Fluorescence microscopy, in particular immunofluorescence microscopy, has been used extensively for the assessment of kidney function and pathology for both research and diagnostic purposes. The development of confocal microscopy in the 1950s enabled imaging of live cells and intravital imaging of the kidney; however, confocal microscopy is limited by its maximal spatial resolution and depth. More recent advances in fluorescence microscopy techniques have enabled increasingly detailed assessment of kidney structure and provided extraordinary insights into kidney function. For example, nanoscale precise imaging by rapid beam oscillation (nSPIRO) is a super-resolution microscopy technique that was originally developed for functional imaging of kidney microvilli and enables detection of dynamic physiological events in the kidney. A variety of techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) enable assessment of interaction between proteins. The emergence of other super-resolution techniques, including super-resolution stimulated emission depletion (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM), has enabled functional imaging of cellular and subcellular organelles at ≤50 nm resolution. The deep imaging via emission recovery (DIVER) detector allows deep, label-free and high-sensitivity imaging of second harmonics, enabling assessment of processes such as fibrosis, whereas fluorescence lifetime imaging microscopy (FLIM) enables assessment of metabolic processes.
Microscopic imaging has revolutionized our understanding of kidney structure and physiology.
Kidney physiology is determined by dynamic processes and, although understanding of kidney structures at super-resolution is needed to understand kidney function, knowledge of kidney structure per se cannot explain kidney function.
Only a few super-resolution microscopy techniques, including super-resolution stimulated emission depletion (STED) microscopy and nanoscale precise imaging by rapid beam oscillation (nSPIRO), can simultaneously provide high-resolution structural information and insights into kidney dynamics.
Imaging of individual cells in tissues requires methods that are capable of imaging deep into the tissue, such as two-photon excitation and autofluorescence methods, imaged using methodologies such as the deep imaging via emission recovery (DIVER) detector; single photon excitation using dyes and immunofluorescence approaches cannot penetrate deep into live tissue.
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Colin, J. R. S. Multiphoton microscopy: a personal historical review, with some future predictions. J. Biomed. Opt. 25, 1–11 (2020).
Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Sheppard, C., Gannaway, J., Kompfner, R. & Walsh, D. The scanning harmonic optical microscope. IEEE J. Quantum Electron. 13, 912–912 (1977).
Blaine, J. et al. Dynamic imaging of the sodium phosphate cotransporters. Adv. Chronic Kidney Dis. 18, 145–150 (2011).
Digman, M. A., Caiolfa, V. R., Zamai, M. & Gratton, E. The phasor approach to fluorescence lifetime imaging analysis. Biophys. J. 94, L14–L16 (2008).
Dobrinskikh, E. et al. Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells. Am. J. Physiol. Cell Physiol. 304, C561–C573 (2013).
Lanaspa, M. A. et al. Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi. Am. J. Physiol. Renal Physiol. 292, F230–F242 (2007).
Jaykumar, A. B., Caceres, P. S., Sablaban, I., Tannous, B. A. & Ortiz, P. A. Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy. Am. J. Physiol. Renal Physiol. 310, F183–F191 (2015).
Gudgin, E., Lopez-Delgado, R. & Ware, W. R. The tryptophan fluorescence lifetime puzzle. A study of decay times in aqueous solution as a function of pH and buffer composition. Can. J. Chem. 59, 1037–1044 (1981).
Leblond, F., Davis, S. C., Valdés, P. A. & Pogue, B. W. Pre-clinical whole-body fluorescence imaging: review of instruments, methods and applications. J. Photochem. Photobiol. B 98, 77–94 (2010).
Parasassi, T., Gratton, E., Yu, W. M., Wilson, P. & Levi, M. Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys. J. 72, 2413–2429 (1997).
Dieterich, P., Klages, R., Preuss, R. & Schwab, A. Anomalous dynamics of cell migration. Proc. Natl Acad. Sci. USA 105, 459–463 (2008).
Dietrich, C. et al. Lipid rafts reconstituted in model membranes. Biophys. J. 80, 1417–1428 (2001).
