Hisanao Hazama

Integrated ultraviolet and tunable mid-infrared laser source for analyses of proteins

Mass spectrometry using matrix-assisted laser desorption/ionization (MALDI) technique is one of the most widely used method to analyze proteins in biological research fields. However, it is difficult to analyze insoluble proteins which have important roles in researches on disease mechanisms or in developments of drugs by using ultraviolet (UV) lasers which have commonly been used for MALDI. Recently, a significant improvement in MALDI process of insoluble proteins using a combination of a UV nitrogen laser and a tunable mid-infrared (MIR) free electron laser (FEL) was reported. Since the FEL is a very large and expensive equipment, we have developed a tabletop laser source which can generate both UV and tunable MIR lasers. A tunable MIR laser (5.5-10 &mgr;m) was obtained by difference frequency generation (DFG) between a Nd:YAG and a tunable Cr:forsterite lasers using two AgGaS2 crystals. The MIR laser can generate pulses with an energy of up to 1.4 mJ at a repetition rate of 10 Hz. A UV laser was obtained by third harmonic generation of a Nd:YAG laser splitted from that used for DFG. A time interval between the UV and the MIR laser pulses can be adjusted with a variable optical delay.

A novel photodynamic therapy for drug-resistant prostate cancer cells using porphyrus envelope as a novel photosensitizer

BACKGROUND In the clinic, it is often very difficult to treat drug-resistant advanced prostate cancer by conventional treatments. Photodynamic therapy (PDT) is a minimally invasive treatment that takes advantage of photochemical reactions between a photosensitizer and light. On the basis of several of its key characteristics, PDT is considered to be a promising novel method for treating drug-resistant prostate cancer. OBJECTIVES For effective treatment of drug-resistant prostate cancer, we developed a novel agent termed porphyrus envelope, which was produced from PpIX lipid and hemagglutinating virus of Japan envelope (HVJ-E). MATERIALS AND METHODS We inserted PpIX lipid into HVJ-E by centrifugation, and used the resultant porphyrus envelope in PDT of two drug-resistant prostate cancer cell lines, PC-3 and PC-3-DR. RESULTS Porphyrus envelope enhanced uptake of PpIX, and cytotoxicity of PDT, relative to free PpIX lipid or PpIX induced by 5-ALA. CONCLUSION PDT using porphyrus envelope has potential as a method for treating drug-resistant prostate cancer.

Localization-dependent cell-killing effects of protoporphyrin (PPIX)-lipid micelles and liposomes in photodynamic therapy.

The protoporphyron (PPIX)-lipid (PL-C17) liposomes were successfully prepared from the corresponding micelles by post-inserted method. Both the PL-C17 micelles and liposomes were distributed in plasma membrane and cytoplasm after incubation of the cells with PL-C17 liposomes for 1h. They translocated from plasma membrane into a certain organelle in the cells after incubation in the photosensitizer-free medium. Higher photo-cytotoxicity was observed in the PL-C17 micelles and liposomes localized in plasma membrane in comparison with those localized in the cytoplasm under light irradiation. The LDH assay revealed that cytopathic damages of the plasma membrane were observed in the PL-C17 micelles and liposomes highly localized in plasma membrane. The fluorescent intensity of the calcein-encapsulating DOPC liposomes post-inserted with PL-C17 increased after light irradiation, suggesting that the membrane disruption is possibly caused by oxidation of membrane lipids with ROS generated from photosensitizers and affects the photo-cytotoxicity in PDT.

Development of a stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer

A novel stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch as well as microdot patterns with a 5 μm dot diameter and a 10 μm pitch made with rhodamine B were clearly observed. The estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Separating stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of the ions was successfully demonstrated by a microdot pattern made with two different dyes, crystal violet and methylene blue. Stigmatic ion images of a microdot pattern made with crystal violet were observed after circulation in MULTUM-IMG, and the pattern of the ion image was maintained after ten cycles in MULTUM-IMG. A section of a mouse brain stained with crystal violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue agreed well with the optical microphotograph of the hippocampus for the same section.

