Light Emitting Diodes

Laser Med Sci. 2012 Jul 20. [Epub ahead of print]

Effect of laser and LED phototherapies on the healing of cutaneous wound on healthy and iron-deficient Wistar rats and their impact on fibroblastic activity during wound healing.

Oliveira Sampaio SC, de C Monteiro JS, Cangussú MC, Pires Santos GM, Dos Santos MA, Dos Santos JN, Pinheiro AL.

Source

Center of Biophotonics, School of Dentistry, Federal University of Bahia, Av. Araújo Pinho, 62, Canela, Salvador, BA, 40110-150, Brazil, susanasampaio2006@yahoo.com.br.

Abstract

Iron deficiency impairs the formation of hemoglobin, red blood cells, as well the transport of oxygen. The wound healing process involves numerous functions, many of which are dependent on the presence of oxygen. Laser has been shown to improve angiogenesis, increases blood supply, cell proliferation and function. We aimed to study the effect of ?660 nm laser and ?700 nm light-emitting diode (LED) on fibroblastic proliferation on cutaneous wounds on iron-deficient rodents. Induction of iron anemia was carried out by feeding 105 newborn rats with a special iron-free diet. A 1?×?1 cm wound was created on the dorsum of each animal that were randomly distributed into seven groups: I, control anemic; II, anemic no treatment; III, anemic?+?L; IV, anemic?+?LED; V, healthy no treatment; VI, healthy?+?laser; VII, healthy?+?LED (n?=?15 each). Phototherapy was carried out using either a diode laser (?660 nm, 40 mW, 10 J/cm(2)) or a prototype LED device (?700?±?20 nm, 15 mW, 10 J/cm(2)). Treatment started immediately after surgery and was repeated at 48-h interval during 7, 14, and 21 days. After animal death, specimens were taken, routinely processed, cut, stained with hematoxylin-eosin, and underwent histological analysis and fibroblast counting. Significant difference between healthy and anemic subjects on regards the number of fibroblast between treatments was seen (p?<?0.008, p?<?0.001). On healthy animals, significant higher count was seen when laser was used (p?<?0.008). Anemic subjects irradiated with LED showed significantly higher count (p?<?0.001). It is concluded that the use of LED light caused a significant positive biomodulation of fibroblastic proliferation on anemic animals and laser was more effective on increasing proliferation on non-anemics.

Ann Dermatol Venereol. 2009 Oct;136 Suppl 6:S351-8.

Light-emitting diodes (LED)

[Article in French]

Cartier H, Le Pillouer-Prost A, Grognard C.

hcartier@hotmail.com

LED home-use is now widely spread. In dermatology, numerous reports have stated their results for many indications: wound healing process, rejuvenation, acne and, of course, photodynamic therapy. Nevertheless, fluence, pulse duration and color of the LED are so variable as it is difficult to bring well codified results. But how should you not be interested in this field? It is already any more a near future but well and truly a therapeutic reality…

Growth Horm IGF Res. 2009 Jun;19(3):274-9. Epub 2008 Dec 16.

Modulation of rat pituitary growth hormone by 670 nm light.

Hymer WC, Welsch J, Buchmann E, Risius M, Whelan HT.

Centralized Biological Laboratory, Penn State University, University Park, PA 16802-4803, USA. wch@psu.edu

In rat pituitary somatotrophs, cytochrome oxidase is co-packaged with growth hormone (GH) in some storage granules. Because this enzyme is thought to be the molecular photoacceptor of red-near infrared light, and because exposure of diverse tissue systems to 670 nm visible light affects their biological responses (e.g., wound healing), we tested the idea that exposure of rat pituitary cells, rat hemi-pituitary glands and rat pituitary homogenates to 670 nm light in vitro might alter GH storage and/or release. In this report we offer evidence to show that light treatment (670 nm, 80s, intensity 50 mW/cm(2), energy density 4 J/cm(2)) up-regulates GH release, in part by breakdown of intracellular, oligomeric GH as determined by gel filtration chromatography.

J Mol Cell Cardiol. 2009 Jan;46(1):4-14. Epub 2008 Sep 30.

Near infrared light protects cardiomyocytes from hypoxia and reoxygenation injury by a nitric oxide dependent mechanism.

