It is demonstrated that a highly sensitive synchroscan streak camera technique for picosecond time-resolved fluorescence spectroscopy is very useful for the investigation of fast dynamic behaviours of biomolecular systems. The technique is applied for studies of 1)incorporation and excretion processes of hematoporphyrin derivative (HpD) into and from malignant tumor cells in vitro and 2)relaxation processes from the electronic excited-state of nucleic acidic bases (9-methyladenine: 9Me-A), as follows.
1)HpD study: The results show that the aggregate component of HpD which has a fast fluorescence lifetime of 100 ps and a red-shifted band of -660 nm in comparison with a monomer band accumulates more and more in the cells with the increase of the incubation time. The aggregate excretes from the cells in the very different manner from the monomer. The mechanisms are discussed.
2)9Me-A study: The result shows diffusion-free intermolecular excimer formation in liquid aqueous solution of 9Me-A. The excimer fluorescence (330 ps lifetime) appars at 380 nm, at longer wavelength than the monomer fluorescence (5 ps lifetime). The excimer is formed via a weakly coupled stacking dimer composed of ground-state monomers.

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Recently, laser has been widely utilized in the field of medicine and surgery. However, laser applications are very rare in cardiovascular surgery throughout the world. In this paper, 1) myocardial revascularization by laser to save severely ill patient for whom CABG can not be carried out,because of diffuse stenosis of the coronary artery, 2) vascular anastomosis including CABG by laser, 3)laser angioplasty with vascular endoscope in the fields of the coronary-and peripheral arteries are described from standpoints of experimental and clinical studies in detail.

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Using spectroscopic, tissue culture and biochemical analysis, HPLC, and electromicroscopic techniques and so on, basic investigations of photodynamic therapy (PDT) using hematoporpyrin derivative (HpD) have been performed. HpD may make an aggregated form after cellular incorporation which bound to mainly mitochondria and cell membrane. The aggregated HpD exerted strong photodynamic reactions and produced the damage of protein synthesis followed by DNA synthesis disturbance. The PDT with argon-dye laser (ADL), 630nm, was performed on superficial tumors of the penis and bladder. Less than 1.5cm tumors in size achieved to CR using the irradiation of output 200-300mW/cm² via a 400um quartz fiber for more than 10min. Integral whole bladder wall PDT was used for CIS of the bladder with the light dose of 10-20J/cm² and CR was obtained in most cases. A gold vapor laser (GVL) was studied in terms of tissue penetration and tumor destruction. The results obtained supported the superiority of GVL over ADL in enhancing PDT effect and in introducing 43°C hyperthermia.

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Laser Raman spectroscopy, which is a powerful nondestructive structural probe for biological molecules and objects, has been recently applied to medical problems. Rapid progress in multichannel detector gives considerable promise to laser Raman spectroscopy as a diagnostic or analitical tool in medicine.
We present here two examples of medical application of multichannel laser Raman spectroscopy. One is cataract diagnosis by Raman spectroscopy, and the other is nondestructive analysis of gallstones by the same technique.
With regard to the cataract diagnosis, we recently succeeded in obtaining Raman spectrum of an excised lens with only 1 mW and 1 second of laser irradiation (488.0nm). This success strongly suggests that one can detect the Raman signal directly from a human eye lens without any damage to the eye.
Concerning the second application we so far examined about 10 gallstones by the use of the Raman microprobe, and it turned out that the combined Raman microprobe-FT-IR microscope method is very powerful for studying the microstructure of various gallstones.

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Since 1978, our group has examined porphyrin derivatives based on hemoglobin and chlorophyll and their metal complexes (200 in all) through the cooperation of Sakata et all, of Toyohakka Kogyo. We have previously found and reported that side chain structure and tumor tissue accumulation are related.
Based on the datas obtained, our study now been forcused on making the following substances available.
a) Bacterio-pheophorbide (λ=755nm) which has an absorption band shifted to longer wavelength than that of HPD or DHE, so that deeper tissue penetration of photo-radiation can be achieved.
b) Highly tumor-seeking radiopharmaceuticals for scintigraphic studies that have advantages over Ga-67 citrate which is used clinically in the world.
c) New cancer-targetting anti-tumor chemicals which have accumulate and remain in cancer tissue.
In this congress, I would like to report our results of investigation.

