Optical penetration depth. 

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... photons must be absorbed by a molecular chromophore or photoacceptor. Light, at appropriate doses and wavelengths, is absorbed by chromophores such as porphyrins, flavins, and other light-absorbing entities within the mitochondria and cell membranes of cells. A growing body of evidence suggests that photobiomodulation mechanism is ascribed to the activation of mitochondrial respiratory chain components resulting in the initiation of a cascade of cellular reactions. It has been postulated that photoacceptors in the red to NIR region are the terminal enzyme of the respiratory chain cytochrome c oxidase with 2 copper elements. The first absorption peak is in the red spectrum and the second peak in the NIR range. Seventy-five years ago, Otto Warburg, a German biochemist, was given a Nobel prize for his ingenious work unmasking the enzyme responsible for the critical steps of cell respiration, especially cytochrome oxidase governing the last reaction in this process. Two chemical quirks are exploited: carbon monoxide (CO) that can block respiration by binding to cytochrome oxidase in place of oxygen, and a flash of light that can dis- place it, allowing oxygen to bind again. Nowadays, it has been reported that cells often use CO and, to an even greater extent, nitric oxide (NO) binding to cytochrome oxidase to hinder cell respiration. 2 Mitochondria harbor an enzyme that synthesizes NO. So why would cells go out of their way to produce NO right next to the respiratory enzymes? Evolution crafted cytochrome oxidase to bind not only to oxygen but also to NO. One effect of slowing respiration in some locations is to divert oxygen elsewhere in cells and tissues, preventing oxygen sinking to dangerously low levels. Fireflies use a similar strategy to flash light (see section “Pulsing and Continuous Modes”). Respiration is about generating energy but also about generating feedback that allows a cell to monitor and respond to its environment. When respiration is blocked, chemical signals in the form of free radicals or reactive oxygen species are generated. Free radicals had a bad reputation, but now they can be considered signals. The activity of many proteins, or transcription factors, depends, at least in part, on free radicals. 3 These include many proteins such as those involved in the p53 cell-signaling pathway. Further, to bring free radical leak under control, there is a cross-talk, known as retrograde re- sponse, between the mitochondria and genes in the nucleus for which we are just beginning to explore the mechanism at play. 4,5 If we can better modulate this signaling, we might be able to influence the life or death of cells in many pathologies as it is more and more demonstrated in its antiaging effects on collagen metabolism. A recent discovery has revealed that NO eliminates the LLLT-induced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. 6 Cells use NO to regulate respiratory chain processes, resulting in a change in cell metabolism. In turn, in LED-exposed cells like fibroblasts increased ATP production, modulation of reactive oxygen species (such as singlet oxygen species), reduction and prevention of apoptosis, stimu- lation of angiogenesis, increase of blood flow, and induction of transcription factors are observed. These signal transduction pathways lead to increased cell proliferation and migra- tion (particularly by fibroblasts), modulation in levels of cytokines (eg, interleukins, tumor necrosis factor- ␣ ), growth factors and inflammatory mediators, and increases in anti- apoptotic proteins. 7 The photodissociation theory incriminating NO as one of the main players suggests that during an inflammatory process, for example, cytochrome c oxidase is clogged up by NO. LED therapy would photodissociate NO or bump it to the extracellular matrix for oxygen to bind back again to cytochrome c oxidase and resume respiratory chain activity. Un- derstanding the mechanisms of cutaneous LED-induced specific cell-signaling pathway modulation will assist in the future design of novel devices with tailored parameters even for the treatment of degenerative pathologies of the skin. In LED, the question is no longer whether it has biological effects but rather what the optimal light parameters are for different uses. Biological effects depend on the parameters of the irradiation such as wavelength, dose (fluence), intensity (power density or irradiance), irradiation time (treatment time), continuous wave or pulsed mode, and for the latter, pulsing patterns. In addition, clinically, such factors as the frequency, intervals between treatments and total number of treatments are to be considered. The prerequisites for effective LED clinical response are discussed hereafter. Light is measured in wavelengths and is expressed in units of nanometers (nm). Different wavelengths have different chromophores and can have various effects on tissue (Fig. 6). Wavelengths are often referred to using their associated color and include blue (400-470 nm), green (470-550 nm), red (630-700 nm) and NIR (700-1200) lights. In general, the longer the wavelength, the deeper the penetration into tissues. 