Zhang R, Mio Y, Pratt PF, Lohr N, Warltier DC, Whelan HT, Zhu D, Jacobs ER, Medhora M, Bienengraeber M.

Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53326, USA.

Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.

Brain Res. 2008 Dec 3;1243:167-73. Epub 2008 Sep 30.

Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.

Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Parkinson’s disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.

Neuroscience. 2008 Jun 2;153(4):963-74. Epub 2008 Mar 26.

Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Liang HL, Whelan HT, Eells JT, Wong-Riley MT.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Parkinson’s disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson’s disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson’s disease by energizing the cells and increasing their viability.

Neuroscience. 2006 May 12;139(2):639-49. Epub 2006 Feb 7.

Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis.

Liang HL, Whelan HT, Eells JT, Meng H, Buchmann E, Lerch-Gaggl A, Wong-Riley M.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Near-infrared light via light-emitting diode treatment has documented therapeutic effects on neurons functionally inactivated by tetrodotoxin or methanol intoxication. Light-emitting diode pretreatment also reduced potassium cyanide-induced cell death, but the mode of death via the apoptotic or necrotic pathway was unclear. The current study tested our hypothesis that light-emitting diode rescues neurons from apoptotic cell death. Primary neuronal cultures from postnatal rat visual cortex were pretreated with light-emitting diode for 10 min at a total energy density of 30 J/cm2 before exposing to potassium cyanide for 28 h. With 100 or 300 microM potassium cyanide, neurons died mainly via the apoptotic pathway, as confirmed by electron microscopy, Hoechst 33258, single-stranded DNA, Bax, and active caspase-3. In the presence of caspase inhibitor I, the percentage of apoptotic cells in 300microM potassium cyanide was significantly decreased. Light-emitting diode pretreatment reduced apoptosis from 36% to 17.9% (100 microM potassium cyanide) and from 58.9% to 39.6% (300 microM potassium cyanide), representing a 50.3% and 32.8% reduction, respectively. Light-emitting diode pretreatment significantly decreased the expression of caspase-3 elicited by potassium cyanide. It also reversed the potassium cyanide-induced increased expression of Bax and decreased expression of Bcl-2 to control levels. Moreover, light-emitting diode decreased the intensity of 5-(and -6) chloromethy-2′, 7-dichlorodihydrofluorescein diacetate acetyl ester, a marker of reactive oxygen species, in neurons exposed to 300 microM potassium cyanide. These results indicate that light-emitting diode pretreatment partially protects neurons against cyanide-induced caspase-mediated apoptosis, most likely by decreasing reactive oxygen species production, down-regulating pro-apoptotic proteins and activating anti-apoptotic proteins, as well as increasing energy metabolism in neurons as reported previously.

Photomed Laser Surg. 2006 Apr;24(2):121-8.

Clinical and experimental applications of NIR-LED photobiomodulation.

Desmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM, Buchmann EV, Connelly MP, Dovi JV, Liang HL, Henshel DS, Yeager RL, Millsap DS, Lim J, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.

Department of Clinical Laboratory Sciences, University of Wisconsin-Milwaukee, 53226, USA.

This review presents current research on the use of far-red to near-infrared (NIR) light treatment in various in vitro and in vivo models. Low-intensity light therapy, commonly referred to as “photobiomodulation,” uses light in the far-red to near-infrared region of the spectrum (630-1000 nm) and modulates numerous cellular functions. Positive effects of NIR-light-emitting diode (LED) light treatment include acceleration of wound healing, improved recovery from ischemic injury of the heart, and attenuated degeneration of injured optic nerves by improving mitochondrial energy metabolism and production. Various in vitro and in vivo models of mitochondrial dysfunction were treated with a variety of wavelengths of NIR-LED light. These studies were performed to determine the effect of NIR-LED light treatment on physiologic and pathologic processes. NIRLED light treatment stimulates the photoacceptor cytochrome c oxidase, resulting in increased energy metabolism and production. NIR-LED light treatment accelerates wound healing in ischemic rat and murine diabetic wound healing models, attenuates the retinotoxic effects of methanol-derived formic acid in rat models, and attenuates the developmental toxicity of dioxin in chicken embryos. Furthermore, NIR-LED light treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. The experimental results demonstrate that NIR-LED light treatment stimulates mitochondrial oxidative metabolism in vitro, and accelerates cell and tissue repair in vivo. NIR-LED light represents a novel, noninvasive, therapeutic intervention for the treatment of numerous diseases linked to mitochondrial dysfunction.

