Multiphoton Microscopy of Collagen Structure in Ex Vivo Human Skin Following Electrochemical Therapy.

OBJECTIVES: Injury to healthy dermis and the dermoepidermal junction initiates a robust healing process consisting of fibrous tissue overgrowth, collagen deposition, and scar formation. The conventional management of scars and other skin injuries has largely relied upon surgical soft tissue transfer to resurface and/or replace damaged and dysmorphic tissue with new skin. However, these strategies are invasive, expensive, and may further exacerbate integumentary injury. In this study, we examine the creation of in situ redox generated pH changes in fresh human skin. We believe this process of "electrochemical therapy" (ECT) leads to changes in collagen matrix structure. Our objective is to map local tissue pH landscapes and image changes in collagen structure of non-injured skin following ECT. STUDY DESIGN: Ex vivo human study involving ECT of human skin. METHODS: Remnant fresh ex vivo human facial skin from facelift operations was enveloped in saline-soaked gauze for a maximum of 2 hours prior to ECT and imaging. ECT was performed by inserting platinum-plated needle electrodes connected to a DC power supply. Voltage (4, 5, or 6 V) and time (3, 4, or 5 minutes) were varied systematically. High frequency ultrasound (25 MHz) was performed immediately after ECT on each sample. Treated samples were also imaged using multiphoton microscopy (MPM) with second harmonic generation (SHG) to specifically visualize collagen fibers in the dermis. The pH landscapes were mapped using indicator dyes in bisected specimens and the MPM images were compared with histologic findings. RESULTS: Above 4 V and 3 minutes, a profound reduction in dermal collagen SHG signal was observed at the anode. Although there was less blunting of SHG signal seen at the cathode, a decrease in the fluorescence of the dermoepidermal junction was observed. The pH application suggests ECT spatial selectivity and a direct relationship between voltage and application time. Ultrasound demonstrated gas formation between the anode and cathode, which is consistent with ECT's mechanism of action. Importantly, these electrochemical changes occurred without disrupting dermal and epidermal histologic architecture. CONCLUSION: ECT alters tissue pH leading to dermal collagen structural change. These results offer additional insight into the translational potential of ECT to locally remodel the soft-tissue matrix. Future directions aim to expand into a skin injury model to determine if similar collagen effects are observed in vivo. ECT is incredibly inexpensive (~$5) and may be a means to treat soft tissue injuries using simple needle-based devices and DC battery power supplies. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas.

Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.

A murine model of inflammation-induced cerebral microbleeds.

BACKGROUND: Cerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are pathological substrates of cerebral microbleeds. The existing CMH animal models are ß-amyloid-, hypoxic brain injury-, or hypertension-induced. Recent evidence shows that CMH develop independently of hypoxic brain injury, hypertension, or amyloid deposition and CMH are associated with normal aging, sepsis, and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent clinical studies show a link between systemic inflammation and CMH. Hence, we hypothesize that inflammation induces CMH development and thus, lipopolysaccharide (LPS)-induced CMH may be an appropriate model to study cerebral microbleeds. METHODS: Adult C57BL/6 mice were injected with LPS (3 or 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. At 2 or 7 days after the first injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (sub-acute) hemorrhages, respectively. Brain tissue ICAM-1, IgG, Iba1, and GFAP immunohistochemistry were used to examine endothelium activation, blood-brain barrier (BBB) disruption, and neuroinflammation. MRI and fluorescence microscopy were used to further confirm CMH development in this model. RESULTS: LPS-treated mice developed H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. No surface and negligible H&E-positive CMH were observed in saline-treated mice (n = 12). LPS (3 mg/kg; n = 10) produced significantly higher number, size, and area of H&E-positive CMH at 2 days. LPS (1 mg/kg; n = 9) produced robust development of PB-positive CMH at 7 days, with significantly higher number and area compared with saline (n = 9)-treated mice. CMH showed the highest distribution in the cerebellum followed by the sub-cortex and cortex. LPS-induced CMH were predominantly adjacent to cerebral capillaries, and CMH load was associated with indices of brain endothelium activation, BBB disruption, and neuroinflammation. Fluorescence microscopy confirmed the extravasation of red blood cells into the brain parenchyma, and MRI demonstrated the presence of cerebral microbleeds. CONCLUSIONS: LPS produced rapid and robust development of H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. The ease of development of both H&E- and PB-positive CMH makes the LPS-induced mouse model suitable to study inflammation-induced CMH.