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Since 1980, we have been studying the effect of light on experimental and clinical wound healing. Particulary its effect according to the difference of light has been studied. In October 1982, the first case underwent the light irradiation therapy. A man had a chronic ulcer of the left 1st toe due to Buerger's disease for 5 months. On the 14th day after 6 treatmemt of irradiation his ulcer completely healed. During this treatment, the patient noticed that the pain completely disappeared after 2 treatments. We believed light irradiation stimulated tissues injured and leaded some good clinical results.
Some investigations concerning mechanism for these effects; i. e. perifereal circulation, cell proliferation, histology of the granulation, chemistry and so on. From these investigations, periferal circulation was improved when the patients suffered from vascular disorders. Furthermore, irradiated G₀G₁ cells increase their electrophoretic mobility but irradiated G₂M cells decrease their mobility. Some results suggest light irradiation comtributes homeostasis of living cells, tissues, and bodies.And some investigation concerning the light soucees, i. e. wave length, energy density and polarization. As a result, linear poralization and right circular poralization improved wound healing, but incoherent light itself does not improve wound healing. We are strongly suggested the role of coherency is very important to do light irradiation on the living body.

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The ability of the turnable dye laser lithotriptor which was developed for the fragmentation of urinary calculi was examined and then the lithotriptor was used clinically.
The destructive power of the shock wave produced by this laser lithotriptor was approximately 1/100 weaker than the shock wave produced by an electro-hydraulic lithotriptor, but this laser system fragmented almost all stones except cystine stones.
Regarding the effects of laser energy on biological tissue, it caused only epithelial ablation in organs such as the ureter and bladder which are fibrous and hypovascular.
Based on the experimental results, cases of upper urinary calculi were treated transurethrally and percutanuously by this laser system and almost satisfactory results were obtained. Our experience suggests that this system should be used through a fine flexible ureteroscope in cases of the transurethral removal of stones.

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Hematoporphyrin derivative photodynamic therapy is evolving as a local treatment for neoplastic disease. Generally argon dye or gold vapor lasers have been used as light sources. We employed an excimer dye laser in anticipation of greater effects. The BALB/C mice receiving i. v. injections of HpD in dose of 5 mg/kg body weight were used in this study. At first, the photodynamic effects of an argon dye laser and excimer dye laser were compared at the same energy in terms of the morphological changes in transplanted tumor of m-KSA cell in the following study. Next, to determine the best conditions for irradiation by the excimer dye laser, tumor-bearing mice were treated with different total doses (10, 30, 50 J/sq cm), dose rates (1, 3, 6 mJ/sq cm), and frequencies (5, 15, 30hz) of light exposure. We obtained necrotic changes in the tumor at depths of up to only 4 mm on average using the argon dye laser (200 mw × 250 sec, 50 J/sq cm). On the contrary, tumoral necrosis at the depth of over 15mm was obtained with the excimer dye laser (6 mJ/puls 5hz 50J/sq cm). Our results showed that the effects of excimer dye laser are related to total power dose and rate dose but not to frequency.

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Cytological changes caused by hematoporphyrin derivative and Laser photoradiation were investigated both in vitro and in vivo.
In clinical materials the changes of lung cancer cells after laser photoradiation were examined and mouse kidney sarcoma cells were studied by phase contrast microscopy and electron microscopy. And immunocytochemical staining for keratin, CEA and CA19-9 with ABC method was performed.
Light-microscopically, the initial morphological changes, swelling of cytoplasm, eosinophilic staining of cytoplasm were occured after 10 mimutes laser photoradiation. An electron microscopy study showed that initial changes occured at the mitochondria. Immunocytochemically, Keratin staining showed marked changes.