Giral, H. et al. Role of PDZ domain containing 1 (PDZK1) in apical membrane expression of renal Na-coupled phosphate (Na/Pi) transporters. J. Biol. Chem. 286, 15032–15042 (2011).
Inoue, M. et al. Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity. J. Biol. Chem. 279, 49160–49171 (2004).
Ruan, Q., Cheng, M. A., Levi, M., Gratton, E. & Mantulin, W. W. Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). Biophys. J. 87, 1260–1267 (2004).
Suzuki-Inoue, K. et al. Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets. J. Biol. Chem. 285, 24494–24507 (2010).
Custer, M., Lotscher, M., Biber, J., Murer, H. & Kaissling, B. Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am. J. Physiol. 266, F767–F774 (1994).
Giral, H. et al. NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli. J. Biol. Chem. 287, 35047–35056 (2012).
Hernando, N. et al. PDZ-domain interactions and apical expression of type IIa Na/P(i) cotransporters. Proc. Natl Acad. Sci. USA 99, 11957–11962 (2002).
Lanzano, L. & Gratton, E. Orbital single particle tracking on a commercial confocal microscope using piezoelectric stage feedback. Methods Appl. Fluoresc. 2, 024010 (2014).
Virkki, L. V., Forster, I. C., Hernando, N., Biber, J. & Murer, H. Functional characterization of two naturally occurring mutations in the human sodium-phosphate cotransporter type IIa. J. Bone Miner. Res. 18, 2135–2141 (2003).
Wade, J. B. et al. Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am. J. Physiol. Cell Physiol. 285, C1494–C1503 (2003).
Weinman, E. J. et al. PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP. Am. J. Physiol. Renal Physiol. 297, F1560–F1565 (2009).
Devi, S. et al. Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus. Nat. Med. 19, 107–112 (2012).
Hall, A. M. & Molitoris, B. A. Dynamic multiphoton microscopy: focusing light on acute kidney injury. Physiology 29, 334–342 (2014).
Hall, A. M., Schuh, C. D. & Haenni, D. New frontiers in intravital microscopy of the kidney. Curr. Opin. Nephrol. Hypertens. 26, 172–178 (2017).
Kang, J. J., Toma, I., Sipos, A., McCulloch, F. & Peti-Peterdi, J. Quantitative imaging of basic functions in renal (patho)physiology. Am. J. Physiol. Renal Physiol. 291, F495–F502 (2006).
Nakano, D. & Nishiyama, A. Multiphoton imaging of kidney pathophysiology. J. Pharmacol. Sci. 132, 1–5 (2016).
Sandoval, R. M. & Molitoris, B. A. Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology. Methods 128, 20–32 (2017).
Camirand, G. et al. Multiphoton intravital microscopy of the transplanted mouse kidney. Am. J. Transpl. 11, 2067–2074 (2011).
Hall, A. M., Crawford, C., Unwin, R. J., Duchen, M. R. & Peppiatt-Wildman, C. M. Multiphoton imaging of the functioning kidney. J. Am. Soc. Nephrol. 22, 1297–1304 (2011).
Peti-Peterdi, J., Toma, I., Sipos, A. & Vargas, S. L. Multiphoton imaging of renal regulatory mechanisms. Physiology 24, 88–96 (2009).
Small, D. M., Sanchez, W. Y., Roy, S., Hickey, M. J. & Gobe, G. Multiphoton fluorescence microscopy of the live kidney in health and disease. J. Biomed. Opt. 19, 1–14 (2014).
Wang, E., Sandoval, R. M., Campos, S. B. & Molitoris, B. A. Rapid diagnosis and quantification of acute kidney injury using fluorescent ratio-metric determination of glomerular filtration rate in the rat. Am. J. Physiol. Renal Physiol. 299, F1048–F1055 (2010).
Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).
Gustafsson, N. et al. Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat. Commun. 7, 12471 (2016).
Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
Levi, M., Wilson, P. V., Cooper, O. J. & Gratton, E. Lipid phases in renal brush border membranes revealed by Laurdan fluorescence. Photochem. Photobiol. 57, 420–425 (1993).