In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin.

We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 µmoles/10(6) cells/h.

Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells.

Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.

Dynamics of nevus development implicate cell cooperation in the growth arrest of transformed melanocytes.

Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.

Melanocytes are pigment-producing cells found throughout the skin. Mutations that activate a gene called BRAF cause these cells to divide and produce melanocytic nevi, also known as "moles". These mutations are oncogenic, meaning they can cause cancer. Indeed, BRAF is the most commonly mutated gene in melanoma, a deadly skin cancer that arises from melanocytes. Yet, moles hardly ever progress to melanoma. A proposed explanation for this behavior is that, once activated, BRAF initiates a process called "oncogene-induced senescence" in each melanocyte. This process, likened to premature aging, is thought to be what causes cells in a mole to quit dividing. Although this hypothesis is widely accepted, it has proved difficult to test directly. To investigate this notion, Ruiz-Vega et al. studied mice with hundreds of moles created by the same BRAF mutation found in human moles. Analyzing the activity of genes in individual cells revealed that nevus melanocytes that have stopped growing are no more senescent than other skin cells, including non-mole melanocytes. Ruiz-Vega et al. then analyzed the sizes at which moles stopped growing, estimating the number of cells in each mole. The data were then compared with the results of a simulation and mathematical modeling. This revealed that any model based on the idea of cells independently shutting down after a number of random events could not reproduce the distribution of mole sizes that had been experimentally observed. On the other hand, models based on melanocytes acting collectively to shut down each other's growth fit the observed data much better. These findings suggest that moles do not stop growing as a direct result of the activation of BRAF, but because they sense and respond to their own overgrowth. The same kind of collective sensing is observed in normal tissues that maintain a constant size. Discovering that melanocytes do this not only sheds light on why moles stop growing, it could also help researchers devise new ways to prevent melanomas from forming.

Electrochemical degradation and saponification of porcine adipose tissue.

Body contouring achieved via subcutaneous adipose tissue reduction has notably advanced over the past century, from suction assisted lipectomy to techniques with reduced degrees of invasiveness including laser, radiofrequency, high frequency focused ultrasound, cryolipolysis, and drug-based injection approaches. These costly techniques have focused on damaging adipocyte cell membranes, hydrolyzing triglycerides (TGs), or inducing apoptosis. Here, we present a simple, low-cost technique, termed electrochemical lipolysis (ECLL). During ECLL, saline is injected into the subcutaneous adipose tissue, followed by insertion of needle electrodes and application of an electrical potential. Electrolysis of saline creates localized pH gradients that drive adipocyte death and saponification of TGs. Using pH mapping, various optical imaging techniques, and biochemical assays, we demonstrate the ability of ECLL to induce acid and base injury, cell death, and the saponification of triglycerides in ex vivo porcine adipose tissue. We define ECLL's potential role as a minimally-invasive, ultra-low-cost technology for reducing and contouring adipose tissue, and present ECLL as a potential new application of an emerging electrochemical redox based treatment modality.

The biophysical effects of localized electrochemical therapy on porcine skin.

