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BACKGROUND: Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to accelerate diabetic wound healing in preclinical and clinical studies. Mitochondrial dysfunction and oxidative stress play key roles in impaired diabetic wound healing, and the effect of PBM on the metabolic state of diabetic wounds remains to be elucidated. METHODS: In this study, a custom-designed in vivo fluorescence imaging technique was used to quantitatively assess the effect of FR-PBM on the mitochondrial bioenergetics of diabetic wounds. The intrinsic fluorescence of two mitochondrial co-enzymes, nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), was monitored to quantify the redox ratio (RR) (NADH/FAD) of wounds over time. RESULTS: Using an excisional model of wound healing, we demonstrated that 670 nm (FR) PBM improved mitochondrial bioenergetics and stimulated the rate of wound healing in diabetic db/db mice. Wound closure and the RR of diabetic wounds in response to 670 nm PBM (4.5 J/cm(2), 60 mW/cm(2) for 90 s per day, 5 days/week) were compared to the sham-treated group. At day 9 of post-wounding, we observed a 43% decrease in the wound area and a 75% increase in RR in FR-treated diabetic mice compared to sham-treated diabetic mice. CONCLUSIONS: We conclude that the increase in mitochondrial RR and the related decrease in oxidative stress may be an important factor in FR-PBM mediated acceleration of wound healing in diabetic mice.

Optical cryo-imaging captured the metabolic redox state (NADH/FAD) of rat kidneys, quantitatively, post-uninephrectomy. The result shows a more oxidized mitochondrial bioenergetics of the remaining kidney due to increased oxidative stress.

Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA–LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm−1), proteins (1662 and 1608 cm−1) and fatty acids (2854, 2923, 2956 and 3012 cm−1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.

Diabetes is known to cause delayed wound healing, and extremity diabetic ulcers may end with lower limb amputations and mortalities. We examine the role of mitochondrial metabolism in the diabetic mice wound healing. On-line label-free optical images of metabolic indices (including NADH and FAD) were captured in the in vivo wounds over 5 days. The results suggest that diabetes alters mitochondrial redox state in wounds (%78 oxidized state) over time due to higher oxidative stress.

Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion (O[Formula: see text] production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O[Formula: see text] production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O[Formula: see text] production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O[Formula: see text]. After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O[Formula: see text] fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O[Formula: see text] production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O[Formula: see text] production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O[Formula: see text] production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O[Formula: see text] production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.

Hereditary Hemorrhagic Telangiectasia-1 (HHT-1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT-1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild-type and Eng +/- mice. Kidneys and eyes from wild-type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/- mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis.

Objective: In this study, the metabolic state of the heart tissue is studied in a rodent model of ischemia and reperfusion (IR) in rats exposed to irradiation injury using a cryofluorescence imaging technique. Mitochondrial metabolic state is evaluated by autofluorescence of mitochondrial metabolic coenzymes NADH and FAD. The redox ratio (NADH/FAD) is used as a biochemical/metabolic marker of oxidative stress, before, during and after IR. Materials and methods: Hearts were extracted from non-irradiated (control) and irradiated rats (Irr) given 15 Gy whole thorax irradiation rats (WTI). After 35 days, before the onset of radiation pneumonitis, these two groups of hearts were subjected to one of three treatments; Time control (TC; hearts perfused for the duration of the protocol without ischemia or IR), 25 minutes ischemia with no reperfusion and 25 minutes ischemia followed by 60 minutes reperfusion (IR). Hearts were removed from the Langendorff perfusion system and immediately snap frozen in liquid N2 to preserve the metabolic state after injury; 3-dimensional (3D) cryo-fluorescent imager was used to obtain in fixed time NADH and FAD fluorescence images and their distribution across the entire ventricles. In this study, a 30-μm axial resolution was used resulting in 550 cross-section images per heart. The 3D images of the redox ratio and their respective histograms were calculated in the six groups of hearts. Results: We compared the mean values of the redox ratio in each group, which demonstrate a reduced mitochondrial redox state in both irradiated and non-irradiated ischemic hearts and an oxidized mitochondrial redox state for both irradiated and non-irradiated ischemia-reperfusion hearts compared to control hearts. For non-irradiated hearts, ischemia and IR injuries resulted respectively in 61% increase and 54% decrease in redox ratio when compared with TC. For irradiated hearts, ischemia and IR injuries resulted respectively in 90% increase and 50% decrease in redox ratio when compared to TC. Conclusion: The cryoimager is able to quantify ischemia and IR injuries. Cryoimaging is a unique 3D imaging tools that provides quantitative measurement of tissue metabolic state. Hearts that underwent irradiation indicates a more oxidized metabolic state in the tissue. This change persists across all three treatments.

Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time-lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 ( Nox4 ) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4 , resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS Nox4−/− ) rats. SS Nox4−/− rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS Nox4−/− rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67 phox , and p22 phox were found to be significantly lower ( P Nox4−/− compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS Nox4−/− rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS Nox4−/− rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67phoxsubunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67phoxwas rendered functionally inactive by zinc finger nuclease mutation of the gene (SSp67 phox-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly ( P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SSp67 phox-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios ( P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SSp67 phox-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SSp67 phoxrats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2in the medulla of SS rats. We conclude that the p67phoxsubunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

The objective of this work is to assess oxidative stress levels in salt-sensitive hypertension animal model using 3D optical cryoimager to image mitochondrial redox ratio. We studied Dahl salt-induced (SS) rats, and compared the results with a consomic SS rat strain (SSBN13). The SSBN13 strain was developed by the introgression of chromosome from the Brown Norway (BN) rat into the salt-sensitive (SS) genetic background and exhibits significant protection from salt induced hypertension 1 . These two groups were fed on a high salt diet of 8.0% NaCl for one week. Mitochondrial redox ratio (NADH/FAD=NADH RR), was used as a quantitative marker of the oxidative stress in kidney tissue. Maximum intensity projected images and their corresponding histograms in each group were acquired from each kidney group. The result showed a 49% decrease in mitochondrial redox ratio of SS compared to SSBN13 translated to an increase in the level of oxidative stress of the tissue. Therefore, the results quantify oxidative stress levels and its effect on mitochondrial redox in salt sensitive hypertension.

Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell and modulating the injuries. However, the generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. This study present a novel protocol to quantify the dynamic of the mitochondrial superoxide as a precursor of reactive oxygen species. To regulate the mitochondrial superoxide level, metabolic perturbation was induced by administration of potassium cyanide (KCN). The presented method was able to monitor and measure the superoxide production rate over time. Our results demonstrated that the metabolic inhibitor, potassium cyanide (KCN) induced a significant increase in the rate of superoxide production in mitochondria of fetal pulmonary artery endothelial cells (FPAEC). Presented method sets the stage to study different ROS mediated injuries in vitro.

This study investigates the effect of hypertension on mitochondrial superoxide production using time lapse microscopy.

Deterioration in mitochondrial function leads to hepatic ischemia and reperfusion injury (IRI) in liver surgery and transplantation. 3D optical cryoimaging was used to measure the levels of mitochondrial coenzymes NADH and FAD, and their redox ratio (NADH/FAD) gave a quantitative marker for hepatocyte oxidative stress during IRI. Using a rat model, five groups were compared: control, ischemia for 60 or 90 minutes (Isc60, Isc90), ischemia for 60 or 90 minutes followed by reperfusion of 24 hours (IRI60, IRI90). Ischemia alone did not cause a significant increase in the redox ratio; however, the redox ratio in both IRI60 and IRI90 groups was significantly decreased by 29% and 71%, respectively. A significant correlation was observed between the redox ratio and other markers of injury such as serum aminotransferase levels and the tissue ATP level. The mitochondrial redox state can be successfully measured using optical cryoimaging as a quantitative marker of hepatic IR injury.

This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% ( ± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys ( P = 0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) ( P = 0.045) and 14% (±0.5) ( P = 0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels.

Oxidative stress (OS), which increases during retinal degenerative disorders, contributes to photoreceptor cell loss. The objective of this study was to investigate the changes in the metabolic state of the eye tissue in rodent models of retinitis pigmentosa by using the cryofluorescence imaging technique. The mitochondrial metabolic coenzymes NADH and FADH 2 are autofluorescent and can be monitored without exogenous labels using optical techniques. The NADH redox ratio (RR), which is the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), was used as a quantitative diagnostic marker. The NADH RR was examined in an established rodent model of retinitis pigmentosa (RP), the P23H rat, and compared to that of control Sprague-Dawley (SD) rats and P23H NIR treated rats. Our results demonstrated 24% decrease in the mean NADH RR of the eyes from P23H transgenic rats compared to normal rats and 20% increase in the mean NADH RR of the eyes from the P23H NIR treated rats compared to P23H non-treated rats.

