Multiphoton fluorescence microscopy of the live kidney in health and disease

Abstract

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.

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis

“…13–14 However, fluorescence lifetime imaging (FLIM) for either labeled samples or tissue autofluorescence has never been employed for characterizing fibrosis in kidneys. A phasor approach to FLIM imaging along with SHG generation can quantify fibrosis.…”

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures

“…6,7 However, the emission wavelengths of many endogenous fluorophores overlap, and this limits the ability to identify individual species with certainty. 8,9 In contrast, the fluorescence lifetimes of these same endogenous fluorophores are significantly different and can be separated by phasor analysis. 5,10 Accordingly, fluorescence lifetime imaging microscopy (FLIM) offers the ability to distinguish a number of intrinsic fluorophores despite their overlapping emission spectra.…”

Visualizing liver anatomy, physiology and pharmacology using multiphoton microscopy

“…The most commonly used fluorophores in MPM have excitation spectra in the range of 400–500 nm, whereas the laser used to excite the TPEF lies in the 700∼1000 nm range. As shown in Table , a number of endogenous molecules in the liver can generate fluorescence, which is a hindrance of traditional fluorescence microscopy, yet is used as an advantage in TPEF imaging . The metabolic coenzymes nicotinamide adenine dinucleotide hydride (NADH) is a respiratory substrate for complex I of the mitochondrial electron transport chain, which is an endogenous fluorophore in the cytoplasm of hepatocytes.…”

“…Because NAD + , the oxidized form of NAD(P)H, has no fluorescence, changes in the fluorescence intensity of NAD(P)H can provide valuable information regarding cell metabolism. Flavin adenine dinucleotide (FAD) is only fluorescent in the oxidative states, and can provide further information of the cellular redox state . The most common optical method for metabolic imaging is the “redox ratio,” which is the ratio of the fluorescence intensity of FAD and NAD(P)H. The redox ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply .…”

“…Flavin adenine dinucleotide (FAD) is only fluorescent in the oxidative states, and can provide further information of the cellular redox state . The most common optical method for metabolic imaging is the “redox ratio,” which is the ratio of the fluorescence intensity of FAD and NAD(P)H. The redox ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply . A decrease in the redox ratio or the fluorescence intensity of NAD(P)H is usually observed in cancer cells or injured cells.…”