Effect of photodynamic therapy with 5-aminolevulinic acid and EDTA-2Na against mixed infection of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The increasing abundance of drug-resistant bacteria is a global threat. Photodynamic therapy is an entirely new, non-invasive method for treating infections caused by antibiotic-resistant strains. We previously described the bactericidal effect of photodynamic therapy on infections caused by a single type of bacterium. We showed that gram-positive and gram-negative bacteria could be killed with 5-aminolevulic acid and 410 nm light, respectively. However, clinically, mixed infections are common and difficult to treat. OBJECTIVE: We investigated the bactericidal effects of photodynamic therapy on mixed infections of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: We compared bacterial growth with and without photodynamic therapy in vitro. Then, in vivo, we studied mixed infections in a mouse skin ulcer model. We evaluated the rates of ulcer area reduction and transitions to healing in treated and untreated mice. In addition, a comparison was made between PDT and existing topical drugs. RESULTS: We found that photodynamic therapy markedly reduced the growth of both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, in culture, and it reduced the skin ulcer areas in mice. PDT was also more effective than existing topical medicines. CONCLUSION: This study showed that photodynamic therapy had antibacterial effects against a mixed infection of gram-positive and gram-negative bacteria, and it promoted skin ulcer healing. These results suggested that photodynamic therapy could be effective in both single- and mixed-bacterial infections.

Deep learning based depth map estimation of protoporphyrin IX in turbid media using dual wavelength excitation fluorescence.

This study presents a depth map estimation of fluorescent objects in turbid media, such as biological tissue based on fluorescence observation by two-wavelength excitation and deep learning-based processing. A U-Net-based convolutional neural network is adapted for fluorophore depth maps from the ratiometric information of the two-wavelength excitation fluorescence. The proposed method offers depth map estimation from wide-field fluorescence images with rapid processing. The feasibility of the proposed method was demonstrated experimentally by estimating the depth map of protoporphyrin IX, a recognized cancer biomarker, at different depths within an optical phantom.

Transient simulation of laser ablation based on Monte Carlo light transport with dynamic optical properties model.

Laser ablation is a minimally invasive therapeutic technique to denature tumors through coagulation and/or vaporization. Computational simulations of laser ablation can evaluate treatment outcomes quantitatively and provide numerical indices to determine treatment conditions, thus accelerating the technique's clinical application. These simulations involve calculations of light transport, thermal diffusion, and the extent of thermal damage. The optical properties of tissue, which govern light transport through the tissue, vary during heating, and this affects the treatment outcomes. Nevertheless, the optical properties in conventional simulations of coagulation and vaporization remain constant. Here, we propose a laser ablation simulation based on Monte Carlo light transport with a dynamic optical properties (DOP) model. The proposed simulation is validated by performing optical properties measurements and laser irradiation experiments on porcine liver tissue. The DOP model showed the replicability of the changes in tissue optical properties during heating. Furthermore, the proposed simulation estimated coagulation areas that were comparable to experimental results at low-power irradiation settings and provided more than 2.5 times higher accuracy when calculating coagulation and vaporization areas than simulations using static optical properties at high-power irradiation settings. Our results demonstrate the proposed simulation's applicability to coagulation and vaporization region calculations in tissue for retrospectively evaluating the treatment effects of laser ablation.

Increased fluorescence observation intensity during the photodynamic diagnosis of deeply located tumors by fluorescence photoswitching of protoporphyrin IX.

Significance: Photobleaching of the photosensitizer reduces fluorescence observation time and the intensity of fluorescence emitted for tumor detection during 5-aminolevulinic acid-based photodynamic diagnosis. Aim: This study aims to utilize the concept of fluorescence photoswitching, which uses the fluorescence emission from photosensitizer excitation followed by the simultaneous excitation of the photosensitizer and its photoproduct to increase the fluorescence detection intensity during PDD of deeply located tumors. Approach: The fluorescence photobleaching of protoporphyrin IX (PpIX) and the formation of its photoproduct, photoprotoporhyrin (Ppp), caused by exposure to 505 nm light were investigated in solution, ex vivo, and in vivo, and the fluorescence photoswitching was analyzed. The fluorescence observations of PpIX and Ppp were performed with 505 and 450 or 455 nm excitation, respectively, which is the suited wavelength for the primary excitation of each fluorophore. Results: Fluorescence photoswitching was observed in all forms of PpIX investigated, and the fluorescence photoswitching time, fluorescence intensity relative to the initial PpIX and Ppp intensity, and fluorescence intensity relative to PpIX after photobleaching were obtained. The dependence of the fluorescence photoswitching time and intensity on the irradiation power density was noted. A fluorescence intensity increase between 1.6 and 3.9 times was achieved with simultaneous excitation of PpIX and Ppp after fluorescence photoswitching, compared with the excitation of PpIX alone. Conclusions: We have demonstrated the potential of fluorescence photoswitching for the improvement of the fluorescence observation intensity for the PDD of deeply located tumors.

