‹Purpose› To evaluate the value of the photodynamic diagnosis (PDD) of bladder cancer with intravesical instillation of 5-aminolevulinic acid (5-ALA).
‹material and method› In 115 patients with bladder cancer, PDD was performed. Two to two half hours prior to transurethral biopsy 15.g 5-ALA dissolved in 50 ml of 8.4% sodium hydrogen carbonate (NaHCO₃) solution was instilled intravesically. The level in images of the fluorescence were compared with pathological result. The area under the receiver operative characteristic curve (AUC) in PDD was compared with that in white light endoscopy.
‹Result› Among the 807 specimens obtained by transurethral biopsy, 276 specimens (34.2%) were pathologically diagnosed malignant epithelium, including 58 specimens (7.2%) with carcinoma in situ and 47 specimens (5.8%) with dysplasia were detected pathologically, with a sensitivity of 94.5%, a specificity of 60.6% and a predictive accuracy of 66.6%. The AUC in blue light endoscopy was more than that in white light endoscopy in not only all cases (p=0.002) but also in polypoid lesion (p=0.001), non-polypoid lesion (p<0.001), non-papillary lesion (p<0.001), new cases (p=0.003) and recurrent case (p=0.226). Procedures were well tolerated by all patients with mild bladder irritability but no systemic side effect.
‹Conclusion› Photodynamic diagnosis with intravesically applied 5-ALA is effective in detecting bladder cancer, and is expected to become a standard in the detection program.

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Nanoparticles have been proposed as potential carriers for photosensitizers. Novel silica nanoparticles made of organosilica (Organo-NPs) have been successfully synthesized by using a St?ber method. We compared the Organo-NPs to classical silica nanoparticles made of tetraethoxysilane (TEOS NPs). The Organo-NPs were well dispersed in solution and have unique surface properties such as thiol residues on the surface and reduced zeta potential compared with TEOS NPs. The Organo-NPs with fluorescent dye distributed throughout the particle were synthesized by a method that deposited fluorescent dye on the silica network via a succinimidyl ester reaction with (3-aminopropyl) trimethoxysilane and via a maleimide reaction with organosilica. The resulting Organo-NPs with fluorescent were bright, non-aggregated in solution, and photostable. The fluorescence intensity and photostability of fluorescent Organo-NPs were sufficient for detection as a single fluorescent particle using flow cytometry and fluorescence microscopy. Protein-modified Organo-NPs were easily prepared by absorption and by maleimide coupling. The Organo-NPs did not exhibit death or cytotoxic findings on in vivo injection. The Organo-NPs containing photosensitizers showed phototoxic activity in culture cells. We demonstrate the usefulness of the Organo-NPs and their surface properties, and discuss their advantages for biological applications and possibility for photodynamic therapy.

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Non-muscle invasive bladder cancer (NMIBC) is known as a high-risk tumor for intravesical recurrence after transurethral resection of bladder tumor (TURBT) since residual tumor around the surgical margin, concomitant small tumor, or flat tumor like carcinoma in situ (CIS) is often overlooked under the conventional cystoscopic observation. The positive rate of cytology for detection and follow-up is usually very low in low-grade NMIBC. We report herein our clinical results of photodynamic diagnosis using 5-aminolevulinic acid (5-ALA)-induced fluorescence cystoscopy and cytology. Thirty-four patients suspected of having NMIBC underwent fluorescence cystoscopy-assisted TURBT. Histopathologically, 29 patients had urothelial carcinoma and 5 were diagnosed as having non-carcinoma tissues (papilloma, squamous metaplasia, hyperplasia or inflammatory granulation). Pathological examination of 223 specimens obtained from 29 urothelial carcinoma patients revealed that the sensitivity and specificity of fluorescence cystoscopy were 96% and 73%, respectively. Four CIS specimens among 107 fluorescence-positive cancer specimens and 8 dysplasia specimens among 30 fluorescence-positive non-cancer specimens were overlooked with the conventional cystoscopy. Only one patient showed irritable bladder of grade 1 associated with 5-ALA. 5-ALA-induced fluorescence cytology and flow cytometry to detect malignant cells exfoliated in the urine samples of 20 patients showed higher positive rates even in low-grade or low-stage NMIBC than the conventional cytology. Fluorescence cystoscopy-assisted TURBT is a safe and reliable surgical modality for NMIBC. Fluorescence cytology and flow cytometry also may be promising tools for detection of malignant cells exfoliated in the urine.

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Photodynamic therapy (PDT) involves the administration of a photosensitizer followed by local illumination of a tumor with light of the appropriate wavelength to activate a specific drug. Efficacy is high for small superficial tumors and, except for temporary skin photosensitization, there are no long-term side effects if appropriate protocols are followed. However, an inherent consequence of PDT is local hypoxia. Hypoxia is the major stimulus for vascular endothelial growth factor (VEGF) production and secretion. VEGF could contribute to tumor survival and regrowth and therefore could be one of the factors impairing PDT from achieving its full tumoricidal potential. Therefore, an understanding of tumor cell responses to PDT will help in the design of new interventions and potentially improve long-term survival of PDT treated patients. It is well known that the cells in tumor tissue are heterogeneous in terms of morphology and differentiation status, even if the tumor consists of progeny developed from a single neoplastic cell. In this study, we found that morphologically heterogeneous tumor cell clones had differing photosensitizer (talaporfin sodium) uptake and PDT sensitivity. Morphologically heterogeneous clones were formed from a cultured cell line (KYSE70) established from a single human esophageal carcinoma tissue. Five subclones were separated from a single clone of KYSE70 cells. Three subclones were mainly flat and diffusive, while the other two formed mounds. Some subclones were more PDT-sensitive than the other subclones, although the intracellular uptake of talaporfin sodium was lower. Although talaporfin sodium could be effectively taken up by some subclones, these subclones were not effectively PDT sensitive, irrespective of cell morphology. The data demonstrated that a single tumor cell has the potential to produce progeny that are heterogeneous in terms of morphology and PDT sensitivity, irrespective of intracellular talaporfin sodium concentration. Further improvements in the clinical application of PDT for heterogeneous tumor cells are expected, since PDT may be ultimately useful as a clinical tumor therapy.

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Raman spectroscopy has been used as a powerful technique to investigate molecular structures and their conditions and environments due to its capability of detecting molecular vibrations in a sample. In the recent developements in optical microscopy, Raman scattering has been utilized to simultaneouly image and analyze the chemical components in a sample, which allows us to obtain molecular information in the sample without any labeling. Coherent-anti Stokes Raman scattering and surface-enhanced Raman scattering provide higher sensitivity in Raman microscopy and boost the capability of Raman imaging for various biological applications.

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Raman microscopy visualizes molecular spices without any staining, because molecular vibrations that all molecules have are sensitive to molecular spices. We developed a real-time CARS (coherent anti-Stokes Raman scattering, which is one of nonlinear Raman scattering) microscopy system. We demonstrate three dimensional lipid distribution in a cell, laser-induced disruption of a lipid rich organelle, and plasma membrane repairing of disrupted plasma membrane.

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2-color infrared (IR) super-resolution microscopy enables us to detect IR absorption with resolution of less than 1 μm, which is equivalent to that of fluorescence microscope. To prove that this microscopy can be utilized as cell analysis, we attempted to detect IR absorption inside A549 cells at IR wavelength of about 3 μm. When stained, we achieved to detect the IR absorption of protein molecules bound to fluorescent molecules. When non-stained, we achieved to detect the IR absorption of flavin molecules using auto-fluorescence. The microscopy is applicable to biochemical analysis inside cells based on molecular structure and thereby e.g. to cell selection.

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Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multi-photon excitation induced by an ultra-short pulse near infrared laser light, is one of the most promising candidates for functional analysis of cells and biomolecules in intact living organs or tissues. Two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over a long observation period with excellent spatial resolution. Furthermore, two-photon microscopy facilitates the investigation of the molecular basis and the pathology underlying exocytosis. This review describes both the current and anticipated capabilities of two-photon microscopy, based on discussions of previous publications and recently obtained data.

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Imaging of fluorescence lifetime has received much attention in recent years for investigating biological systems. Fluorescence intensity depends on concentration of chromophore, excitation intensity, and optical conditions, whereas fluorescence lifetime is an inherent property of a chromophore and therefore independent of the factors that limit fluorescence intensity measurements. Fluorescence lifetime imaging (FLIM) techniques therefore provide more quantitative information on the cellular environment compared with fluorescence intensity imaging. In the present review, we briefly introduce the physical properties of fluorescence lifetime and show the FLIM images of living cells expressing green fluorescent protein mutants.

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