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The past decade has seen a resurgence of interest in photodynamic therapy (PDT), whereby malignant lesions are illuminated by lamps or endoscopically delivered laser beams after administration of malignant tissue-specific photosensitizers. Interest has heightened due to the first report of 5-year disease-free survival of a case of lung cancer treated only by this modality. However controversy has remained as to the exact site of uptake of the photosensitizers and also the effect of PDP at the cellular level.
Movement of mitochondria was reported based on the movement of granules in organella in the 19th century and was demonstrated. in detail by phase microscopy in the year 1954. No report, however, has demonstrated any difference in the mitochondrial movement of normal and tumor cells.
In order to observe possible differences in metabolic activity between normal cells and tumor cells, the mitochondrial movements of normal cells and tumor cells to which those normal cells had been transformed were compared. Photosensitizers which have been reported to have a high affinity for mitochondria induce degeneration and destruction of mitochondria through the production of singlet oxygen. We examined the degree of damage to mitochondria, as indicated by the diminution of mitochondrial movement, and also the exact site of uptake of the photosensitizer, by allowing a photosensitizer to be taken up by tumor cells and applying light.
Our results suggest that there is a significantly greater amount of movement by mitochondria. in malignant cells than in normal cells, that different photosensitizers have different intracellular uptake sites and that this can affect the effects on cells containing such photosensitizers when light is applied.

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We have been developing a photodynamic cancer diagnosis and treatment system.This system consists of an excimer laser pumped dye laser which emits beams on wavelengths of 405 and 630 nm, and an image and spectroscopic analyzer of the endoscopic findings. In diagnosis, the 405 nm laser beam is applied to the tissue to detect the fluorescence of HpD. At the same time, the light of an Xe lamp is also applied to enable observation. When light intensities of the laser and the Xe lamp are strong, HpD deteriorates rapidly. The HpD fluorescence becomes weak and changes its spectral shape. In this case the analysis does not lead to a conclusive diagnosis. We investigated this phenomenon using two kinds of samples: HpD absorbed filter papers and tissues from an experimental cancer mouse which was injected with HpD beforehand. For the cancer tissue, the intensity of the fluorescence after 14 minutes became 1/4 of the initial value at 60,000 lux Xe light illumination.

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There has been increasing interest in the usage of porphyrins for tumor localization and therapy. Hematoporphyrin derivative is well-known tumor-specific photosensitizer. Hematoporphyrin derivative has been used clinically as a photosensitizer in photodynamic therapy (PDT). PDT is performed after hematoporphyrin derivative i. v. injection. Therefore, hematoporphyrin derivative may interact with serum components. We investigated the binding affinity of hematoporphyrin derivative to albumin, on of the serum components. Most hematoporphyrin derivative binds to albumin among all the serum protein in vitro. Therefore, alubumin binding hematoporphyrin derivative decreased according to the time after injection. This decreasing present were 19.6%, but other serum components were not change. In stead of albumin binding hematoporphyrin derivative, tumor binding hematoporphyrin derivative were increased to time after injection. These result suggest that albumin may be hematoporphyrin derivative carrier protein.

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Characteristic electron spin resonance (ESR) spectrum was detected after photodynamic therapy (PDT) which used porphyrin polymer (N-HpD) and low power of Argon laser. This ESR spectrum was as triplet signal between 3rd and 4th of Mn²⁺ signal. And it was considered that this signal orignated from R₂NO radical which was a product of N-HpD after PDT.
The strength of ESR spectrum was correlative with the volume of N-HpD that was absorbed into cells.
Basing on above results, we measured the ESR spectrum after PDT of normal gastric mucosa of rat and the subcutaneous transplanted tumor of mouse.
The triplet signal about gastric mucosa revealed the highest peak on 24 hours' radiation of Argon laser after N-HpD administration in abdominal cavity of rats. Next, the triplet signal about subcutaneous tumors of mice revealed the highest peak on 24 hours' radiation after N-HpD administration.
From above data, it is suggested that the measurement of ESR spectrum will make a deal about the mechanism and the suitable condition of PDT.

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This study was carried out to investigate the effect of photodynamic therapy (PDT) for hepatoma.
Cultured HC-4 tumor cell was used as an object of this study. 1) in vitro
PDT was performed for HC-4 tumor cell with HpD and an argon dye laser and the effect was evaluated by Cr release assay. After 12 and 24 hours, PDT showed cytocydal effect with an efficient difference. And the results have tendency to depend on the dose of HpD and irradiation of the argon dye laser. 2) in vivo
HC-4 cells were transplanted to bach of nude mouse and PDT was performed. In control, HpD only and irradiation of the argon dye laser only, the tumor showed normal growth. But in PDT, the tumor was necrotized and showed no growth.
As a result of this, potency of the PDT for hepatoma was supported.

