Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.     

Energy-linked regulation of glucose and pyruvate oxidation in isolated perfused rat heart. Role of pyruvate dehydrogenase.

1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart. 2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation. The time course and percentage changes of the inhibition of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused heart. 3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, alpha-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD+ ratio. In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking. The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD+. The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.     

A reflectance spectrophotometer-surface fluorometer suitable for monitoring changes in hemoprotein spectra and fluorescence of flavins and nicotinamide nucleotides in intact tissues

A spectrophotometer-fluorometer was constructed for simultaneous measurement of reflectance spectrum changes and surface fluorescence of intact tissues and organs. The apparatus employs monochromators for dual-wavelength spectrophotometry and optical filters for fluorometry. Three light beams are time-shared by a novel type of modulator to hit the same area of the tissue to be measured. The analog circuitry was controlled by a digital logic circuitry clocked by an optically derived frequency based on the angular velocity of the rotating light modulator and produced a maximum of three sets of differences in the parameters measured. The optical design has the advantage of allowing the use of monochromators rather than filters in a multichannel apparatus. The electronics accept a wide range of light modulation frequencies without difficulties in the phasing of the detector gates, so that signal sampling can be optimized for the recording of fast and slow phenomena. The apparatus was used successfully for the simultaneous monitoring of the mitochondrial redox state and tissue oxygenation in isolated perfused rat hearts.     

Energy-linked regulation of glucose and pyruvate oxidation in isolated perfused rat heart. Role of pyruvate dehydrogenase

1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart.2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation.The time course and percentage changes of the inhibition of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused heart.3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, α-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD⁺ ratio.In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking.The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD⁺.The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.     

High-resolution visualization of oxygen distribution in the liver in vivo

Microcirculatory failure after stress events results in mismatch in oxygen supply and demand. Determination of tissue oxygen distribution in vivo may help elucidate mechanisms of injury, but present methods have limited resolution. Male Sprague-Dawley rats were anesthetized, prepared for intravital microscopy, and received intravenously the oxygen-sensitive fluorescent dye Tris(1,10-phenanthroline)ruthenium(II) chloride hydrate [Ru(phen)3(2+)]. An impaired hepatic oxygen distribution was induced by either phenylephrine or hemorrhage. Intensity of Ru(phen)3(2+) fluorescence was compared with NADH autofluorescence indicating changes in the mitochondrial redox potential. Ethanol was injected to affect the NADH-to-NAD+ ratio without altering the P(O2). Infusion of Ru(phen)3(2+) resulted in a heterogeneous fluorescence under baseline conditions reflecting the physiological acinar P(O2) distribution. A decrease in oxygen supply due to phenylephrine or hemorrhage was paralleled by an increase in Ru(phen)3(2+) and NADH fluorescence reflecting an impaired mitochondrial redox state. Ethanol did not alter Ru(phen)3(2+) fluorescence but increased NADH fluorescence indicating independence of P(O2) and redox state imaging. Intravenous administration of Ru(phen)3(2+) for intravital videomicroscopy represents a new method to visualize the hepatic tissue P(O2). Combined with NADH autofluorescence, it provides additional information regarding the tissue redox state.     

(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.

In vivo analysis of the metabolic state of tissue by means of reduced nicotinamide adenine dinucleotide (NADH) fluorimetry is disturbed by tissue movements and by hemodynamic and oximetric effects. These factors cause changes in the absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to compensate measured NADH fluorescence intensities for these effects. In this paper we show on theoretical grounds that the ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compensatory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflectance images, using a CCD camera with a biprism configuration. The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood-perfused myocardium.     

Imaging mitochondrial redox potential and its possible link to tumor metastatic potential

Cellular redox states can regulate cell metabolism, growth, differentiation, motility, apoptosis, signaling pathways, and gene expressions etc. A growing body of literature suggest the importance of redox status for cancer progression. While most studies on redox state were done on cells and tissue lysates, it is important to understand the role of redox state in a tissue in vivo/ex vivo and image its heterogeneity. Redox scanning is a clinical-translatable method for imaging tissue mitochondrial redox potential with a submillimeter resolution. Redox scanning data in mouse models of human cancers demonstrate a correlation between mitochondrial redox state and tumor metastatic potential. I will discuss the significance of this correlation and possible directions for future research.     