Photomed Laser Surg. 2005 Jun;23(3):268-72.

Effects of 670-nm phototherapy on development.

Yeager RL, Franzosa JA, Millsap DS, Angell-Yeager JL, Heise SS, Wakhungu P, Lim J, Whelan HT, Eells JT, Henshel DS.

School of Public and Environmental Affairs, Indiana University, Bloomington, IN 47405, USA. rlyeager@indiana.edu

OBJECTIVE: The objective of the present study was to assess the survival and hatching success of chickens (Gallus gallus) exposed in ovo to far-red (670-nm) LED therapy. BACKGROUND DATA: Photobiomodulation by light in the red to near-infrared range (630-1000 nm) using low-energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing and improve recovery from ischemic injury. The mechanism of photobiomodulation at the cellular level has been ascribed to the activation of mitochondrial respiratory chain components resulting in initiation of a signaling cascade that promotes cellular proliferation and cytoprotecton. MATERIALS AND METHODS: Fertile chicken eggs were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. In ovo survival and death were monitored by daily candling (after Day 4). RESULTS: We observed a substantial decrease in overall and third-week mortality rates in the light-treated chickens. Overall, there was approximately a 41.5% decrease in mortality rate in the light-treated chickens (NL: 20%; L: 11.8%). During the third week of development, there was a 68.8% decrease in the mortality rate in light-treated chickens (NL: 20%; L: 6.25%). In addition, body weight, crown-rump length, and liver weight increased as a result of the 670-nm phototherapy. Light-treated chickens pipped (broke shell) earlier and had a shorter duration between pip and hatch. CONCLUSION: These results indicate that 670-nm phototherapy by itself does not adversely affect developing embryos and may improve the hatching survival rate.

J Biol Chem. 2005 Feb 11;280(6):4761-71. Epub 2004 Nov 22.

Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase.

Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, Kane M, Whelan HT.

Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. mwr@mcw.edu

Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 microm KCN. It significantly reduced neuronal cell death induced by 300 microm KCN from 83.6 to 43.5%. However, at 1-100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins.

 

Biomed Eng Online. 2004; 3: 9.
Published online 2004 Mar 22. doi:  10.1186/1475-925X-3-9

Safety assessment of near infrared light emitting diodes for diffuse optical measurements

Alper Bozkurtcorresponding author1 and Banu Onaral1
corresponding authorCorresponding author.
Alper Bozkurt: ude.lexerd@trukzob.repla; Banu Onaral: ude.lexerd@larano.unab
Author information ? Article notes ? Copyright and License information ?
Received 2004 Jan 30; Accepted 2004 Mar 22.

Background

New medical applications using optical measurement techniques are emerging rapidly. These methods are used to study the content of biological pigments and tissue structures by analyzing the absorption and scattering of the induced light. When visible or near infrared light at specific wavelengths in the window of 600 to 950 nm (fig. ?(fig.1)1) is shone through the tissue, information about the amount of blood chromophores such as oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) can be obtained. This constitutes an important measure of the hemodynamic state of the tissue [1], and is the principle upon which pulse oximetry is based.

Figure 1

The optical window used in diffuse optical measurements

The hazard potential of the near-infrared light should be considered from two perspectives: eye hazards and skin hazards. The effect of NIR light on eye and skin are studied separately since the eye lens focuses the light on the retina. Focused light is stronger in terms of irradiance than non-focused light. Hence, injury potential increases with focusing. Here, we concentrate on effects of NIR light on skin, since the light source is attached to the skin during diffuse optical measurements.

The main cause of the potential hazard for NIR light shone through the skin is the heating effect. When NIR light is emitted by an LED in direct contact with the skin, there are two sources of heat: radiated NIR light energy absorbed by the skin, and conducted energy, which is caused by the temperature increase in the semiconductor junction inside the LED. Temperature increase due to the radiated energy has been quantified to be less than 0.5°C [2]. However, the effect of conducted heat due to the temperature increase in the semiconductor junction has not yet been reported for diffuse optical measurement applications.