Ex vivo efficacy evaluation of laser vaporization for treatment of benign prostatic hyperplasia using a 300-W high-power laser diode with a wavelength of 980 nm

BACKGROUND AND OBJECTIVE Laser vaporization of the prostate is considered to be a promising treatment for benign prostatic hyperplasia (BPH), and efficiency of vaporization and hemostasis are both important parameters for such treatment. In this study, we used a high-power laser diode with a wavelength of 980 nm to obtain high vaporization efficiency with good hemostasis. The objective of this study is to evaluate the efficacy of laser vaporization for treatment of BPH in ex vivo experiments using a 300-W high-power laser diode with a wavelength of 980 nm quantitatively. MATERIALS AND METHODS An ex vivo experimental setup simulating clinical treatment situation was constructed. Bovine prostate tissue was used as a sample. The power setting was 100, 150, 200, 250, or 300 W, and the irradiation time was 0.5, 1, or 2 s. After laser irradiation, vaporized and coagulated depths were measured. RESULTS The vaporized depth increased with the laser power and irradiation time, and the results confirmed that the high-power laser diode could efficiently vaporize the prostate tissue. Coagulated depth increased as the laser power became higher. CONCLUSIONS Laser vaporization of prostate tissue using a high-power laser diode with a wavelength of 980 nm represents a promising treatment for BPH; this method exhibits high vaporization efficiency and good hemostasis. However, operators must be aware of the risk of postoperative perforation of the prostatic capsule caused by coagulation of deep regions that cannot be visualized by endoscopic observation.

Endoscopic submucosal dissection using a carbon dioxide laser with submucosally injected laser absorber solution (porcine model)

BackgroundRecently, endoscopic submucosal dissection (ESD) has been performed to treat early gastric cancer. The en bloc resection rate of ESD has been reported to be higher than that of conventional endoscopic mucosal resection (EMR), and ESD can resect larger lesions than EMR. However, ESD displays a higher complication rate than conventional EMR. Therefore, the development of devices that would increase the safety of ESD is desired. Lasers have been extensively studied as a possible alternative to electrosurgical tools. However, laser by itself easily resulted in perforation upon irradiation of the gastrointestinal tract. We hypothesized that performing ESD using a CO2 laser with a submucosal laser absorber could be a safe and simple treatment for early gastric cancer. To provide proof of concept regarding the feasibility of ESD using a CO2 laser with submucosally injected laser absorber solution, an experimental study in ex vivo and in vivo porcine models was performed.MethodsFive endoscopic experimental procedures using a carbon dioxide (CO2) laser were performed in a resected porcine stomach. In addition, three endoscopic experimental procedures using a CO2 laser were performed in living pigs.ResultsIn the ex vivo study, en bloc resections were all achieved without perforation and muscular damage. In addition, histological evaluations could be performed in all of the resected specimens. In the in vivo study, en bloc resections were achieved without perforation and muscular damage, and uncontrollable hemorrhage did not occur during the procedures.ConclusionsEndoscopic submucosal dissection using a CO2 laser with a submucosal laser absorber is a feasible and safe method for the treatment of early gastric cancer.