Endoscopic image-guided laser treatment system based on fiber bundle laser steering.

A miniaturized endoscopic laser system with laser steering has great potential to expand the application of minimally invasive laser treatment for micro-lesions inside narrow organs. The conventional systems require separate optical paths for endoscopic imaging and laser steering, which limits their application inside narrower organs. Herein, we present a novel endoscopic image-guided laser treatment system with a thin tip that can access inside narrow organs. The system uses a single fiber bundle to simultaneously acquire endoscopic images and modulate the laser-irradiated area. The insertion and operation of the system in a narrow space were demonstrated using an artificial vascular model. Repeated laser steering along set targets demonstrated accurate laser irradiation within a root-mean-square error of 28 [Formula: see text]m, and static repeatability such that the laser irradiation position was controlled within a 12 [Formula: see text]m radius of dispersion about the mean trajectory. Unexpected irradiation on the distal irradiated plane due to fiber bundle crosstalk was reduced by selecting the appropriate laser input diameter. The laser steering trajectory spatially controlled the photothermal effects, vaporization, and coagulation of chicken liver tissue. This novel system achieves minimally invasive endoscopic laser treatment with high lesion-selectivity in narrow organs, such as the peripheral lung and coronary arteries.

Nonlinear absorption-based analysis of energy deposition in melanosomes for 532-nm short-pulsed laser skin treatment.

BACKGROUND AND OBJECTIVES: The clinical use of 532-nm short-pulsed lasers has provided effective treatment of epidermal pigmented lesions. However, the detection of significant differences in treatment effects between picosecond and nanosecond lasers has still varied among clinical studies. For robust evaluation of the differences based on the treatment mechanism, this study presents a nonlinear absorption-based analysis of energy deposition in melanosomes for 532-nm short-pulsed laser treatment. STUDY DESIGN/MATERIALS AND METHODS: Nonlinear absorption by melanin is modeled based on sequential two-photon absorption. Absorption cross-sections and nonradiative lifetimes of melanin, which are necessary for the nonlinear absorption-based analysis, are determined from transmittance measurement. Using the model and parameters, energy deposition in melanosomes was calculated with varying fluence and pulse width settings, including actual clinical parameters. RESULTS: The energy deposition in melanosomes increased with shorter laser pulses, and subnanosecond laser pulses were found to be most efficient. The comparison of energy deposition calculated using clinical parameters demonstrated the differences in treatment effects between picosecond and nanosecond lasers reported in clinical studies. CONCLUSION: The nonlinear absorption-based analysis provides quantitative evidence for the safety and efficacy evaluation of short-pulsed laser treatments, which may lead to the establishment of numerical indices for determining treatment conditions. Future studies considering the effects of the surrounding tissue on energy deposition in melanosomes will be needed.

Demonstration of an optical dentin hardness measuring device using bovine dentin with different demineralization times.

