important implications in wound healing, aging and cancer development. Our objective was to determine whether irradiation using a 980-nm laser affects the presence of myofibroblasts and cellular contractility using an attached collagen lattice model. Fibroblasts in type I collagen lattices were allowed to generate tension for 5 days in the presence or absence of laser light stimulation on day 4. Immunostaining was used to determine the total number of cells in a representative image and percentage of myofibroblasts in the same image; tension generation was determined by releasing tension and measuring diameter change over time. One treatment demonstrated a slight lattice contraction increase over control, correlated with increased cell number. Myofibroblast percentage was low and was not correlated with lattice contraction. More experiments are necessary due to limited sample size and lack of large deviations from the control.

We report a two-photon (TP) absorbing molecular probe 1,4-bis(4'-(N,N-bis(6''-(N,N,N-trimethylammonium)hexyl)amino)-styryl)benzene tetrabromide (C1) and its interaction with cells upon encapsulation with polymeric vesicles. Two-photon microscopy (TPM) revealed that the free C1 specifically could bind to the plasma membrane and shows bright TP emission. However, C1 encapsulated with polymeric vesicles internalized into the cytosol. In addition, fluorescence quantum efficiency and TP cross section of encapsulated C1 enhanced by 2-fold. These results not only show useful guidelines for the development of efficient TP probes, but also underscore the possibility of using this type of nanostructure for intracellular delivery of the bioactive therapeutics.

In this paper, near-infrared (NIR) light responsive polyelectrolyte multilayer capsules (PE/Au NRs@Dox) were successfully synthesized by layer-by-layer techniques. Gold nanorods with aspect ratio of 3.9, and a longitudinal absorbance peak at 765nm were prepared using seed-mediated method which used the cationic surfactant cetyltrimethylammonium bromide (CTAB) as a soft template and structure-guiding agent. Sequential layers of polyanions (sodium polystyrene sulfonate (PSS)) and polycations (poly diallyldimethylammonium chloride (PDADMAC)) were followed by adsorption of three BSA/PSS bilayers. The doxorubicin (Dox) is loaded by the electrostatic interaction. The release of Dox from the swelling polyelectrolyte complex PE/Au NRs@Dox was induced by the heat generated from gold nanorods under laser irradiation. Moreover, the in vitro cytotoxicity of PE/Au NRs@Dox and the doxorubicin release from the composites were investigated.

Our objective is to test the effect of pulsed (Q-switched) and continuous wave (CW) laser light at wavelength of 532nm on the viability of free-living stationary phase bacteria with and without gentamicin (an antibiotic) treatment. Free living stationary phase gram negative bacteria (Pseudomonas aeruginosa strain PAO1) was immersed in Luria Broth (LB) solution and exposed to Q-switched and CW lasers with and without the addition of the antibiotic gentamicin. Cell viability was determined at different time points. Laser treatment alone did not reduce cell viability compared to untreated control and the gentamicin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamicin treatment, however, resulted in a synergistic effect and viability was reduced by 8 log's for P. aeruginosa PAO1.

Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψ_m) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immunity in mammals. Laser irradiation has been found to produce photobiological effects with evidence of interference with organic functions. In this study, we focused our attention on the effects of He-Ne laser on the phagocytic activity of macrophages, the regulation mechanism of phagocytosis was also discussed. Our results indicated that Low-power laser irradiation can enhance the phagocytosis of macrophage through activation of Src.

The cytochemical analysis of the aerobic and anaerobic bactericidal, phagocytic activity, phagocytosis completeness, as well as membrane topology and rigidity has been performed with the Scanning probe microscopy (Solver Pro, NT-MDT, Russia) in neutrophils of the patients with cervical carcinoma treated with the Erbium laser radiation at different intensity. The obtained data reveal a significant effect of the femtosecond laser radiation on neutrophil morphofunctional state in patients with cervical cancer. The efficiency of the femtosecond radiation depends on its intensity and clinical stage of the cervical cancer.

Oral cancer has becomes the most prominent male cancer disease due to the local betel nut chewing habit combing with smoking and alcohol-drinking lifestyle. In order to minimize the systemic phototoxic effect of 5-aminolevulinic acid (ALA), this study was designed to use a topical ALA-mediated PDT for treatment of DMBA-induced hamster buccal pouch cancerous lesions. DMBA was applied to one of the buccal pouches of hamsters thrice a week for 8 to 10 weeks. Precancerous lesions were induced and proven by histological examination. These DMBA-induced cancerous lesions were used for testing the efficacy of topical ALA -mediated PDT. We found that ALA reached its peak level in cancerous lesions about 2.5 hrs after topical application of ALA gel. The precancerous lesions in hamsters were then treated with topical ALA -mediated PDT with light exposure dose of 75 and 100 J/cm² using LED 635 nm Wonderlight device. It is suggesting that optimization of the given light dose is critical to the success of PDT results.