Two types of sources are employed to radiate light in NIR range: LEDs and lasers. Standards specific to the risk of the radiated energy for LEDs in direct contact with the skin have not yet been developed. However, standards that govern safety regulations of laser applications can be used for LED source as well [3].

Beyond a certain power level, radiated light energy has the potential to cause serious injuries. This property becomes beneficial in surgical operations for incision and ablation of tissue parts. The thresholds of the damages that can ensue from light energy strongly depend on wavelength, spot size and pulse length of the light [3]. Thus, the effect of each factor should be analyzed to assess the safety of the NIR light.

Light absorption in the tissue varies depending on the wavelength of the light radiated. The most significant effect of the absorbed power is surface heating, which may cause serious burn injuries. This effect is due to absorption of light by water, which has been the key concept behind the tissue ablation and incision using light energy. Especially, wavelengths longer than 950 nm are strongly absorbed by water. However water is almost transparent to the light in NIR range (fig. ?(fig.1).1). In healthy individuals, NIR light is mostly absorbed by blood pigments in deep tissue which does not heat up due to constant circulation. Hence, light in the NIR region does not present the risk of tissue damage that occurs at wavelengths longer than 950 nm which have been used for laser surgery [3]. NIR wavelengths are not ionizing hence do not carry the risk of altering genetic information as may be caused by ultraviolet light.

LEDs and lasers differ in spot size. Spot size defines how well light energy is focused into a single spot. Lasers are inherently collimated. Thus, different spot sizes can be achieved. However, LED light diverges as it leaves the source, so it is difficult to focus all energy into a spot. Different spot sizes may be achieved by means of additional optical apparatus, which is rarely applied in current practice. Hence, spot size is not an issue in LED safety considerations.

Pulse length or in other words light emission duration is another factor determining the potential risk of the radiated light energy. A given amount of energy is turned into more powerful pulses as pulse length decreases. Nanosecond range or shorter pulses are used for ablation in laser surgery operations. The needed short time duration cannot be achieved by LEDs because of the slow response time of the semiconductor junction [4]. Therefore, LEDs do not carry the risk caused by short powerful bursts.

Standards specific to the use of LEDs in direct contact with the skin have not yet been proposed. Though there are some efforts by International Commission on NonIonizing Radiation Protection (ICNIRP), the International Electrotechnical Commission (IEC) and American National Standards Institute (ANSI) to develop regulation about LED hazards, most efforts have been concentrated on eye injury due to radiated energy [5]. LEDs have been regarded safe for eye exposure by a number of studies in the literature [6,7] from a radiated energy perspective. The combined – radiated plus conducted – heating effect of the LEDs on human skin tissue has not been reported yet. In this paper the combined heating effect of the NIR LED in direct contact with skin is analyzed and tested.

Methods

The measurement setup is illustrated in figure ?figure22 for in-vitro and figure ?figure33 for in-vivo experiments. An LED (L730-805-850-40B32 from Epitex Inc.), that could emit NIR light at three wavelengths of 730, 805, and 850 nm, was employed as a NIR light source. These wavelengths are in the middle of the optical window described in figure ?figure1.1. The LED was inserted in a cushioning material that is attached to the surface of the absorbing medium via a medically approved double-sided sticky tape (Adchem Inc.) to ensure good optical contact. An optical phantom with an absorption coefficient of 0.08 cm-1 and reduced scattering coefficient of 12.5 cm-1 is used as the light-absorbing medium. A thermocouple needle with a 0.1 mm diameter was inserted between the source and absorbing medium to monitor the temperature change continuously. The needle was connected to the thermocouple device (Sper Scientific Ltd.). Data was sent to a PC by using RS-232 connection to be stored and plotted continuously. “Testlink 1.1.0.0” software provided by the thermocouple company is used to plot and store the data with sampling frequency of 1 Hz and temperature resolution of 0.1°C.

Figure 2

Setup to measure the heating effect of the semiconductor in vitro
Figure 3

Description of the in-vivo experimental setup

In order to compensate for the effect of the ambient temperature, the ambient temperature was also continuously recorded on the tissue phantom using a second thermocouple 6 cm away. The change in the baseline due to drifts in ambient temperature was corrected after the experiment.