Significance: The increase in root caries is a serious problem as society ages. Root caries is diagnosed by inspection and palpation, which are qualitative. A method to objectively and quantitatively evaluate the progress of root caries in a clinical setting is strongly desired. The root caries could be diagnosed by measuring hardness because dentin becomes softer as the caries progresses. Vickers hardness has been customarily used as an indicator of tooth hardness. However, this method cannot be used to in vivo teeth because the teeth must be dried prior to measurement to make the indentation. A hardness meter using an indenter with light for tooth monitoring (HAMILTOM) is proposed as an optical device. HAMILTOM could measure hardness of teeth in wet condition as a dark area while applying a load to dentins without drying. Therefore, HAMILTOM may realize hardness measurements of in vivo teeth in a clinical setting quantitatively. Aim: The aim of our study is to demonstrate the optical dentin hardness measuring device HAMILTOM using bovine dentin with different demineralization times and to evaluate the correlation between the dark areas measured by HAMILTOM and the Vickers hardness measured by the Vickers hardness tester. Approach: The samples were 20 bovine dentins. They were demineralized by a lactic acid solution with different times and divided into groups 1 and 2 of 10 samples each. In both groups, the dark areas and Vickers hardness were measured for each sample. Group 1 was used to obtain a calibration curve to calculate Vickers hardness from the dark area. Group 2 was used to validate the calibration curve obtained from the dentin samples of group 1. Results: The areas appearing black without a total internal reflection of the indenter measured by HAMILTOM increased as the demineralization time increased. Additionally, the Vickers hardness of group 2 calculated by the dark areas of group 2 and the calibration curve obtained in group 1 and the Vickers hardness of group 2 measured by the Vickers hardness tester were strongly correlated with a determination coefficient of 0.99. Conclusions: The results demonstrate that HAMILTOM may be a suitable alternative to the conventional method. Unlike the conventional method, which cannot be used for in vivo teeth, HAMILTOM holds potential to quantitatively evaluate the progress of caries in in vivo teeth.

Mathematical modelling for antimicrobial photodynamic therapy mediated by 5-aminolaevulinic acid: An in vitro study.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) using aminolaevulinic acid (ALA) is a promising alternative to antibiotic therapy. ALA administration induces protoporphyrin IX (PpIX) accumulation in bacteria, and light excitation of the accumulated PpIX generates singlet oxygen to bacterial toxicity. Several factors, including drug administration and light irradiation conditions, contribute to the antibiotic effect. Such multiple parameters should be determined moderately for effective aPDT in clinical practice. METHODS: A mathematical model to predict bacterial dynamics in ALA-aPDT following clinical conditions was constructed. Applying a pharmacokineticspharmacodynamics (PK-PD) approach, which is widely used in antimicrobial drug evaluation, viable bacteria count by defining the bactericidal rate as the concentration of singlet oxygen produced when PpIX in bacteria is irradiated by light. RESULTS: The in vitro experimental results of ALA-aPDT for Pseudomonas aeruginosa demonstrated the PK-PD model validity. The killing rate has an upper limit, and the lower power density for a long irradiation time can suppress the viable bacteria number when the light dosages are the same. CONCLUSIONS: This study proposed a model of bacterial viability change in ALA-aPDT based on the PK-PD model and confirmed, by in vitro experiments using PA, that the variation of bacterial viability with light-sensitive substance concentration and light irradiation power densities could be expressed. Further validation of the PK-PD model with other gram negative and gram positive strains will be needed.

Effect of Q-switched Er:YAG laser irradiation on bonding performance to dentin surface.

The use of Q-switched erbium:yttrium-aluminum-garnet laser (Er:YAG laser), which have much less thermal effects than conventional Er:YAG lasers, has been proposed mainly in the medical field. The purpose of this study was to evaluate the bonding ability of dentin after Q-switched Er:YAG laser irradiation.The effects of dentin irradiation with Q-switched and conventional lasers were evaluated in terms of dentin morphology, roughness, hardness, elemental content, and resin bonding strength. Q-switched Er:YAG laser at average power densities of 20, 40, and 60 W/cm2 and conventional Er:YAG laser at 909 W/cm2 were used, and their performance was compared with that of the untreated group. Significant differences (p<0.05) were observed between 20 W/cm2 and the other groups in term of surface roughness and surface hardness. The resin adhesion of the 20 W/cm2 group was significantly higher than that of the other groups (p<0.05).

Open study of photodynamic therapy for skin ulcers infected with MRSA and Pseudomonas aeruginosa.