Digman, M. A. & Gratton, E. Lessons in fluctuation correlation spectroscopy. Annu. Rev. Phys. Chem. 62, 645–668 (2011).
Digman, M. A., Stakic, M. & Gratton, E. in Methods in Enzymology Vol. 518 (ed. Tetin S. Y.) 121–144 (Academic, 2013).
Elson, E. L. Fluorescence correlation spectroscopy: past, present, future. Biophys. J. 101, 2855–2870 (2011).
Haustein, E. & Schwille, P. Fluorescence correlation spectroscopy: novel variations of an established technique. Annu. Rev. Biophys. Biomol. Struct. 36, 151–169 (2007).
Müller, J. D., Chen, Y. & Gratton, E. in Methods in Enzymology Vol. 361 (eds Marriott, G. & Parker, I.) 69–92 (Academic, 2003).
Murer, H., Hernando, N., Forster, I. & Biber, J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol. Rev. 80, 1373–1409 (2000).
Levi, M. et al. Mechanisms of phosphate transport. Nat. Rev. Nephrol. 15, 482–500 (2019).
Wilmes, A. et al. Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity. Toxicol. In Vitro 25, 613–622 (2011).
Rayner, S. G. et al. Reconstructing the human renal vascular–tubular unit in vitro. Adv. Healthc. Mater. 7, e1801120 (2018).
Sakolish, C. et al. Technology transfer of the microphysiological systems: a case study of the human proximal tubule tissue chip. Sci. Rep. 8, 14882–14882 (2018).
Sakolish, Courtney M. & Mahler, G. J. A novel microfluidic device to model the human proximal tubule and glomerulus. RSC Adv. 7, 4216–4225 (2017).
Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).
Bunt, G. & Wouters, F. S. FRET from single to multiplexed signaling events. Biophys. Rev. 9, 119–129 (2017).
Villar, V. A. M. et al. Novel role of sorting nexin 5 in renal D1 dopamine receptor trafficking and function: implications for hypertension. FASEB J. 27, 1808–1819 (2012).
Miles, J. et al. Time resolved amplified FRET identifies protein kinase B activation state as a marker for poor prognosis in clear cell renal cell carcinoma. BBA Clin. 8, 97–102 (2017).
Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456 (2001).
Manzo, C. & Garcia-Parajo, M. F. A review of progress in single particle tracking: from methods to biophysical insights. Rep. Prog. Phys. 78, 124601 (2015).
Magde, D., Elson, E. & Webb, W. W. Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705–708 (1972).
Lanzano, L. et al. Nanometer-scale imaging by the modulation tracking method. J. Biophotonics 4, 415–424 (2011).
Bacia, K., Kim, S. A. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nat. Methods 3, 83–89 (2006).
Rossow, M. J., Sasaki, J. M., Digman, M. A. & Gratton, E. Raster image correlation spectroscopy in live cells. Nat. Protoc. 5, 1761–1774 (2010).
Labilloy, A. et al. Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease. Mol. Genet. Metab. 111, 184–192 (2014).
Zhong, Z. H. et al. Insulin binding monitored by fluorescence correlation spectroscopy. Diabetologia 44, 1184–1188 (2001).
Blaine, J. et al. PTH-induced internalization of apical membrane NaPi2a: role of actin and myosin VI. Am. J. Physiol. Cell Physiol. 297, C1339–C1346 (2009).
Lanzano, L. et al. Differential modulation of the molecular dynamics of the type IIa and IIc sodium phosphate cotransporters by parathyroid hormone. Am. J. Physiol. Cell Physiol. 301, C850–C861 (2011).
Khundmiri, S. J. et al. Structural determinants for the ouabain-stimulated increase in Na-K ATPase activity. Biochim. Biophys. Acta 1843, 1089–1102 (2014).
Klingner, C. et al. Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells. J. Cell Biol. 207, 107–121 (2014).