The heating of the thermocouple needle itself due to NIR absorption was tested in parallel. To do this, NIR light was applied to the needle where the contact to LED was avoided by means of an optical filter which only allows NIR light (NIR-pass filter). It has been observed that the NIR radiation does not significantly heat the thermocouple.

As discussed earlier, the LED source produces two types of heating effects due to conducted and radiated energy. Conducted heat due to the semiconductor junction is measured in-vitro using a NIR-pass filter as shown in figure ?figure2.2. In this setup, measurements are performed with and without NIR-pass filter. The NIR-pass filter does not heat up due to NIR light absorption since it is transparent to NIR light. When filter is used, the thermocouple measures the temperature increase due to NIR light absorption. When filter is not used, the temperature increase is caused by the combined heating effect of semiconductor junction and NIR light absorption. Therefore, the difference between two readings gives the temperature increase solely due to semiconductor junction heating.

The temperature increase of the semiconductor junction depends on the effective (root mean square) dissipated power of the LED. Different effective powers can be obtained by changing the emitted waveform. DC, pulsating waveform with 12.5 ms pulse duration and 33.3 % duty cycle and sinusoidal waveform with 1 kHz frequency were tested in-vitro to study the effect of different waveforms. A single wavelength of 730 nm was used. Peak irradiances between 25 and 50 mW/cm2 were employed. The crest factor for pulsating waveform and sinusoidal waveform is 1.73 and 1.41, respectively.

For the pulsating waveform, effective power can be controlled by varying the pulse duration, duty cycle, irradiance and using single or multiple wavelengths. A function generator coupled to the LED driver was used to generate pulsating LED light to study the effect on the temperature of the semiconductor junction. Single wavelength of 730 nm was used. Temperature increase for pulse durations of 12.5, 50, 100 and 150 ms, duty cycles of 25 %, 33.3%, 50% and 75% were tested in-vitro. For these duty ratios crest factors are 2, 1.73, 1.41 and 1.15 respectively. The peak value of the pulses was selected to be 37.5 mW/cm2. This is the power range generally used in diffuse optical measurement applications.

Temperature increase is also affected by the number of different wavelengths used. In continuous wave diffuse optical applications, generally two wavelengths are used to resolve the absorption by two chromophores. Therefore, we tested for the effect of using two wavelengths in-vitro. Wavelengths of 730 and 850 nm were time multiplexed in a way that only one wavelength was turned on at a time. Pulse duration was 12.5 ms and duty cycle was 33.3% which corresponds to a crest factor of 1.73. Peak irradiance was 37.5 mW/cm2.

In addition to square pulses, 730 nm and 850 nm wavelengths with 1 kHz sinusoidal waveform and 90° phase shift were used in-vitro to study the heating effect of the quadrature phase-modulated system. The peak irradiance of 37.5 mW/cm 2 with a crest factor of 1.73 and wavelengths of 730 and 850 nm were employed as in the case of square pulses.

The combined effect of the radiated and conductive heat was also tested with an in-vivo set up on three Caucasian adult subjects. All in-vivo studies were carried out under Institutional Review Board (IRB) approval. The LED was directly coupled to the surface of a human arm to observe the in-vivo effect as described in figure ?figure3.3. The change in the surface temperature directly beneath the LED was recorded continuously for 12.5 ms pulses with 37.5 mW/cm2 irradiance and 33.3% duty cycle.

During in-vivo experiments, the LED was coupled to the skin using a cushioning material attached to the skin by a medically graded double-sided sticky tape. It is well known that heat increase of the skin is dissipated by sweating mechanism and by heat exchange with air as well as convection via blood flow [8]. The cushioning material that is used to attach the LED to the skin causes pressure and isolation of skin tissue from ambient air. In addition, it blocks sweating by closing the pores and hence contributes to the temperature increase. This was monitored with a secondary thermocouple attached to the skin a few centimeters away from the LED source using the same kind of cushioning material.