BACKGROUND: Minimally-invasive methods to treat scars address a common pathway of altering collagen structure, leading to collagen remodeling. OBJECTIVE: In this study, we employed in situ redox chemistry to create focal pH gradients in skin, altering dermal collagen, in a process we refer to as electrochemical therapy (ECT). The effects of ECT to induce biochemical and structural changes in ex vivo porcine skin were examined. METHODS: During ECT, two platinum electrodes were inserted into fresh porcine skin, and following saline injection, an electrical potential was applied. pH mapping, high frequency ultrasonography, and two photon excitation microscopy and second harmonic generation (SHG) microscopy were used to evaluate treatment effects. Findings were correlated with histology. RESULTS: Following ECT, pH mapping depicted acid and base production at anode and cathode sites respectively, with increasing voltage and application time. Gas formation during ECT was observed with ultrasonography. Anode sites showed significant loss of SHG signal, while cathode sites showed disorganized collagen structure with fewer fibrils emitting an attainable signal. Histologically, collagen denaturation at both sites was confirmed. CONCLUSION: We demonstrated the production of in situ acid and base in skin occurring via ECT. The effects chemically and precisely alter collagen structure through denaturation, giving insight on the potential of ECT as a simple, low-cost, and minimally-invasive means to remodel skin and treat scars.

Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media.

We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 microm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-microm excitation in nonlinear optical microscopy.

Developing compact multiphoton systems using femtosecond fiber lasers.

We implement a fiber-delivered compact femtosecond fiber laser at 1,030-nm wavelength in multiphoton imaging. The laser pulse duration is 150 fs, the average power is 200 mW, and the repetition rate is 40 MHz. The laser measures 200 x 160 x 45 mm in size and its output is delivered through a photonic bandgap fiber. Intrinsic second-harmonic generation signal is excited from rat tail tendon and human skin samples. Two-photon excited fluorescence signal is obtained from human skin tissues stained with exogenous fluorophore. Our results show that femtosecond fiber lasers at 1030-nm wavelength have significant potential in developing compact, all-fiber-based, portable multiphoton systems and endoscopes.

In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam.

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Brain Endothelial Erythrophagocytosis and Hemoglobin Transmigration Across Brain Endothelium: Implications for Pathogenesis of Cerebral Microbleeds.

Peripheral endothelial cells are capable of erythrophagocytosis, but data on brain endothelial erythrophagocytosis are limited. We studied the relationship between brain endothelial erythrophagocytosis and cerebral microhemorrhage, the pathological substrate of MRI-demonstrable cerebral microbleeds. To demonstrate the erythrophagocytic capability of the brain endothelium, we studied the interactions between brain endothelial cells and red blood cells exposed to oxidative stress in vitro, and developed a new in vitro cerebral microbleeds model to study the subsequent passage of hemoglobin across the brain endothelial monolayer. Using multiple approaches, our results show marked brain endothelial erythrophagocytosis of red blood cells exposed to oxidative stress compared with control red blood cells in vitro. This brain endothelial erythrophagocytosis was accompanied by passage of hemoglobin across the brain endothelial monolayer with unaltered monolayer integrity. In vivo and confocal fluorescence microscopy studies confirmed the extravasation of RBC exposed to oxidative stress across brain endothelium. These findings, demonstrating erythrophagocytosis mediated by the brain endothelial monolayer and the subsequent passage of iron-rich hemoglobin in vitro and RBC in vivo, may have implications for elucidating mechanisms involved in the development of cerebral microbleeds that are not dependent on disruption of the microvasculature.

Limiting glioma development by photodynamic therapy-generated macrophage vaccine and allo-stimulation: an in vivo histological study in rats.

Immunotherapy of brain tumors involves the stimulation of an antitumor immune response. This type of therapy can be targeted specifically to tumor cells thus sparing surrounding normal brain. Due to the presence of the blood-brain barrier, the brain is relatively isolated from the systemic circulation and, as such, the initiation of significant immune responses is more limited than other types of cancers. The purpose of this study was to show that the efficacy of tumor primed antigen presenting macrophage (MaF98) vaccines can be increased by: (1) photodynamic therapy (PDT) of the priming tumor cells and (2) intracranial injection of allogeneic glioma cells directly into the tumor site. Experiments were conducted in an in vivo brain tumor development model using Fischer rats and F98 (syngeneic) and BT4C (allogeneic) glioma cells. The results showed that immunization with Ma (acting as antigen-presenting cells), primed with PDT-treated tumor cells (MaF98), significantly slowed but did not prevent the growth of F98-induced tumors in the brain. Complete suppression of tumor development was obtained via MaF98 inoculation combined with direct intracranial injection of allogeneic glioma cells. No deleterious effects were noted in any of the animals during the 14-day observation period.