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We investigated the anti-tumoral effect of intratumoral injection using nude mice with subcutaneously transplantated MKN-45 gastric cancer followed by exposure to red light in order to avoid phototoxic reaction after intravenous administration of hematoporphyrin derivatives (HpD). Histopathological findings revealed that necrosis was uniform in the group administered intraperitoneally but it was not so in the group administered intratumorally. We conclude that anti-tumoral effect of intraperitoneal injection was superior to that of intratumoral injection but still significant even in the intratumoral injection, based on the results of calculated the necrotic volume(%) by means of Kontron MOP-AM03 instrument in each group.

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Concentration of HpD polymer and monomer in tumor tissue and liver of rabbits was examined 24, 48 and 72h. after 5mg/kg intravenous administration. Concentration of HpD polymer was higher than that of the monomer.
Tissue damage in rats and dogs was examined after PDT with N₂ and Argon dye laser. Liver damage upon laser irradiation was deeper after injection of monomer in comparison with polymer.

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Hematoporphyrin derivatives (HpD) and photofrin II have been studied extensively for photochemical therapy. Recently newly prepared porphyrin oligomer (average molecular weight, 3000) were measured and compared with those of HpDs. As the fluorescence intensity of the porphyrin oligomer was very small in aqueous solution, the porphyrin oligomer may be coagulated. In micellar solution and in the tumor cell, however the fluorescence intensity increased sharply and was easily detected. Triplet life time of the porphyrin oligomer was longer than HpDs. The porphyrin oligomer seems to be suitable for photochemical therapy.

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To treat Liver cancer with PDT, it is essential to accumulate much more photosensitizer in the to than in normal Liver tissue. We succeeded in accumulating a sensitizer selectively in liver tumor by intraarterial administration of pheophorbide a dissolved in Lipiodol. With this technique and activation by 670 nm Light of Nd-YAG dye laser, we produced severe PDT necrosis in Liver tumor of the rat without damage of surrounding Liver tissue.

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Pheophorbide-a, a group of photosensitizer, and Nd: YAG laser equipped Q-switch were tested for photodynamic activity against transplantable FANFT-induced urotherial tumors growing in female C3H/He mice. Histologically larger areas of tumor cell necrosis on the slide spiecimen were observed 7 days after completion of therapy when compaired with control. This study demonstrates the potential of the new photodynamic cancer therapy.

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The authors developed a new photodynamic therapy using phoephorbide-A and Nd: YAG Laser (acoustic Q switch). In vitro studies were done using rat T 9 glioma and human glioma cells (T 98 G). The survival rates after PDT at 37.0°C were 68.8% (T 9, 20 min) and 0.4% (T 98 G, 5 min). Histological examination of irradiated rat T 9 tumor showed wide vascular damage just after PDT. This portion eventually became necrotic in one week. Acuostic Q switched Nd: YAG Laser penetrated deeper into the cat brain tissue and showed flat light distribution.

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We measured the decay time of the fluorescence in methylene blue solution for elucidating the generation of the singlet oxygen. And using the duodenal mucosa of the rat, we studied the damage effect of the singlet oxygen which was produced from the methylene blue.

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The study of photodynamic therapy (PDT) has been given increasing attention, especially the use of hematoporphyrin derivative (HpD). Recently, Mono-L-serinyl chlorin e6 (NIPPON PETROCHEMICAL CO., LTD.) has been developed as a new photosensitizer for PDT. This material is considered to be useful for PDT due to its greater absorption band in the wavelengths longer than these of HpD. We studied the distribution Mono-L-serinyl chlorin e6 (20mg/kg weight) was administered intravenously via the tail of tumor-bearing mice. The relative intensity of fruolescence from a tumor and other tissues were accumulated most at 3hrs after in jection. And at 24hrs after injection, the intensity from other tissues decreased by about 20% of the intensity from the tumor at 3hrs after injection. Additionaly, mice were illuminated 24hrs after injection, the mice received Mon-L-serinyl chlorin e6 showed hardly skin damage versus mice received PHOTOFRIN II of skin damage.
These results suggest that Mono-L-serinyl chlorin e6 may be used in photodynamic therapy at an early stage.