In addition to the test setup, the device currently used in Drexel University and University of Pennsylvania with the purpose of functional optical brain imaging using NIR light [9,10] was studied for the heating effect during actual operation with human subjects. The device employs the same kind of LED as used in in-vitro studies. In these experiments, a standard duty cycle of 8.3%, pulse duration of 12.5 ms and irradiance of 37.5 mW/cm2 were used. The crest factor is 3.47 for such a pulse shape. The temperature rise in the semiconductor junction was tested in-vitro. The combined heating effect on skin due to both NIR absorption and the heating of semiconductor junction was experimented in-vivo on the human arm and forehead.

In each experiment, data were collected for 35 minutes. The temperature increase after 30 minutes of operation was assumed as the steady state temperature. Data points corresponding to 5 minutes following the 30th minute of the operation were avera ged to determine the resulting temperature increase.

An alternative way to analyze the temperature increase due to absorption of LED emitted NIR light is to compare it with sun emitted NIR light. With the geographic conditions defined by United States Committee on Extension to the Standard Atmosphere, for the 48 contiguous states of the USA over a period of one year as an inclined plane at 37° tilt toward the equator, facing the sun; i.e., the surface normal points to the sun at an elevation of 48.81° above the horizon [11], the American Society for Testing and Materials (ASTM) developed and defined a standard terrestrial solar spectral irradiance distribution called ASTM G159 [12]. This standard can be used to calculate the average NIR irradiance emitted by the sun and absorbed by human skin. The average power delivered by the NIR part of the sunlight calculated using this standard is 50 mW/cm2.

Results

A typical temperature increase due to the combined effect of conducted and radiated heats of the LED is plotted in figure ?figure4.4. As seen in this figure, the rise in temperature reached an asymptotical steady state by the 30th minute of operation. This was observed in all of the experiments conducted.

Figure 4

(in vitro) Sample temperature increase result for an experiment

It has been observed that, for a given peak irradiance level, the single wavelength DC waveform increased the temperature of the semiconductor junction more than pulsed and modulated waveforms during in-vitro experiments (fig. ?(fig.5).5). Pulsed and sinusoidal waveforms caused similar temperature increases. Increase in the peak irradiance of the LED caused higher elevation in the temperature.

Figure 5

(in vitro) Temperature increase with varying peak irradiance levels for DC, pulsating and modulated waveforms

The effect of varying pulse characteristics on the temperature of the semiconductor junction can be observed in in-vitro experiment results. As pulse duration, duty cycle and irradiance were increased the temperature elevation was observed (figure ?(figure5,5, ?,66).

Figure 6

(in vitro) Temperature increase with varying duty cycles and pulse durations

730 nm and 850 nm wavelengths have similar temperature increase effects on the semiconductor junction when they are used one at a time in both sinusoidal and pulsed waveforms. Using both wavelengths simultaneously in a time-switched pulsed manner caused a temperature increase that is approximately equal to the addition of the effect of each wavelength. When two wavelengths with sinusoidal waveform having 90° phase shift are used simultaneously, temperature increase was approximately the summation of the effect of each wavelength. (fig. ?(fig.77)

Figure 7

(one wavelength vs. two wavelength) Temperature increase for pulsating and phase modulated waveforms

In-vivo and in-vitro results demonstrated a similar trend for increasing irradiance (fig. ?(fig.8).8). In-vivo temperature increase results were approximately 1°C higher than in-vitro results. The temperature increase due to the cushioning material used to attach the light source was 0.5 ± 0.1°C, which was caused by blocking skin-air heat exchange and sweating.

Figure 8

(in vivo vs. in vitro) Temperature increase for set up in fig ?fig33 and for brain-imaging device

Results of in-vitro and in-vivo experiments, where the functional near infrared brain imaging device of Drexel University was used, can be found in figure ?figure88 also. During the in-vivo experiments, temperature rise was observed as in the case of in-vitro results; however the temperature increase was 1–1.5°C higher than in the in-vitro results.

Discussion

Under normal conditions, the main potential hazard of NIR light is caused by tissue heating as a result of light absorption by the skin. When an LED is used as the source of the NIR light in direct contact with the skin, an additional temperature increase occurs due to the heating effect of the semiconductor junction. The combined heating must be considered to assess the safety risk. An understanding of the separate contributions of these two effects will significantly help the design of new generation diffuse optical measurement systems.

