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2.3 Methods CHAPTER 2 2.3.1 Subjects This study was conducted under the ethics approval of the Human Research Ethics Committee at the Australian National University (protocol no: 2015/288). Nine live human participants were recruited (6 males & 3 females; age = 26.9 ± 2.1 years) and classified into three different skin tones (light, medium and dark). Subjects were asked to fill out a questionnaire to determine their Fitzpatrick skin type (Fitzpatrick, 1988). Light skin toned participants were all Caucasian (Fitzpatrick skin type scores: 9, 11, 12); medium skin toned participants were of Iranian, Eastern European and Afghan descent (Fitzpatrick skin type scores: 16, 18 and 23 respectively); dark skin toned participants were of Indian and Sri Lankan descent (Fitzpatrick skin type scores: 25, 30 and 33 respectively). Human tissues from 6 cadavers (donation to the Australian National University Medical School Anatomy program) were also included in this study (terminal age = 81.3 ± 4.3 years). Sixteen sites of the limbs and cranium were tested (Figure 2.1a). The upper limb sites included the shoulder joint, upper arm and elbow joint; the lower limb sites included the wrist (distal radioulnar joint), the thumb (1st metacarpophalangeal joint) and the middle finger (3rd metacarpophalangeal joint). The lower limb sites included thigh, knee, calf, and ankle. At the elbow, knee and ankle, measurements were performed in 2 planes, sagittal (anterior-posterior), and coronal (lateral-medial). In the arm and thigh, two measurements were performed; one which the light path included the bony structures (humerus, femur), and the other which was limited to the soft tissues (biceps brachii, hamstring muscles). Cranium measurements were obtained through the dura at the inion or at bregma from human cadavers. 2.3.2 LED irradiation and measurement A custom-built 660 nm LED device, with varying output intensities ranging from 15-500 mW/cm2 measured at the aperture window, was situated on each testing site. A light probe tuned to 670 nm (Thorlabs, PM100D) was placed on the opposing side of the respective body part (Figure 2.1a). For cranium measurements, a smaller custom-built 670 nm light probe was used as previously described (Hu et al., 2016). The LED was placed either at the Bregma or the inion and the measurement was taken transcranially with the light probe placed on the dura on the opposing side of the LED. Within this light probe, light was detected by a photodiode chip (surface area, 0.62 mm2; maximal response (>95 %) to 630-685 nm; ODD-660W, Opto Diode Corp.) and then amplified (AD8304, Analog Devices). The readings were then calibrated against the Thorlab light probe. There was negligible difference when the tuneable light probe was set at 670 or 660 nm, however it was necessary to match the readings to the custom light probe which was fixed at 670 nm. Both probes measure the amount of 670 nm light in μW/cm2. Readings ≥ 0.1 μW/cm2 were considered as positive for penetration. The thickness of each testing site was measured using digital V ernier callipers. Minimum force was applied to avoid squeezing and multiple measurements were taken to ensure accuracy and consistency. Ambient lights were kept to a 39PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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