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Tissue concentrations of AlCl-tetrasulphonated phthalocyanine(AlPCS) were determined at various intervals following intraperitoneal injection using KK-47 bladder cancer transplanted into nude mice. Concentrations of AlPCS, extracted from the tumor and normal tissues, the liver, spleen, kidney, lung, muscle and skin were calculated on the basis of their fluorescence intensities at 675nm when excited at 610nm. The following results were obtained. The values in the tumor were 12.6pg/g, 7.6pg/g 6.5pg/g, 6.1p.g/g and 4.4pg/g at 3, 24, 48, 96 and 168hr respectively. The concentrations of ALPCS were not greater in the tumor than those in the liver at all times. The values in the tumors at 24, 48, 96 and 168hr were slightly decreaced in comparison with that at 3hr. But the decrease of the values in the liver ocurred with increasing time after injection at a relatively higher rate in comparison with those in the tumor. A tendencies of retaining in the tumor was noted as comparied with the values in the kidney, spleen, lung, muscle and skin. AlPCS may be a promising new photosensitizer for photodynamic therapy.

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Recently some investigations have focussed on phthalocyanines as promising photosensitizers in photodynamic therapy. In the present study, cellular uptake and photodynamic cell killing ability of AlCl tetrasulphonated phthalocyanine (PC) were compared with those of hematoporphyrin derivative (HpD) and its polymerized form (HpD polymer; MW: 12000). KK-47 bladder cancer cells were incubated with each of the dyes dissolved in growth medium for different times. The dye uptakes were qualified by measuring the fluorescence intensities of the dye-incorporating cells. The results indicated that the uptake of HpD, polymer was the highest. Survival of the cells incubated with each dye for 1 hr, followed by argon-dye laser irradiation(0.1mW/sqcm) was evaluated by using a colony forming assay. The data showed that 1) photodynamic cell killing ability of the dyes increased with increasing laser irradiation time and 2) PC was the most effective photosensitizer among three dyes.

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Thermocouples are practically useful and usually used to measure the temperature of lased tissues. The thermocouple, however, can not always indicate the actual temperature of lased tissues, particurally in the case of penetrable laser light, because the sensor absorbs directly the laser light penetrated into a tissue. Therefore, it is necessary to make a correction for the indicated valuein this measurement. In this experiment, Nd:YAG laser irradiated vertically on the surface of the liver, and the temperature on the same point of lased surface of the liver were measured simultaneously by the and infrared thermometer.The temperature measured by the were compared to the actual value of temperature measured by infrared thermometer.It's impossible to derive the information for the correction of the indicated values by thermocouple. Therefore, the peak temperature in tissue was estimated by the characteristics of the temperature measured with a thermocouple after laser irradiation.

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Previous studies have shown that local hyperthermia using Nd: YAG laser (laserthermia) is a safer and more reliable method for treating depressed early gastric cancer than both contact and non-contact laser irradiation. This study compares laserthermia with conventional hyperthermia transmitted by a metal probe.
1) In vivo studies on carcinoma-bearing mice suggest that laserthermia is more effective than conventional hyperthermia.
2) In vitro studies on cultivated rat liver cells revealed a cytocidic effect of laser light even at temperatures below 38°C
These findings indicate that laserthermia has a synergistic effect of laser light and hyperthermia.

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The usefulness of laser hyperthermia using Nd: YAG Laser was studied experimentally and clinically to treat deep seated brain tumor. Histological study was done to compare the changes following conventional interstitial laser irradiation and laser hyperthermia. Moderate to marked edema was seen 12-48 hrs after conventional laser irradiation. No obvious edema was seen following laser hyperthermia. Temperature profile was studied. Trypan blue injection study was also done and it revealed destruction of BBB after laser hyperthermia. Four patients with brain tumors were treated with laser hyperthermia using Nd: YAG Laser. Follow up CT scan revealed disappearance of tumor in three patients and decrease in volume in one patient. Long term neurological follow up reealed no deterioration. Laser hyperthermia using Nd: YAG Laser is easy and safe to use and it is beneficial to treat deep seated brain tumor.

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To use laser hyperthermia (laserthermia) as a form of cancer therapy in the field of oral surgery, we have conducted several basic studies. Using normal tongue of rabbits, we recently investigated the differences between hyperthermia by laser light and by other means. More specifically, we experimentally compared the effects of the artifical sapphire probe commonly used for laserthermia and a new titanium probe of the same form which tansmits heat but not laser light.
Results:
1) Ultrasonography showed that laserthermia penetrated deeper into the tissue than conventional hyperthermia.
2) Histro-pathological examination revealed that laserthermia has greater invasiveness than conventional hyperthermia.
3) Biochemical examination revealed that laserthermia has greater change than conventional hyperthermia on lipid peroxide levels.