Oxidative stress (OS), which increases during diabetes, exacerbates the development and progression of diabetes complications including renal vascular and proximal tubule cell dysfunction. The objective of this study was to investigate the changes in the metabolic state of the tissue in diabetic mice kidneys using fluorescence imaging. Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH-2 (Flavin Adenine Dinucleotide) are autofluorescent and can be monitored without exogenous labels by optical techniques. The ratio of the fluorescence intensity of these fluorophores, (NADH/FAD), called the NADH redox ratio (RR), is a marker of metabolic state of a tissue. We examined mitochondrial redox states of kidneys from diabetic mice, Akita/+ and its control wild type (WT) for a group of 8- and 12-week-old mice. Average intensity and histogram of maximum projected images of FAD, NADH, and NADH RR were calculated for each kidney. Our results indicated a 17% decrease in the mean NADH RR of the kidney from 8-week-old mice compared with WT mice and, a 30% decrease in the mean NADH RR of kidney from12-week-old mice compared with WT mice. These results indicated an increase in OS in diabetic animals and its progression over time. Thus, NADH RR can be used as a hallmark of OS in diabetic kidney allowing temporal identification of oxidative state.

Chronic hyperglycemia during diabetes leads to increased production of reactive oxygen species (ROS) and increased oxidative stress (OS). Here we investigated whether changes in the metabolic state can be used as a marker of OS progression in kidneys. We examined redox states of kidneys from diabetic mice, Akita(/+) and Akita(/+);TSP1(-/-) mice (Akita mice lacking thrombospondin-1, TSP1) with increasing duration of diabetes. OS as measured by mitochondrial redox ratio (NADH/FAD) was detectable shortly after the onset of diabetes and further increased with the duration of diabetes. Thus, cryo fluorescence redox imaging was used as a quantitative marker of OS progression in kidneys from diabetic mice and demonstrated that alterations in the oxidative state of kidneys occur during the early stages of diabetes.

The objective of this study was to demonstrate the utility of optical cryoimaging and fluorometry to evaluate tissue redox state of the mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide) in intact rat lungs. The ratio (NADH/FAD), referred to as mitochondrial redox ratio (RR), is a measure of the lung tissue mitochondrial redox state. Isolated rat lungs were connected to a ventilation-perfused system. Surface NADH and FAD fluorescence signals were acquired before and after lung perfusion in the absence (control perfusate) or presence of potassium cyanide (KCN, complex IV inhibitor) to reduce the mitochondrial respiratory chain (state 5 respiration). Another group of lungs were perfused with control perfusate or KCN-containing perfusate as above, after which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. Results demonstrate that lung treatment with KCN increased lung surface NADH signal by 22%, decreased FAD signal by 8%, and as result increased RR by 31% as compared to control perfusate (baseline) values. Cryoimaging results also show that KCN increased mean lung tissue NADH signal by 37%, decreased mean FAD signal by 4%, and increased mean RR by 47%. These results demonstrate the utility of these optical techniques to evaluate the effect of pulmonary oxidative stress on tissue mitochondrial redox state in intact lungs.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury (ALI) in adults and bronchopulmonary dysplasia (BPD) in premature infants. Bacterial infection and oxygen toxicity, which result in pulmonary vascular endothelial injury, contribute to impaired vascular growth and alveolar simplification seen in the lungs of premature infants with BPD. Hyperoxia induces ALI, reduces cell proliferation, causes DNA damage and promotes cell death by causing mitochondrial dysfunction. The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto-fluorescent mitochondrial metabolic coenzymes, NADH and FAD in four different groups of rats. The ratio of these fluorescence signals (NADH/FAD), referred to as NADH redox ratio (NADH RR) has been used as an indicator of tissue metabolism in injuries. Here, we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide (LPS) exposure in neonatal rat lungs. We examined the tissue redox states of lungs from four groups of rat pups: normoxic (21% O2 ) pups, hyperoxic (90% O2 ) pups, pups treated with LPS (normoxic + LPS), and pups treated with LPS and hyperoxia (hyperoxic + LPS). Our results show that hyperoxia oxidized the respiratory chain as reflected by a ~ 31% decrease in lung tissue NADH RR as compared to that for normoxic lungs. LPS treatment alone or with hyperoxia had no significant effect on lung tissue NADH RR as compared to that for normoxic or hyperoxic lungs, respectively. Thus, NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions: hyperoxia and LPS exposure.

Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11 ± 0.03 in the SD normal and 0.841 ± 0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypox- emia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low- temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). (DOI: 10