The experiments by Ito et al [2], which employed laser light with irradiance levels and wavelength similar to the above experiments, created a temperature increase of 0.3 ± 0.2°C at the depth of 0.5 mm. This is similar to our findings during in-vitro experiments. In those experiments, they used optical fibers to insulate skin from junction temperature to measure the increase in temperature created only by NIR light absorption.

In in-vitro experiments, we studied the effect of the semiconductor junction. In some cases, the temperature increase due to semiconductor junction can be minimized by using thermal insulators such as double walled glass window. Alternatively, we demonstrated that using sinusoidal or pulsating waveforms results in less temperature rise compared to the case when DC waveform is used for a given amount of power, since the idle duration between two pulses creates a cooling effect. It has also been also observed that the degree of cooling can be controlled by varying the pulse duration, duty cycle, and irradiance, and using single or multiple wavelengths. This is due to the fact that effective (root mean square) dissipated power, which determines the temperature increase, depends on these parameters. As pulse duration, duty cycle and irradiance increase, the effective dissipated power increases causing the temperature to rise.

Further, the same argument applies to heating due to absorption of NIR light by the absorbing medium. During the experiments, although peak irradiance of the pulsating waveforms radiated by LED was between 25 and 50 mW/cm2, the average irradiance was between 12 and 25 mW/cm2. This is comparable to the irradiance of the NIR region of the sunlight, which is about 50 mW/cm2.

It should be noted that temperature increase can be controlled by pulse parameters, while factors such as signal to noise ratio and temporal resolution also depends on these parameters. Therefore, when designing continuous wave diffuse optical measurement systems trade-off between those factors should be considered.

When multiple wavelengths are considered, different wavelengths are emitted by separate semiconductor junctions encapsulated in a single LED package. The semiconductor junction for one wavelength functions independent of others. Thus, the heat generated by each of the LEDs is independent. It follows that, when two wavelengths are employed together, the expected effect is approximately the superposition of temperature increases observed when they are used separately. This is also supported by experimental results.

Temperature increases due to the semiconductor junction itself varies depending on various LED parameters such as the material used to manufacture the semiconductor junction and the geometry of the package. We used an AlGaAs LED that encapsulates 12 different diodes mounted with AlN heat sink pedestal on TO-5 stem and sealed with a flat glass can. Different temperature increases may be observed for other off-the-shelf LEDs. Therefore, one has to be careful about the quantitative results about the temperature increase due to the semiconductor junction documented in this study. However, qualitative interpretation of the results reported ab ove is valid for all LEDs.

In-vivo experiments demonstrate that the temperature increase on human arm and forehead is 1.5 ± 0.5°C greater than in-vitro results. The first reason for this difference is the temperature increase contributed by absorption of NIR light by tissue that was neglected in in-vitro experiments. In the in-vitro experiments, the only source of the heat was semiconductor junction.

In summary, the observed temperature increase in in-vivo experiments is contributed by heating of the semiconductor junction, heating due to absorption of NIR light and the effect of isolation by the cushioning material. The relative contribution of each heating can be estimated. Based on our experiments and the experiments of Ito et al. [2], temperature increase due to NIR light absorption is less than 0.5°C. The contribution of the cushioning material, on the other hand, is also less than 0.5°C. Thus, the temperature increase due to semiconductor junction is in the range of 1 to 10°C. Therefore, the major source of temperature increase is the semiconductor junction.

When NIR light emitted by LED attached to the skin is shone to the tissue, the main hazard is due to heating, which may cause burn injuries [13,14]. It is a well-known fact that the probability of cell death increases when the cell temperatures are sustained above 41°C [8]. This value is also the limit value for pulse oximetry applications [15]. Therefore, temperature should be kept under this temperature in order to avoid any burn injury.

Finally, body temperature varies according to the location of the measurement. For instance, the average skin temperature on the surface of the arm is 32°C whereas it is around 35°C on the forehead. Thus, the maximum temperature increase can be 9°C on the arm whereas it is 6°C in the forehead not to exceed the limit of 41°C. According to this, the maximum allowable temperature increase depends on the location of the application.