BACKGROUND: Drug-resistant bacterial infections are a global problem. Novel treatment methods that simultaneously control infection and promote wound healing without leading to new resistant bacteria are needed. Photodynamic therapy (PDT) is a useful antibiotic-free treatment approach. Our previous studies have shown that PDT for skin ulcers infected with methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) can achieve infection control and promoting wound healing in vitro and in vivo murine model. Here, we investigated the safety and effectiveness of PDT with 5-aminolevulinic acid (ALA-PDT) for human skin ulcers infected with MRSA and PA. METHODS: ALA-PDT with macrogol ointment containing 0.5% ALA-HCl and 0.005% EDTA-2Na (wavelength 410 nm, 10 J/cm2) was performed on consecutive days in patients aged ≥20 years who had skin ulcers infected with MRSA and PA. RESULTS: Six of our seven patients showed a clear tendency for ulcer area reduction to ≤60% of that measured at baseline. ALA-PDT was judged to be completely safe in all patients; only one patient had an increase in bacterial count. CONCLUSIONS: ALA-PDT is safe and effective for MRSA and PA infected skin ulcers to control and heal wound.

Mass Spectrometric Analysis of the Photobleaching of Protoporphyrin IX Used in Photodynamic Diagnosis and Therapy of Cancer.

Photobleaching and photoproduct formations are considered essential phenomena in improving the efficacy of photodynamic diagnosis and therapy (PDD and PDT). We investigated the photobleaching of protoporphyrin IX (PpIX) by measuring its concentration with mass spectrometry (MS). The reduction in the concentration of PpIX dissolved in dimethyl sulfoxide was measured during PDD and PDT conditions using lasers with wavelengths of 405 and 635 nm, respectively, at a power density of 10, 50 or 100 mW/cm2 . The obtained results were compared with the results of conventional fluorescence spectroscopy and previously reported results. Our results demonstrate the variation in the MS-based photobleaching coefficient of PpIX with the power density, while the fluorescence-based photobleaching coefficient was independent of the power density. The results of MS also show faster photobleaching of PpIX in comparison with that obtained from fluorescence. The difference may be attributed to the change in the fluorescence quantum yield of PpIX with its concentration and the effect of fluorescence emission from the PpIX photoproducts. Thus, an MS-based investigation of the photobleaching poses to be a more stable investigation form. Our finding highlights the importance of recognizing the potential significance of these discoveries in the PDD and PDT dosimetry and efficacy.

Incident Fluence Analysis for 755-nm Picosecond Laser Treatment of Pigmented Skin Lesions Based on Threshold Fluences for Melanosome Disruption.

BACKGROUND AND OBJECTIVES: In this study, the threshold fluences for disrupting the melanosomes for pigmented skin lesion treatment were determined using a 755-nm picosecond laser for clinical use. Based on the melanosome disruption thresholds, incident fluences corresponding to the target lesion depths were evaluated in silico for different laser spot sizes. STUDY DESIGN/MATERIALS AND METHODS: Melanosome samples were isolated from porcine eyes as alternative samples for human cutaneous melanosomes. The isolated melanosomes were exposed to 755-nm picosecond laser pulses to measure the mean particle sizes by dynamic light scattering and confirm their disruption by scanning electron microscopy. The threshold fluences were statistically determined from the relationships between the irradiated fluences and the mean particle sizes. Incident fluences of picosecond laser pulses for the disruption of melanosomes located at different depths in skin tissue were calculated through a light transport simulation using the obtained thresholds. RESULTS: The threshold fluences of 550- and 750-picosecond laser pulses were determined to be 2.19 and 2.49 J/cm2 , respectively. The numerical simulation indicated that appropriate incident fluences of picosecond laser pulses differ depending on the depth distribution of the melanosomes in the skin tissue, and large spot sizes are desirable for disrupting the melanosomes more deeply located within the skin tissue. CONCLUSION: The threshold fluences of picosecond laser pulses for melanosome disruption were determined. The incident fluence analysis based on the thresholds for melanosome disruption provides valuable information for the selection of irradiation endpoints for picosecond laser treatment of pigmented skin lesions. Lasers Surg. Med. © 2021 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Measurement of absorption and reduced scattering coefficients in Asian human epidermis, dermis, and subcutaneous fat tissues in the 400- to 1100-nm wavelength range for optical penetration depth and energy deposition analysis (Errata).

The errata correct errors that appeared in Table 2 of the published article.

Improvement in Ionization Efficiency Using Metal Oxide Nanoparticles in Laser Desorption/Ionization Mass Spectrometry of a Cancer Drug.