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Photodynamic therapy (PDT) for malignant tumors is performed increasingly throughout the world.
However, detailed information concerning the optimum power density, light dose and thermal effects are unknown. Therefore the authors investigated tumor response and tumor temperature during PDT using transplanted B-16 melanoma in mice. A thermister probe was fixed at a depth of 2mm from the tumor surface. At the site, the tumor temperature rose 8°C at 150mW/cm², 13°C at 300mW/cm², 21°C at 450mW/cm²2, 28°C at 600mW/cm² above the base line value. A tumor response study showed increased effects with increased power density. Mice receiving a high power density (450mW/cm², 600mW/cm²) without photosensitizer indicated same result. The presence of thermal effects was suggested in the high power density group of PDT. The increased light dose showed improvement of results over the low power density group (150mW/cm², 300mW/cm²).

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In an attempt to obtain basic data for endoscopical photodynamic therapy with hyperthermia, antitumor effect of this combination therapy have been studied using human hepatocellular carcinoma cells as well as its transplanted tumors.
In vitro the results indicated that the cytocidal effect of PDT with hyperthermia is five times as more as that of hyperthermia alone, two times as more as that of PDT alone.
In vivo antitumor effect was assessed by relative mean tumor volumes (T/C ratios) after 1 week of the treatment. T/C ratios were control group 100%, PDT group 41.2%, hyperthermia group 34.7% combination group 14.0%, respectively.
These results suggest that PDT with hyperthermia has more antitumor effect than PDT or hyperthermia alone.

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In 1984 we developed laser hyperthermia (laserthermia), a system for transmitting low power Nd: YAG laser energy (2.0W) through an interstitial probe for local hyperthermia (43-44°C). Evaluating the effect of laserthermia, we histo-pathologically investigated the resected stomachs after preoperative pilot treatments on 20 patients with early gastric carcinoma. In 17 of 20 cases (85%) the bottom of the laserthermia induced ulcer showed no residual carcinoma, indicating the efficacy of treatment.
In 1986 photodynamic therapy (PDT) using a Q-switched Nd: YAG pulse laser and pheophorbide a (Ph-a) as a photosensitizer was developed. In vitro studies conducted after laser irradiation with and without Ph-a at 36.5°C±0.5°C for 15 min. showed survival rates of human colon adenocarcinoma of 27.2% and 65.3% respectively (control 89.7%). In vivo studies revealed that tumor bearing mice treated with Ph-a intraperitoneal injection and laser irradiation maintained a noticably elevated serum lipid peroxide level for at least one week, and that an 84% reduction in tumor size occured one wk. after irradiation. The results suggested that photo-chemical reaction occurred in cultured: carcinoma cells and tumor bearing mice, and lipid peroxide played an important role in PDT using an Nd: YAG pulse laser and ph-a.

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We tried dye laser photocoagulation with 590nm and 630nm beams directed to the lesions with subretinal hemorrhage in the monkey eyes. When photocoagulation was made by 590nm beam no coagulation was produced in the pigment epithiium and choroid,on the other hand, 630nm beam produced a coagulation effect of the level of pigment epithlium and choroid.
Furthermore,we compared effects of 590nm and 630nm beams for branch retinal vein occlusion. As a result, 630nm beam was more effective than 590nm beam for the photocoagulation of retina and choroid in the case of subretinal hemorrhage.

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We made the following experiments to compare and discuss the effects of laser iridotomy with various wavelength.
1) Laser photocoagulations were made on brown polyester sheets in piles with various wavelength of argon, krypton and dye lasers, and the transmission of the laser beams were evaluated.
2) Laser iridotomy was performed on rabbit's iris with various wavelength. Furthermore, laser iridotomy with dye laser (orange, 590nm) was performed on 9 eyes of 9 patients clinically.
Consequently, we have successfully performed laser iridotomy with dye laser (590nm) by fewer numbers of photocoagulations than argon laser.