Effects of tissue absorbance on NAD(P)H and Indo-1 fluorescence from perfused rabbit hearts

The effects of tissue optical absorbance on intracellular NAD(P)H and Indo-1 fluorescence emission have been evaluated in the perfused rabbit heart. These results demonstrate that the tissue optical absorbance significantly modifies the emission characteristics of these fluorophores. This tissue 'inner filter' effect, observed with both probes, changed as a function of tissue oxygenation and redox state in a wavelength-dependent manner. Pathlength calculations from these results indicate that this inner filter effect could occur with a mean pathlength of 310 microns due to the extremely high extinction coefficient of heart tissue. It is concluded that tissue optical absorbance significantly affects the fluorescent emission characteristics of both intrinsic and extrinsic probes in the intact heart, under a variety of conditions. Several potential methods of correcting for these tissue inner filter effects are discussed.     

Correlations between redox-state potential changes in different tissues and the heart frequency in vivo

Redosis evoked in different tissues by methylene-blue or menadione (oxidants), resulted in an increase in heart frequency, while oxidosis evoked by thiamine or cysteine (reductants) diminished the frequency. In isolated organ tissues where compensatory redox feed back overshoots are rarely to develop, owing to the low redox buffer capacity and lack of the influence of nervous and humoral factors, the heart frequency decreased in response to direct oxidosis induced by the application of oxidants, and increased following reductant application; this suggested an environmental type redox regulatory influence of the agents rather than specific action of the agents. This environmental type effect can result from direct action on isolated organs, or from direct and indirect actions in vivo. An increased redox-state potential resulted in decreased heart frequency and inversely. In a pathological situation provoked by complete strangulation of aortae, a significant oxidosis developed in parallel with a decrease in heart frequency. On increasing the redox buffer capacity by application of methylene-blue (oxidant), or thiamine (reductant) both the redox and the resulting heart frequency changes could readily be counteracted. When cigarette smoke was pumped through an intratracheal tube, a significant redosis developed in the heart ventricle in parallel with an increased heart frequency. These data show that regardless of the origin of redox-state potential changes in tissues, a shift to oxidosis decreases and a shift to redosis increases the heart frequency.     

Opposite effects of methylene blue and ascorbate on lipid peroxidation in muscles. Correlation with the redox state. I. Experiments on satisfied frogs

Homogenates of heart, stomach and rectus abdominis muscles of the frog have shown different degrees of malondialdehyde (MDA) formation. MDA content was highest in heart, and lowest in stomach musculature. The resultant tissue redox-state potential (RSP) and redox potential (E'0) in homogenates determined potentiometrically also showed differences with opposite signs in relation to MDA levels. An electron acceptor, methylene blue (MB), decreased but an electron donor, ascorbate (Asc), increased the MDA level in each of the muscles. These effects were dependent upon the concentration of MB and Asc and proportional to the control MDA content in each muscle. Thus an inverse interdependence between MDA level and redox state existed even when a positive change in redox potentials was induced by MB, and also when a negative change was induced by Asc. Since there was a close negative correlation between the changes of MDA concentration and redox potential in the homogenates, it is strongly suggested that the changes of redox state in muscle are implicated in the processes leading to lipid peroxidation (LP).     

Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.     

Effects of perfusion on the viscoelastic characteristics of liver

Accurate characterization of soft tissue material properties is required to enable new computer-aided medical technologies such as surgical training and planning. The current means of acquiring these properties in the in vivo and ex vivo states is fraught with problems, including limited accessibility and unknown boundary conditions in the former, and unnatural behavior in the latter. This paper presents a new testing method where a whole porcine liver is perfused under physiologic conditions and tested in an ex vivo setting. To characterize the effects of perfusion on the viscoelastic response of liver, indentation devices made force and displacement measurements across four conditions: in vivo, ex vivo perfused, ex vivo post perfused, and in vitro on an excised section. One device imposed cyclic perturbations on the liver's surface, inducing nominal strains up to 5% at frequencies from 0.1 to 200 Hz. The other device measured 300 s of the organ's creep response to applied loads, inducing nominal surface stresses of 6.9-34.7 kPa and nominal strains up to 50%. Results from empirical models indicate that the viscoelastic properties of liver change with perfusion and that two time constants on the order of 1.86 and 51.3s can characterize the liver under large strains typical of surgical manipulation across time periods up to 300 s. Unperfused conditions were stiffer and more viscous than the in vivo state, resulting in permanent strain deformation with repeated indentations. Conversely, the responses from the ex vivo perfusion condition closely approximated the in vivo response.     

