In the detection by electron spin resonance (ESR) spectra using the DPPMPO (the latest high sensitivity spin trap agent) of mouse skin after TP fiber laser irradiation, we have succeeded in observation of the ESR spectra of the three. Those were the superoxideanion radical (), hydroxyl radical (OH*) and ascorbyl radical (AsA*). Multiple standard free radicals were generated, and the -value and hyperfine coupling constant (hfcc) of each spin adduct were 3 obtained by the ESR-spin trapping method to identify the various free radicals. The spin trapping agents used were 5-(diphenylphosphinoyl)5-methyl-4,5-dihydro-3H-pyrrole--oxide (DPPMPO). The signal ratio was obtained for each sample by comparison with the signal of MnO, an internal standard. The relative intensity of radicals was calculated by comparison with the third MnO signal intensity. The -value and distance (mT) between the peaks for hfcc were measured by software supplied with the ESR equipment, ESR spectrometer (JEOL, JES-FA200 spectrometer, Tokyo), ESR universal cavity (JEOL, ES-UCX2: TE11 mode cavity) with X-band microwave units (8.750 to 9.650 GHz), an ESR standard marker with manganese oxide (MnO) powder (JEOL DATUM, MO7-FB-4), an aqueous sample cell (JEOL, ES-LC12), sample volume: 20 to 100 μl, a tissue-type: quartz cell (Labotec, Tokyo) with a self-made cover glass (). The spin trapping agents used for ESR were DPPMPO (50 to 500 mM, 10%, dimethyl sulfoxide solution). DPPMPO () was applied to the irradiation spot before laser irradiation. The irradiated skin tissue was immediately removed and placed on an ice-cold plate after being rinsed with ice-cold phosphate-buffered saline (pH 7.2). The skin tissues including the dermis and epidermis were cut to samples measuring 3 to to 20 mm. The slice weight was measured before removal to normalize the ESR signal of each radical. The same spin trap agent (10 to 50 μl) was added to the tissue samples (10 to 50 mg) immediately after being weighed, and after precisely 5 min, samples were measured using ESR. To identify the peaks, the signals were analyzed by specialized analysis software installed in the ESR device (ESR computer software, A-System vl.40 ISAJ, FA-manager vl.20, JES, Tokyo, Japan) to determine the -value and hfcc from the distance between peaks. The data were analyzed using the least significant difference test. One-way analysis of variance was used to determine variance among the data. All data are reported as . Statistical significance was set at .