Conclusions

Standards and regulations are required for every medical technology to protect humans from harmful effects. As of this publication, there are no specific standards that ensure the safety of NIR LEDs used in diffuse optical measurement applications, especially when it is placed in direct contact with the skin. Although it is reasonable to use standard limits for laser sources for optical radiation hazards to the skin in the absence of standards specific to LEDs, there is still a need to establish standards for the conducted heat due to the LED. This is particularly important since improper usage of LEDs may cause burn injuries especially in long term monitoring of vulnerable populations such as newborns. In this study, we have assessed the combined effect of conducted and radiated heat in skin caused by NIR emitting LEDs during diffuse optical measurements. The main source of heating is found to be the temperature increase in the semiconductor junction. Elevations up to 10°C have been observed. The temperature contribution of NIR absorption by the skin is as low as 0.5°C since applied light power is comparable with the power of the NIR region of the sunlight.

When designing diffuse optical systems with LEDs in direct contact with skin, the temperature increase due to semiconductor junction can be minimized by using insulating layers. Alternatively, temperature increase can be controlled by adjusting irradiance, duty cycle and duration of NIR light pulses and frequency of the sinusoids. As discussed in the body of the paper, it is possible to reduce the overall temperature increase down to 1°C by shining light in the form of pulses or sinusoids instead of DC waveform.

Authors’ contributions

AB carried out the in-vivo and in-vitro experiments and drafted the manuscript. BO conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgements

We acknowledge, with thanks, Dr. Sandy Weininger of Food and Drug Administration (FDA) for helpful comments on the manuscript. This work has been sponsored in part by funds from the Defense Advanced Research Projects Agency (DARPA) Augmented Cognition Program and the Office of Naval Research (ONR), under agreement numbers N00014-02-1-0524 and N00014-01-1-0986.

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Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3439-44. Epub 2003 Mar 7.

Therapeutic photobiomodulation for methanol-induced retinal toxicity.

Eells JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT.

Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. jeells@mcw.edu

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/cm(2)), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

J Clin Laser Med Surg. 2003 Aug;21(4):231-5.

A preliminary investigation into light-modulated replication of nanobacteria and heart disease.

Sommer AP, Oron U, Pretorius AM, McKay DS, Ciftcioglu N, Mester AR, Kajander EO, Whelan HT.

Central Institute of Biomedical Engineering, University of Ulm, 89081 Ulm, Germany. samoan@gmx.net

OBJECTIVE: The purpose of this preliminary study is to evaluate the effect of various wavelengths of light on nanobacteria (NB). BACKGROUND DATA: NB and mitochondria use light for biological processes. NB have been described as multifunctional primordial nanovesicles with the potential to utilize solar energy for replication. NB produce slime, a process common to living bacteria. Slime release is an evolutionary important stress-dependent phenomenon increasing the survival chance of individual bacteria in a colony. In the cardiovascular system, stress-induced bacterial colony formation may lead to a deposition of plaque. METHODS: Cultured NB were irradiated with NASA-LEDs at different wavelengths of light: 670, 728 and 880 nm. Light intensities were about 500k Wm(-2), and energy density was 1 x 10(4) J m(-2). RESULTS: Monochromatic light clearly affected replication of NB. Maximum replication was achieved at 670 nm. CONCLUSIONS: The results indicate that suitable wavelengths of light could be instrumental in elevating the vitality level of NB, preventing the production of NB-mediated slime, and simultaneously increasing the vitality level of mitochondria. The finding could stimulate the design of cooperative therapy concepts that could reduce death caused by myocardial infarcts.

Aerosp Am. 2000 Apr;38(4):24-5.

From growing plants to killing tumors.

Flinn ED.

edflinn@pipeline.com

Abstract

NASA: A technique called photodynamic therapy, originally developed for commercial plant growth research on the Space Shuttle, has been used by surgeons in two successful operations for brain tumors. The device uses pin-head-size light emitting diodes (LEDs) that release long, cool, wavelengths of light which activate photosensitive antineoplastic drugs. The device is being adapted to non-space uses through a Small Business Innovation Research grant. The LEDs also are used to treat skin cancer, psoriasis, and rheumatoid arthritis. Research is being conducted regarding LED use in wound healing, tissue growth, and prevention of muscle and bone atrophy in astronauts.