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A new method has been developed to evaluate the retinal blood flow using laser speckle phenomena. A He-Ne laser illuminates the ocular fundus by means of a standard fundus camera. The laser light scattered from the fundus generates the speckling phenomenon in the detecting plane which fluctuates due to red blood cells moving in the retinal vessels. This speckle intensity fluctuation is detected by a photomultiplier and analyzed to obtain the power spectrum. The retinal blood-flow rate is estimated from the distribution of the power spectrum.
In the present method, the size of the probe region at the fundus is defined to be large (3mm φ) in comparison with the diameter of the blood vessels. Then, the analysis of output signals gives the overall estimation of various blood flows existing in the probe region, rather than the absolute determination of both the blood-flow velocity and its direction in a single vessel.
The method is used to evaluate the retinal and choroid blood flows of a rabbit. The experimental results show the usefulness of the method for evaluation of the retinal blood flow.

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The effects of Nd-YAG laser irradiation on carcinogenesis were examined in tongues of Syrian Hamsters treated with 9, 10-dimethyl -1, 2-benzanthracene (DMBA). 0.5% DMBA-acetone solution was applied to the lateral border of the middle third of the tongues three times per week for 12 weeks. Following the procedure, in the experimental group, laser was irradiated on the same part of the tongues, meanwhile, in the control group, incision was made by biopsy forceps on the same regions. The tongues were examined histopathologically at 2, 4, and 8 weeks after operation. The changes of epithelial dysplasia shown in laser wounds seemed to be more severe than that shown in biopsy forceps wounds.

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To catch the 3-D distribution and the affected area of laser light on tissues are the most crucial in clinical application of lasers. The authors have made an effort to observe simultaneously both the light distribution and the affected area of unvisible near-infrared lasers on tissues as visible images using CCD TV camera for the light distribution and a high speed thermograph of real time response. This paper reports on the outline of this technique showing some data examples.

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Study of spontaneous photon emission without external or artificial stimulation in biological tissues and cells in vivo is impeded by the extremely low intensity of such biophoton emission in many subjects and systems of interest. The aim of our research project is the research and development of improved means of detecting these biophoton phenomena and the study of their characteristics. We describe here recent developments in highly sensitive photon counting technique and measurement of ultraweak photon emission. As an illustration, we discuss two areas of current research: 1) Studies of the effects of various solutions of germination promoter on ultraweak photon emission in soybean have been made. Among inhibitors examined, KCN and EtOH were found to enhance photon emission intensity. 2) The effect of environment (oxygen and nitrogen) and scavenger solutions on ultraweak photon emission intensity in soaked soybean was examined and found to be oxygen-dependent. Thus it is thought that the active oxygen species were participated in the ultraweak photon emission of soybeans. The mechanisms underlying these phenomena and the results of further studies will be discussed.

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Ultraweak photon emission phenomena from various living organisms, tissues and cells are recently being recognized and are attracting a great deal of interest. We are currently investigating a highly sensitive ultraweak photon imaging measurement and analysis system based on PIAS and VIMS (Hamamatsu Photonics) for this purpose. We have successfully observed for the first time ultraweak photon counting images of soybean seeds and the root tissues after 1 to 5 days of germination. All images were obtained in complete darkness at room temperature. It is believed that the ultraweak photon emission patterns thus observed is not due to external or artificial stimulation (e. g. fluorescence) but, rather, to intrinsic processes and functions closely related to cell division and growth.

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Macrophages are known to release active oxygen species during phagocytosis or in response to triggering with opsonized zymosan or phorbol myristate acetate. Chemiluminescence intensity from such active species is so low that it has been detected previously by utilizing luminol or lucigenin as a photoamplifier. Using a highly sensitive photon counting system that was developed recently by our group, we detected very weak chemiluminescence from mouse peritoneal macrophages without use of luminol or lucigenin. Protease peptone or OK-432 (Chugai Pharmaceutical Co., Tokyo) was administered intraperitoneally to mice (C3H/He, male, 6 weeks of age) and 72 hours later the induced peritoneal exudate macrophages were collected. Cells were then cultured with or without interferon-gamma (recently reported to be a“macrophage activating factor”) for 24 hours followed by triggering with phorbol myristate acetate. Chemiluminescence intensity of these cells was easily detected even without culturing with interferon-gamma. Chemiluminescence of interferon-gamma cultured cells was markedly augmented when they were induced by OK-432 and decreased to background dark count levels when catalase was added prior to analysis. These results seem to suggest that OK-432 augments macrophage functions.