Photochemical internalization enhanced macrophage delivered chemotherapy.

BACKGROUND: Macrophage (Ma) vectorization of chemotherapeutic drugs has the advantage for cancer therapy in that it can actively target and maintain an elevated concentration of drugs at the tumor site, preventing their spread into healthy tissue. A potential drawback is the inability to deliver a sufficient number of drug-loaded Ma into the tumor, thus limiting the amount of active drug delivered. This study examined the ability of photochemical internalization (PCI) to enhance the efficacy of released drug by Ma transport. METHODS: Tumor spheroids consisting of either F98 rat glioma cells or F98 cells combined with a subpopulation of empty or doxorubicin (DOX)-loaded mouse Ma (RAW264.7) were used as in vitro tumor models. PCI was performed with the photosensitizer AlPcS2a and laser irradiation at 670 nm. RESULTS: RAW264.7 Ma pulsed with DOX released the majority of the incorporated DOX within two hours of incubation. PCI significantly increased the toxicity of DOX either as pure drug or derived from monolayers of DOX-loaded Ma. Significant growth inhibition of hybrid spheroids was also observed with PCI even at subpopulations of DOX-loaded Ma as low as 11% of the total initial hybrid spheroid cell number. CONCLUSION: Results show that RAW264.7 Ma, pulsed with DOX, could effectively incorporate and release DOX. PCI significantly increased the ability of both free and Ma-released DOX to inhibit the growth of tumor spheroids in vitro. The growth of F98 + DOX loaded Ma hybrid spheroids were synergistically reduced by PCI, compared to either photodynamic therapy or released DOX acting alone.

In situ multiphoton optical tomography of hair follicles in mice.

We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations.

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment.

Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.

Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy.

We have developed a multiphoton microscopy (MPM) system using a 12-fs Ti:sapphire laser with adjustable dispersion precompensation in order to examine the impact of pulse duration on nonlinear optical signals. The efficiencies of two-photon-excited fluorescence (TPEF) and second harmonic generation (SHG) were studied for various pulse durations, measured at the sample, ranging from approximately 400 fs to sub-20 fs. Both TPEF and SHG increased proportionally to the inverse of the pulse duration for the entire tested range. Because of improved signal-to-noise ratio, sub-20-fs pulses were used to enhance MPM imaging depth by approximately 160%, compared to 120-fs pulses, in human skin.

Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source.

A 12-fs broadband (100-nm) source is used to combine multiphoton microscopy (MPM) and optical coherence tomography (OCT) in a single platform. An ultrafast Ti:sapphire laser simultaneously provides short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. Using 0.3-microm microspheres and a 63x, 0.95 numerical aperture objective, we demonstrate that MPM and OCT channels are coregistered with lateral resolution of approximately 0.5 microm and axial resolution of approximately 1.5 microm. Preliminary studies of a 3-D organotypic epithelial tissue model show that multiphoton images of fluorescence and second harmonic signals are derived from cellular and extracellular matrix structures, respectively, while OCT images are generated from scattering interfaces due to tissue variations in refractive index. The combined MPM/OCT microscope is capable of providing simultaneous functional and structural information from cells and extracellular matrix and is potentially a powerful tool for studying biological processes in thick tissues.

Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy.

A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.

Measuring Redox Status of Melanoma Cells.

Redox homeostasis plays multiple roles in essentially all aspects of cellular function, and hence, reliable methods for measuring cellular or tissue redox status are key elements in understanding the redox related signal pathways. However, in the free radical biology field, there are many controversies on the methods to measure reactive oxygen species. In this chapter we describe our experience in measuring superoxide, hydrogen peroxide, and a general redox status using redox-sensitive green fluorescence proteins (roGFPs) in human melanoma cells.