Use of noninvasive fluorometry and spectrophotometry to study epithelial metabolism and transport

Various examples illustrating the use of spectrophotometry and fluorometry in epithelia are presented. The first example uses the redox level of cytochrome aa3, measured spectrophotometrically as an index of tissue anoxia in cortical tubules and slices from the rabbit kidney. In the second example the redox level is used to measure the kinetics of aerobic energy production during transition to anoxia in the midgut of the tobacco hornworm. In the third application, the redox level of mitochondrial NADH is measured fluorometrically in a cortical tubule suspension from the rabbit kidney. Inhibition of active transport work causes reduction of NAD whereas increased work elicits oxidation of NAD, both occurring as expected from mitochondrial transitions to a lesser or more active state, respectively. Another use of NADH fluorescence is the determination of the relative effectiveness of metabolic substrates to deliver reducing equivalents to the respiratory chain in a particular tissue. Redox changes in mitochondrial NAD may be used to distinguish between primary metabolic and primary transport effects of hormones, drugs, and changes in the state of the organism. Finally, examples are provided of the use of an intracellular pH-sensitive dye and an extracellular calcium-sensitive dye in kidney tubules.     

Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.     

Alterations of mitochondrial protein metabolism in liver, brown adipose tissue and skeletal muscle. During cold-acclimation.

Incorporation of L-[U-14C] leucine into liver, brown adipose tissue and skeletal muscle mitochondrial proteins was determined in vivo and in vitro during cold-acclimation. Major alterations in mitochondrial protein metabolism were observed in brown adipose tissue and skeletal muscle but not in liver. Immediate cold-exposure is accompanied by an inhibition of the in vivo incorporation of L-[U-14C] leucine into mitochondrial proteins of all tissues. However, during cold-acclimation the incorporation of leucine increases markedly in brown adipose tissue, continues to decrease in skeletal muscle, nut does not change appreciably in the liver. Because increased incorporation of L-[U-14C]-leucine into brown adipose tissue mitochondrial proteins was observed both in vivo and in vitro, it can be concluded that the mitochondrial protein-synthesizing system of this tissue is directly affected by the acclimation process. The observed changes in mitochondrial protein metabolism of brown adipose tissue and skeletal muscle might be responsible for the development of several morphological and biochemical alterations that characterize the establishment in these tissues of the cold-acclimated state.     

Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTracker™ Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.     

Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.     

Alterations of mitochondrial protein metabolism in liver, brown adipose tissue and skeletal muscle during cold-acclimation

Incorporation of -[U-¹⁴C] leucine into liver, brown adipose tissue and skeletal muscle mitochondrial proteins was determined in vivo and in vitro during cold-acclimation. Major alterations in mitochondrail protein metabolism were observed in brown adipose tissue and skeletal muscle but not in liver. Immediate cold-exposure is accompanied by an inhibition of the in vivo incorporation of -[U-¹⁴C] leucine into mitochondrial proteins of all tissues. However, during cold-acclimation the incorporation of leucine increases markedly in brown adipose tissue, continues to decrease in skeletal muscle, nut does not change appreciably in the liver. Because increased incorporation of -[U-¹⁴C] leucine into brown adipose tissue mitochondrial proteins was observed both in vivo and in vitrom it can be concluded that the mitochondrial protein-synthesizing system of this tissue is directly affected by the acclimation process. The observed changes in mitochondrial protein metabolism of brown adipose tissue and skeletal muscle might be responsible for the development of several morphological and biochemical alterations that characterize the establishment in these tissues of the cold-acclimated state.     

In vivo time domain optical imaging of renal ischemia-reperfusion injury: discrimination based on fluorescence lifetime

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.     

Label‐free imaging of redox status and collagen deposition showing metabolic differences in the heart

The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth, there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. In this study, we have employed 2‐photon excitation fluorescence microscopy to define the relationship between two critical metabolic cofactors nicotinamide adenine dinucleotide(P)H and flavin adenine dinucleotide, effectively utilizing a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two‐photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Two‐photon excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualize cardiac and other muscle pathologies.