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Pulse CO₂ laser was used to investigate the cutting mechanism of biotissue by KrF excimer laser. At macroscopic view, pulse CO₂ laser with low repetition rate was experimentally clarified to have same cutting ability to tissue without carbonization as KrF excimer laser. The high peak power laser with short pulse duration is supposed to have a great influence on the unnatural tissue layer near the irradiation surface and its cutting mechanism. The laser absorption by water involving in tissue will be important factor in analyzing the laser cutting mechanism.

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Laser has three main abilities, cutting, coagulation and vaporization. Among them, the most useful effect is cutting which can be also found in other medical equipments. Cutting technique is the important method in modern surgery and is widely used recently. But, such useful laser systems as Nd: YAG laser and CO₂ laser have unfavorable side effects. It is inevitable that these lasers produce remarkable carbonization owing to its strong thermal effect. In this paper we discuss the mechanism of cutting of excimer laser.

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The mechanism of biological tissue cutting was investigated using excimer laser. No or a minimal thermal effect can be observed from the post operative tissues. Many hypotheses have been published about the mechanism of tissue cutting; thermal effect, photon effect, photochemical effect, plasma effect and others. After thermographical analysis with wood and white of an egg, we have been studying this problem using rabbit's and rat's muscle and bone. Postoperative tissues were observed with light and electro microscope. Under the optimum condition, the carbonated layer couldn't be found. With light microscope and SEM, tissue fragments were ablated. With TEM, selective organelle destruction (especially mitochondoria and glycogen granule) were caused. The results show that the first step of tissue cutting mechanism using excimer laser is not due to thermal effect but due to selective organelle destruction, although the whole mechanism contains many phases.

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The mechanism of biological tissue cutting was investigated using excimer laser. No or a minimal thermal effect can be observed from the post operative tissues. Many hypotheses have been published about the mechanism of tissue cutting; thermal effect, photon effect, photochemical effect, plasma effect and others. After thermographical analysis with wood and white of an egg, we have been studying this problem using rabbit's and rat's muscle and bone. Postoperative tissues were observed with light and electro microscope. Under the optimum condition, the carbonated layer couldn't be found. With light microscope and SEM, tissue fragments were ablated. With TEM, selective organelle destruction (especially mitochondoria and glycogen granule) were caused. The results show that the first step of tissue cutting mechanism using excimer laser is not due to thermal effect but due to selective organelle destruction, although the whole mechanism contains many phases.

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To examine the chronic effects of the laser exposure on the brain, CO₂, Nd: YAG, or the combination of both lasers was exposed for one second and specific gravity method was used to measure water contents of material of the lesion 48 hours after the exposure. Nd: YAG laser exposure showed that water contents in the white matter were more extensive than those of the other modes of laser exposure. CO₂ laser exposure showed that water contents in the gray matter were least extensive of the three modes of laser exposure. Effects on the white matter by CO2/YAG coaxial laser exposure were similar to those by CO2 laser exposure.

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Optical measurement of cortical activity in the brain using laser beam has the following advantages compared to white light: (1) laser beam is easy to be focused on the cell stained with voltage-sensitive dye (the beam waist is nearly equal to the wavelength): (2) the light is monochromatic, then, the overlap between the excitation and the emission from the dye stained in the cell is negligible: (3) the optical measurement system of the cortical activity is a small size and a simple design. We propose the system of the laser image mapping applied to the intact brain with the voltage-sensitive dye. An optical fiber with a short length can be used for transmitting the laser light. The beam spot emerged out of the fiber can be controlled by using SELFOC micro-lens. The optical image mapping of cortical activity from a few cells or aggregated cells is produced from the small or large laser beam spot size.

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Recently there are many studies on bio-stimulation effect by low-energy laser light irradiation using several types of animals. However, its characteristic mechanism are as well as not clear yet so that it is important to investigate this effect at biological cell levels. We paid our attention to the change of electric charge on cell surface, which is monitored by measuring cell electrophoretic mobility (E. P. M.), as a first trial of estimating quantitatively bio-stimulation effect. We report here for the first time measured variations of E. P. M. for two kinds of mouse T lymphocytes (CTLL-2 and T cell) after low-energy Ar laser light irradiation of 514.5nm wavelength. It was found that low-energy laser light irradiation causes some influence on the cell electrophoretic mobility of both lymphocytes, although their variations are different.

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