In Taiwan, oral cancer has becomes the fastest growth male cancer disease due to the betel nut chewing habit combing with smoking and alcohol-drinking lifestyle of people. In order to eliminate the systemic phototoxic effect of 5-aminolevulinic acid (ALA), this study was designed to use a topical ALA-mediated PDT for treatment of DMBA-induced hamster buccal pouch precancerous lesions. DMBA was applied to one of the buccal pouches of hamsters thrice a week for 10 to 12 weeks. Cancerous lesions were induced and proven by histological examination. These DMBA-induced cancerous lesions were used for testing the efficacy of topical ALA-mediated PDT. Before PDT, fluorescence spectroscopy was used to determine when ALA reached its peak level in the lesional epithelial cells after topical application of ALA gel. We found that ALA reached its peak level in precancerous lesions about 2.5 hrs after topical application of ALA gel. The cancerous lesions in hamsters were then treated with topical ALA -mediated PDT with light exposure dose of 150 J/cm² using LED 635 nm fiber-guided light device. Visual examination demonstrated that adjuvant topical ALA -mediated PDT group has shown better therapeutic results in compared to those of non-adjuvant topical ALA-mediated PDT group for DMBA-induced hamster buccal pouch precancerous lesions.

Cell adhesion molecule integrin α_vβ₃ is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. In this manuscript, we describe the synthesis of near-infrared (NIR) fluorochrome ICG-Der-02-labeled dimeric cyclic RGD peptides (ICG-Der-02-c(RGDyK)₂) for in vivo tumor integrin targeting. The optical properties and structure of the probe were intensively characterized. Afterwards, the integrin specificity of the fluorescent probe was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous MDA-MB-231 and U87MG tumor targeting. The results indicated that after labeling RGD peptide, the optical properties of ICG-Der-02 showed no obvious change. Besides, in vitro and in vivo tumor targeting experiment indicated that the ICG-Der-02-c(RGDyK)₂ probe with high integrin affinity showed excellent tumor activity accumulation. Noninvasive NIR fluorescence imaging is able to detect tumor integrin expression based upon the highly potent RGD peptide probe.

In human, estrogens play important roles in many physiological processes, and is also found to be connected with numerous cancers. In these diseases, estrogen mediates its effects through the estrogen receptor (ER), which serves as the basis for many current clinical diagnosis. Two forms of the estrogen receptor have been identified, ERα and ERβ, and show different and specific functions. The two estrogen receptors belong to a family of ligand-regulated transcription factors. Estrogen via ERα stimulates proliferation in the breast, uterus, and developing prostate, while estrogen via ERβ inhibits proliferation and promotes differentiation in the prostate, mammary gland, colon, lung, and bone marrow stem cells. MicroRNAs (miRs) are small non-coding RNA molecules that occur naturally and downregulate protein expression by translational blockade of the target mRNA or by promoting mRNA decay. MiR-21 is one of the most studied miRNAs in cancers. MiR-21 is overexpressed in the most solid tumors, promoting progression and metastasis. The miR-21 gene is located on the chromosome 17, in the 10th intron of a protein-coding gene, TMEM49. While, the function of TMEM49 is currently unknown. Our experiment is designed to identity the relationship between miR-21 and ERβ in cancer progression. The human cancer cells were transfected with ERβ. Real-time PCR analysis showed that the expression level of miR-21 was significantly inhibited down by ERβ treatment. As MTT assay showed the tumor cell survival rate was also inhibited significantly. G_o/G_l phase cell cycle arrest was founded and tumor cell apoptosis was induced in ERβ group.

Upconversion nanoparticles (UCNPs) as a new kind of biological luminescence materials have many advantages comparing with organic fluorescence probes and semi-conductive quantum dots, such as sharp fluorescence emission, long emission lifetimes, high optical and chemical stability and low toxicity, especially low auto-fluorescence background and deep tissue penetration under near-infrared (NIR) light excitation for bioimaging. Herein, we demonstrate a facile approach to transfer UCNPs from hydrophobic to hydrophilic and the use of these UCNPs for cell imaging. Oleic acid-capped UCNPs based on NaYF₄ were synthesized and modified with amphiphilic chitosan derivative through hydrophobic interaction. The as-prepared chitosan coated UCNPs with an average diameter of 35 nm were mono-dispersed in aqueous solution and possess good optical properties upon NIR light excitation. Cell viability assays indicated the low cytotoxicity and good biocompatibility of chitosan-coated UCNPs. Cell imaging results demonstrated that chitosan-coated UCNPs had great potential for bioimaing and biolabeling. Our work suggests a feasible method to modify OA-UCNPs with amphiphilic polymer and the promise of chitosan-based UCNPs for bioimaging application.