Katayama, Y. et al. Real-time nanomicroscopy via three-dimensional single-particle tracking. Chemphyschem 10, 2458–2464 (2009).
Lanzano, L. & Gratton, E. Measurement of distance with the nanoscale precise imaging by rapid beam oscillation method. Microsc. Res. Tech. 75, 1253–1264 (2012).
Beaufils, H., Jouanneau, C. & Chomette, G. Kidney and cancer: results of immunofluorescence microscopy. Nephron 40, 303–308 (1985).
D’Agati, V. D. & Mengel, M. The rise of renal pathology in nephrology: structure illuminates function. Am. J. Kidney Dis. 61, 1016–1025 (2013).
McCluskey, R. T. The value of immunofluorescence in the study of human renal disease. J. Exp. Med. 134, 242–255 (1971).
Krane, L. S., Manny, T. B. & Hemal, A. K. Is near infrared fluorescence imaging using indocyanine green dye useful in robotic partial nephrectomy: a prospective comparative study of 94 patients. Urology 80, 110–118 (2012).
Sharkey, J. et al. Imaging technologies for monitoring the safety, efficacy and mechanisms of action of cell-based regenerative medicine therapies in models of kidney disease. Eur. J. Pharmacol. 790, 74–82 (2016).
Unnersjö-Jess, D., Scott, L., Blom, H. & Brismar, H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue. Kidney Int. 89, 243–247 (2016).
Ranjit, S., Malacrida, L., Stakic, M. & Gratton, E. Determination of the metabolic index using the fluorescence lifetime of free and bound NADH in the phasor approach. J. Biophotonics 12, e201900156 (2019).
Ranjit, S., Malacrida, L. & Gratton, E. Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card. Microsc. Res. Tech. 81, 980–989 (2018).
Buch, A. et al. Role of direct immunofluorescence in the diagnosis of glomerulonephritis. Med. J. DY Patil. Vidyapeeth 8, 452–457 (2015).
Mise, K. et al. Clinical implications of linear immunofluorescent staining for immunoglobulin G in patients with diabetic nephropathy. Diabetes Res. Clin. Pract. 106, 522–530 (2014).
Aguilar-Arnal, L. et al. Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species. Proc. Natl Acad. Sci. USA 113, 12715–12720 (2016).
Bird, D. K. et al. Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res. 65, 8766 (2005).
Chance, B. in Methods in Enzymology Vol. 385 (ed. Conn, P. M.) 361–370 (Academic, 2004).
Croce, A. C. & Bottiroli, G. Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur. J. Histochem. 58, 2461 (2014).
Ranjit, S., Datta, R., Dvornikov, A. & Gratton, E. Multicomponent analysis of phasor plot in a single pixel to calculate changes of metabolic trajectory in biological systems. J. Phys. Chem. A 123, 9865–9873 (2019).
Stringari, C. et al. Metabolic trajectory of cellular differentiation in small intestine by phasor fluorescence lifetime microscopy of NADH. Sci. Rep. 2, 568 (2012).
Köhler, S., Winkler, U., Sicker, M. & Hirrlinger, J. NBCe1 mediates the regulation of the NADH/NAD+ redox state in cortical astrocytes by neuronal signals. Glia 66, 2233–2245 (2018).
Ranjit, S., Dvornikov, A., Levi, M., Furgeson, S. & Gratton, E. Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images. Biomed. Opt. Express 7, 3519–3530 (2016).
Ranjit, S., Malacrida, L., Jameson, D. M. & Gratton, E. Fit-free analysis of fluorescence lifetime imaging data using the phasor approach. Nat. Protoc. 13, 1979–2004 (2018).
Skala, M. C. et al. In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J. Biomed. Opt. 12, 024014–024014 (2007).
Skala, M. C. et al. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc. Natl Acad. Sci. USA 104, 19494–19499 (2007).
Stringari, C. et al. Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue. Proc. Natl Acad. Sci. USA 108, 13582–13587 (2011).
Stringari, C., Nourse, J. L., Flanagan, L. A. & Gratton, E. Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential. PLoS One 7, e48014 (2012).