LESS-INVASIVE LASER THERAPY AND DIAGNOSIS USING A TABLETOP MID-INFRARED TUNABLE LASER

Since numerous characteristic absorption lines caused by molecular vibration exist in the mid-infrared (MIR) wavelength region, selective excitation or selective dissociation of molecules is possible by tuning the laser wavelength to the characteristic absorption lines of target molecules. By applying this feature to the medical fields, less-invasive treatment and non-destructive diagnosis with absorption spectroscopy are possible using tunable MIR lasers. A high-energy nanosecond pulsed MIR tunable laser was obtained with difference-frequency generation (DFG) between a Nd:YAG and a tunable Cr:forsterite lasers. The MIR-DFG laser was tunable in a wavelength range of 5.5–10 μm and generated laser pulses with energy of up to 1.4 mJ, a pulse width of 5 ns, and a pulse repetition rate of 10 Hz. Selective removal of atherosclerotic lesion was successfully demonstrated with the MIR-DFG laser tuned at a wavelength of 5.75 μm, which corresponds to the characteristic absorption of the ester bond in cholesterol esters in the atherosclerotic lesions. We have developed a non-destructive diagnostic probe with an attenuated total reflection (ATR) prism and two hollow optical fibers. An absorption spectrum of cholesterol was measured with the ATR probe by scanning the wavelength of the MIR-DFG laser, and the spectrum was in good agreement with that measured with a commercial Fourier transform infrared spectrometer.

Mid-infrared tunable optical parametric oscillator pumped by a Q-switched Tm,Ho:YAG ceramic laser

Since resonant absorption of light caused by a variety of different molecular bond occurs in the mid-infrared (MIR) wavelength region, many applications using tunable MIR lasers have been reported. However, the applicable fields of the MIR tunable lasers have been restricted by their large size and high cost equipments. Therefore, we are developing a compact tunable MIR laser using an optical parametric oscillator (OPO). To obtain a long term stability and a high conversion efficiency, a diode-pumped and Q-switched Tm,Ho:YAG ceramic laser with a wavelength of 2.1 μm was adopted for the pump source. A maximum output energy of 40 mJ was obtained with the Tm,Ho:YAG ceramic laser at a pulse width of 150 ns and a repetition rate of 10 Hz. An experiment was performed using a singly-resonant OPO with a ZnGeP2 crystal pumped by another OPO with a wavelength of 2.1 μm. A threshold pump fluence of 0.2 J/cm2 and a slope efficiency of 60% were obtained at a signal and idler wavelengths of 3.3 and 5.6 μm, respectively. Using these results and a theoretical model calculation, the maximum output energy of MIR-OPO pumped with the Tm,Ho:YAG ceramic laser was estimated to be about 20 mJ.

Comparison of mass spectra of peptides in different matrices using matrix‐assisted laser desorption/ionization and a multi‐turn time‐of‐flight mass spectrometer, MULTUM‐IMG

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.

Selective removal of cholesterol ester in atherosclerotic plaque using nanosecond pulsed laser at 5.75 μm

Laser angioplasty, for example XeCl excimer laser angioplasty, has gained more attention in addition to conventional methods of surgical and interventional treatment of atherosclerotic diseases such as bypass operation and balloon dilatation. Low degrees of thermal damage after ablation of atherosclerotic lesions have been achieved by XeCl excimer laser at 308 nm. However, in most cases, laser ablation is not selective and normal arterial wall is also damaged. To avoid complications such as severe dissections or perforation of the arterial wall in an angioplasty, a laser light source with high ablation efficiency but low arterial wall injury is desirable. At atherosclerotic lesions, cholesterol accumulates on the tunica intima by establishing an ester bond with fatty acids such as oleic acid, and thus cholesterol ester is the main component of atherosclerotic plaques. Mid-infrared pulsed laser at 5.75 μm is selectively well absorbed in C=O stretching vibration mode of ester bonds. The purpose of this study is to determine the effectiveness of nanosecond pulsed laser at 5.75 μm irradiation of cholesterol ester in atherosclerotic plaques. In this study, we used a mid-infrared tunable solid-state laser which is operated by difference frequency generation method, with a wavelength of 5.75 μm, a pulse width of 5 nsec and a pulse duration of 10 Hz. It was confirmed that non-invasive interaction to normal thoracic aortas could be induce by the parameters, the wavelength of 5.75 μm, the average power densities of 35 W/cm2 and the irradiation time under 10 sec. This study shows that nanosecond pulsed laser irradiations at 5.75 μm provide an alternative laser light source as an effectively cutting, less traumatic tool for removal of atherosclerotic plaque.