Mass spectrometry imaging (MSI) without labeling has the potential for faster screening in drug development. Matrix-assisted laser desorption/ionization (MALDI) is typically used, but it has a large matrix size and uneven drug distribution. Surface-assisted laser desorption/ionization (SALDI) using nanoparticles (NPs) may overcome these issues. Here, the influence of NPs, solvent ratio, and order of dropping of NPs on SALDI-MSI of protoporphyrin IX (PpIX), a cancer drug, are reported. A solution of PpIX in a 50% aqueous solution of 50% acetonitrile at a concentration of 10 µM was used. The NPs include ZnO, Fe3O4, and four types of TiO2. The NPs were fabricated by dissolving them on an aqueous 90% acetonitrile solution. Mass spectra were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a 355-nm wavelength. The signal intensity using TiO2 at a 0.5 mg/mL concentration in 50% acetonitrile was increased by 1.6-fold compared to that without TiO2. Changing the solvent to 90% acetonitrile gave a uniform TiO2 distribution and a 9-fold increase in the signal intensity for PpIX. Among the four types of TiO2 with different particle sizes and crystal structures, TiO2 with a smaller particle size and a rutile crystal structure produced the highest signal intensity. Forming a layer on top of the PpIX also resulted in an increased signal intensity. Hence, SALDI using TiO2 provides effective ionization of the drug. In the future, we plan to investigate a spray method for the ionization of PpIX using TiO2 for the MSI of various drugs.

Efficient photodynamic therapy against drug-resistant prostate cancer using replication-deficient virus particles and talaporfin sodium.

To enhance the potency of photosensitizer, we developed a novel photosensitizer, Laserphyrin®-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin®, Meiji Seika Pharma) into hemagglutinating virus of Japan envelope (HVJ-E). In this study, we examined the optimal Laserphyrin® concentration for preparation of Laserphyrin®-HVJ-E which had photocytotoxicity and maintained direct cytotoxicity derived from HVJ-E. Then, potency of Laserphyrin®-HVJ-E and Laserphyrin® were compared in vitro using castration-resistant prostate cancer cell line (PC-3). A laser diode (L660P120, Thorlabs, USA) with a wavelength of 664 nm was used for light activation of Laserphyrin®, which corresponds to an absorption peak of Laserphyrin® and provides a high therapeutic efficiency. The photocytotoxicity and direct cytotoxicity of Laserphyrin®-HVJ-E prepared using various Laserphyrin® concentrations were evaluated using PC-3 cell in vitro. We categorized the treatment groups as Group 1: 50 µL of D-MEM treatment group, Group 2: HVJ-E treatment group, Group 3: Laserphyrin®-HVJ-E treatment group, and Group 4: Laserphyrin® treatment group. Group 3 was subjected to different concentrations of Laserphyrin®-HVJ-E suspension, and all groups were subjected to different incubation periods (24, 48 h), (30 min, 1 h, or 3 h,) respectively, without and after PDT. Laserphyrin®-HVJ-E prepared using 15 mM Laserphyrin® had high photocytotoxicity and maintained HVJ-E's ability to induce direct cytotoxicity. Therapeutic effect of Laserphyrin®-HVJ-E was substantially equivalent to that of Laserphyrin® alone even at half Laserphyrin® concentration. By utilizing Laserphyrin®-HVJ-E, PDT could be performed with lower Laserphyrin® concentration. In addition, Laserphyrin®-HVJ-E showed higher potency than Laserphyrin® by combining cytotoxicities of HVJ-E and PDT.

Enhancement of Ionization Efficiency Using Zeolite in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Multiple Drugs in Cancer Cells (Mass Spectrometry of Multiple Drugs in Cells Using Zeolite).

Combined therapy using photodynamic therapy (PDT) and chemotherapy has been proposed for anticancer-drug-resistant cancer cells. To evaluate the efficacy of such a combined therapy, the uptakes of an anticancer drug and a photosensitizer in cancer cells must be assessed. Mass spectrometry using matrix-assisted laser desorption/ionization can detect multiple drugs simultaneously. Human prostate cancer cells PC-3 or docetaxel-resistant cancer cells PC-3-DR were incubated in a serum-free medium containing a photosensitizer, protoporphyrin IX (PpIX), and an anticancer drug, docetaxel. A zeolite matrix was created by mixing 6-aza-2-thiothymine and NaY5.6 zeolite, and dissolving in water with 50% acetone. Ions were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a wavelength of 355 nm. The cell morphology was preserved by washing the cells with ammonium acetate and drying in a vacuum after drug administration. Protonated PpIX (m/z 563.3) and the sodium adduct ion of docetaxel (m/z 829.9) were obtained from PC-3 cells simultaneously using the zeolite matrix. On the other hand, PpIX was detected but ions originating from docetaxel were not detected from PC-3-DR cells. The result indicated the efficacy of PDT for docetaxel-resistant cancer cells.

Measurement of absorption and reduced scattering coefficients in Asian human epidermis, dermis, and subcutaneous fat tissues in the 400- to 1100-nm wavelength range for optical penetration depth and energy deposition analysis.

SIGNIFICANCE: In laser therapy and diagnosis of skin diseases, the irradiated light distribution, which is determined by the absorption coefficient µa and reduced scattering coefficient µs' of the epidermis, dermis, and subcutaneous fat, affects the treatment outcome and diagnosis accuracy. Although values for µa and µs' have been reported, detailed analysis for Asian skin tissues is still lacking. AIM: We present µa and µs' measurements of Asian skin tissues in the 400- to 1100-nm wavelength range for evaluating optical penetration depth and energy deposition. APPROACH: The measurements with Asian human skin samples are performed employing a double integrating sphere spectrometric system and an inverse Monte Carlo technique. Using the measured parameters, the optical penetration depth and energy deposition are quantitatively analyzed. RESULTS: The µa of the epidermis layer varies among different ethnic groups, while the µa of the other layers and the µs' of all of the layers exhibit almost no differences. The analysis reveals that the optical penetration depth and the energy deposition affect the photodynamic therapy treatment depth and the heat production in skin tissue, respectively. CONCLUSIONS: The experimentally measured values of µa and µs' for Asian skin tissues are presented, and the light behavior in Asian skin tissues is analyzed using a layered tissue model.

Fluorescence detection of deep intramucosal cancer excited by green light for photodynamic diagnosis using protoporphyrin IX induced by 5-aminolevulinic acid: an ex vivo study.

SIGNIFICANCE: The diagnostic depth of photodynamic diagnosis (PDD) for gastric cancer with protoporphyrin IX (PpIX) is limited, which leads to missing intramucosal cancers in screening and surgery. AIM: The reason is that the excitation light, whose wavelength is determined by the highest absorption peak of PpIX (∼405 nm), is strongly attenuated by mucosal tissues. We investigated an excitation wavelength that can extend the diagnostic depth of PpIX fluorescence at the mucosal subsurface. APPROACH: By calculating the depth-dependent intensity of the excitation light in porcine gastric mucosa for each wavelength, relationships among the wavelength, fluorophore depth, and fluorescence intensity were assessed and fluorescence images of PpIX pellets located at different fluorophore depths were compared experimentally by changing the excitation wavelength. RESULTS: The numerical calculation showed that a 505-nm excitation light provided the highest fluorescence intensities at a fluorophore depth deeper than 1.1 mm. In the fluorescence observation, the fluorescence intensities at fluorophore depths of 0 and 1.0 mm at 405 nm were 5.4 × 103 and 1.0 × 103 arb. units, whereas those at 505 nm were 5.3 × 101 and 1.9 × 102 arb. units, respectively. CONCLUSION: The experimental results suggest that the diagnosis depth of PDD with PpIX for intramucosal cancer can be extended by 505-nm excitation light.

Effect of Amplifier Gain on Photoacoustic SNR (Signal to Noise Ratio) in an LED-based Photoacoustic Imaging System.

The possibility of using photoacoustic imaging for functional diagnosis has attracted much attention especially in the clinical field. Among such imaging systems, a system, which offers real-time imaging using compact and low-priced LEDs as a light source, has appeared. Compared to solid state lasers, the LED pulse energy was extremely small, so it had been thought that imaging would be extremely difficult, but by adding a pre-amplifier, real time photoacoustic imaging became possible. However the signal-to-noise ratio (SNR) and the amplifier gain needed for making real time imaging possible have remained unclear. The present study was designed to clarify these data. The results showed that, using a tissue phantom and human fingers, an SNR > 4 and amplifier gain > 80dB were required, and demonstrated why making an image without a pre-amplifier had proved difficult.