Stringari, C., Sierra, R., Donovan, P. J. & Gratton, E. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy. J. Biomed. Opt. 17, 046012 (2012).
Wright, B. K. et al. NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. Biophys. J. 103, L7–L9 (2012).
Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).
Digman, M. & Gratton, E. in Fluorescence Lifetime Spectroscopy and Imaging (eds Marcu, L., French, P. M. W. & Elson, D. S.) 235–248 (CRC, 2014).
Gratton, E. & Jameson, D. M. Phasor approach to fluorescence lifetime imaging microscopy. Lumipedia http://www.lumipedia.org/index.php?title=Phasor_Approach_to_Fluorescence_Lifetime_Imaging_Microscopy (2017).
Campagnola, P. Second harmonic generation imaging microscopy: applications to diseases diagnostics. Anal. Chem. 83, 3224–3231 (2011).
Chen, X., Nadiarynkh, O., Plotnikov, S. & Campagnola, P. J. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat. Protoc. 7, 654 (2012).
Strupler, M. et al. Second harmonic imaging and scoring of collagen in fibrotic tissues. Opt. Express 15, 4054–4065 (2007).
Zoumi, A., Yeh, A. & Tromberg, B. J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl Acad. Sci. USA 99, 11014–11019 (2002).
Ranjit, S. et al. Imaging fibrosis and separating collagens using second harmonic generation and phasor approach to fluorescence lifetime imaging. Sci. Rep. 5, 13378 (2015).
Ranjit, S. et al. Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis. Kidney Int. 90, 1123–1128 (2016).
Crosignani, V., Jahid, S., Dvornikov, A. S. & Gratton, E. A deep tissue fluorescence imaging system with enhanced SHG detection capabilities. Microsc. Res. Tech. 77, 368–373 (2014).
Dvornikov, A., Malacrida, L. & Gratton, E. The DIVER microscope for imaging in scattering media. Methods Protoc. 2, 53 (2019).
Arnesano, C., Santoro, Y. & Gratton, E. Digital parallel frequency-domain spectroscopy for tissue imaging. J. Biomed. Opt. 17, 096014 (2012).
Datta, R., Heylman, C., George, S. C. & Gratton, E. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes. Biomed. Opt. Express 7, 1690–1701 (2016).
Formoso, E., Mujika, J. I., Grabowski, S. J. & Lopez, X. Aluminum and its effect in the equilibrium between folded/unfolded conformation of NADH. J. Inorg. Biochem. 152, 139–146 (2015).
Kolb, D. A. & Weber, G. Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide and oxalate. Biochemistry 14, 4471–4476 (1975).
Lakowicz, J. R., Szmacinski, H., Nowaczyk, K. & Johnson, M. L. Fluorescence lifetime imaging of free and protein-bound NADH. Proc. Natl Acad. Sci. USA 89, 1271–1275 (1992).
Scott, T. G., Spencer, R. D., Leonard, N. J. & Weber, G. Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models. J. Am. Chem. Soc. 92, 687–695 (1970).
Mayevsky, A. & Chance, B. Oxidation–reduction states of NADH in vivo: from animals to clinical use. Mitochondrion 7, 330–339 (2007).
Poyan Mehr, A. et al. De novo NAD+ biosynthetic impairment in acute kidney injury in humans. Nat. Med. 24, 1351–1359 (2018).
Ralto, K. M., Rhee, E. P. & Parikh, S. M. NAD+ homeostasis in renal health and disease. Nat. Rev. Nephrol. 16, 99–111 (2020).
Tran, M. T. et al. PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection. Nature 531, 528–532 (2016).
Hung, C.-H. et al. Crystal structures and molecular dynamics simulations of thermophilic malate dehydrogenase reveal critical loop motion for co-substrate binding. PLoS One 8, e83091 (2013).
Read, J. A., Winter, V. J., Eszes, C. M., Sessions, R. B. & Brady, R. L. Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase. Proteins 43, 175–185 (2001).
Hall, A. M., