The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease.
The liver is important in the biotransformation of various drugs, where hepatic transporters facilitate uptake and excretion. Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery, and the developing oxidative stress can lead to graft failure. We used intravital multiphoton tomography, with fluorescence lifetime imaging, to characterize metabolic damage associated with hepatic I/R injury and to model the distribution of fluorescein as a measure of liver function. In addition to measuring a significant increase in serum alanine transaminase levels, characteristic of hepatic I/R injury, a decrease in the averaged weighted lifetime of reduced nicotinamide adenine dinucleotide phosphate was observed, which can be attributed to a changed metabolic redox state of the hepatocytes. I/R injury was associated with delayed uptake and excretion of fluorescein and elevated area-under-the-curve within the hepatocytes compared to sham (i.e., untreated control) as visualized and modeled using images recorded by intravital multiphoton tomography. High-performance liquid chromatography analysis showed no differences in plasma or bile concentrations of fluorescein. Finally, altered fluorescein distribution was associated with acute changes in the expression of liver transport proteins. In summary, multiphoton intravital imaging is an effective approach to measure liver function and is more sensitive in contrasting the impact of I/R injury than measuring plasma and bile concentrations of fluorescein.
This article [J. Biomed. Opt.. 18, , 061217 (2013)] was originally published online on 27 November 2012 with an error in Fig 4. The label above the right column should read 450–515 nm. The corrected figure is reprinted below.
Ultraviolet radiation from solar exposure is a key extrinsic factor responsible for premature skin aging (i.e., photo-aging). Recent advances using in vivo multiphoton tomography (MPT) demonstrate the efficacy of this approach to assess intrinsic and extrinsic skin aging as an alternative to existing invasive techniques. In this study, we measured changes in epidermal autofluorescence, dermal collagen second harmonic generation (SHG), and the redox state of solar-exposed and solar-protected human skin by MPT with fluorescence lifetime imaging (MPT-FLIM). Twenty-four volunteers across four age categories (20 to 29, 30 to 39, 40 to 49, and 50 to 59 years old; six volunteers each) were recruited for MPT-FLIM imaging of the dorsal (solar-exposed; photo-damaged) and volar (solar-protected) forearm. We demonstrate a higher intensity of dermal collagen SHG within the volar forearm compared to dorsal solar-exposed skin. Redox imaging of each epidermal skin stratum by FLIM demonstrates an increase in fluorescence lifetime in the solar-exposed dorsal forearm that is more apparent in aged skin. The results of this study suggest the redox state of the viable epidermis is a key marker in assessing intrinsic and photo-damage skin aging, in combination with changes in autofluorescence and SHG.
Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be time-consuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging (MPT-FLIM) we assess ischemic necrosis of ex vivo skin by NAD(P)H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media (Dulbecco Modified Eagle Medium) at −20, 4, and 37 °C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD(P)H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime (m) from ~1000 to 2000 ps. Additionally, significant distortions in NAD(P)H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4 °C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.
Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be time-consuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging (MPT-FLIM) we assess ischemic necrosis of ex vivo skin by NAD(P)H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media (Dulbecco Modified Eagle Medium) at −20, 4, and 37 °C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD(P)H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime (m) from ~1000 to 2000 ps. Additionally, significant distortions in NAD(P)H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4 °C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.
Nanoparticles of titanium dioxide (TiO2) and zinc oxide (ZnO) are used in sunscreens as protective compounds against
UV radiation. We investigate these particles from the viewpoint of nanosafety (penetration into skin in vivo, production
of free radicals when UV-irradiated) as well as UV protection. We show that: a) even after multiple applications, the
particles remain within stratum corneum (uppermost skin layer); b) the optimal sizes are 62 nm and 45 nm, respectively
for TiO2 and ZnO particles for 310-nm light and, correspondingly, 122 and 140 nm - for 400-nm radiation; c) in general,
small particles (25 nm in diameter) are more photoactive than the larger ones (400 nm in diameter); however, on the
background if porcine skin in vitro this difference is not seen and is substantially surpassed by skin contribution into
production of free radicals.
KEYWORDS: Skin, Zinc oxide, Nanoparticles, In vivo imaging, Ultraviolet radiation, Luminescence, Scanning electron microscopy, In vitro testing, Absorption, Particles
Zinc oxide (ZnO-nano) and titanium dioxide nanoparticles (20 to 30 nm) are widely used in several topical skin care products, such as sunscreens. However, relatively few studies have addressed the subdermal absorption of these nanoparticles in vivo. We report on investigation of the distribution of topically applied ZnO in excised and in vivo human skin, using multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) and an energy-dispersive x-ray (EDX) technique to determine the level of penetration of nanoparticles into the sub-dermal layers of the skin. The good visualization of ZnO in skin achieved appeared to result from two factors. First, the ZnO principal photoluminescence at 385 nm is in the “quiet” spectral band of skin autofluorescence dominated by the endogenous skin fluorophores, i.e., NAD[P]H and FAD. Second, the two-photon action cross section of ZnO-nano [~0.26 GM; diameter, 18 nm] is high: ~500-fold of that inferred from its bulk third-order nonlinear susceptibility [Im ], and is favorably compared to that of NAD[P]H and FAD. The overall outcome from MPM, SEM, and EDX studies was that, in humans in vivo, ZnO nanoparticles stayed in the stratum corneum (SC) and accumulated into skin folds and/or hair follicle roots of human skin. Given the lack of penetration of these nanoparticles past the SC and that the outermost layers of SC have a good turnover rate, these data suggest that the form of ZnO-nano studied here is unlikely to result in safety concerns.
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