8-10 Depending on the type of tissue, the penetration depth is less than 1 mm at 400 nm, 0.5 to 2 mm at 514 nm, 1 to 6 mm at 630 nm, and maximal at 700 to 900 nm. 10 The various cell and tissue types in the body have their own unique light absorption characteristics, each absorbing light at specific wavelengths. For best effects, the wavelength used should allow for optimal penetration of light in the targeted cells or tissue. Red light can be used successfully for deeper localized target (eg, sebaceous glands), and blue light may be useful for the treatment of skin conditions located within the epidermis in photodynamic therapy (PDT) (eg, actinic keratoses). To reach as many fibroblasts as possible, which is often the aim of LED therapy, a deeply penetrating wavelength is desirable. At 660 nm, for instance, light can achieve such a goal reaching a depth of 2.3 mm in the dermis, therefore covering fibroblasts up to the reticular dermis. The wavelength used should also be within the absorption spectrum of the chromophore or photoacceptor molecule and will often determine for which applications LEDs will be used. Because cytochrome c oxidase is the most likely chromophore in LLLT, 2 absorption peaks are considered in the red ( ϳ 660 nm) and NIR ( ϳ 850 nm) spectra. 6 Two major wavelength boundaries exist for LED applications: at wavelengths Ͻ 600 nm, blood hemoglobin (Hb) is a major obstacle to photon absorption because blood vessels are not compressed during treatment. Futhermore, at wavelengths Ͼ 1000 nm, water is also absorbing many photons, reducing their availability for specific chromophores located, for instance, in dermal fibroblasts. Be- tween these 2 boundaries, there is a valley of LED possible applications (see Fig. 7). The Arndt-Schulz law states that there is only a narrow win- dow of opportunity where you can actually activate a cellular response using precise sets of parameters, i.e. the fluence or dose (see Fig. 8). The challenge remains to find the appropriate combinations of LED treatment time and irradiance to achieve optimal target tissue effects. Fluence or dose is, indi- cated in joules per cm 2 (J/cm 2 ). The law of reciprocity states that the dose is equal to the intensity ϫ time. Therefore, the same exposure should result from reducing duration and increasing light intensity, and vice versa. Reciprocity is as- sumed and routinely used in LED and LLLT experiments. However, the scientific evidence supporting reciprocity in LED therapy is unclear. 11 Dose reciprocity effects were examined in a wound healing model and showed that varying irradiance and exposure time to achieve a constant specified energy density affects LED therapy outcomes. 12 In practice, if light intensity (irradiance) is lower than the physiological threshold value for a given target, it does not produce photostimulatory effects even when irradiation time is extended. Moreover, photoinhibi- tory effects may occur at higher fluences. In Fig. 9, different light delivery patterns are shown. Interestingly, they are all of the same fluence but over time, the energy of photons does not reach the biological targets in the same way. This may alter the LED biological response significantly. The importance of pulsing will be discussed in the next section. Certainly a minimal exposure time per treatment is neces- sary—in the order of several minutes rather than only a few seconds—to allow activation of the cell machinery; other- wise, tissue response is evanescent and no clinical outcome is expected. The ideal treatment time has to be tailored according to the skin condition or degree of inflammation present at the time of treatment. Both pulsed wave and continuous wave (CW) modes are available in LED devices, which add to the medical applica- bility. The influence of CW versus pulsing mode, as well as precise pulsing parameters (eg, duration, interval, pulse per train, pulse train interval), on cellular response has not been fully studied. To date, comparative studies have shown con- flicting results. 13 In our own experience, sequentially pulsed optical energy (proprietary pulsing mode with repeated se- quences of short pulse trains followed by longer intervals) has been shown to stimulate more collagen production than CW mode. 14 Under certain conditions, ultra-short pulses can travel deeper into tissues than CW radiation. 15,16 This is because the first part of a powerful pulse may contain enough photons to take all chromophore molecules in the upper tissue layer to excited states, thus literally opening a road for itself into tissue. Moreover, too long a pulse may produce cellular ex- haustion whereas too short a pulse may deliver insufficient energy for a biologic effect to ...

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... is measured in wavelengths and is expressed in units of nanometers (nm). Different wavelengths have different chro- mophores and can have various effects on tissue (Fig. 6). Wavelengths are often referred to using their associated color and include blue (400-470 nm), green (470-550 nm), red (630-700 nm) and NIR (700-1200) lights. In general, the longer the wavelength, the deeper the penetration into tis- sues. 8-10 Depending on the type of tissue, the penetration depth is less than 1 mm at 400 nm, 0.5 to ...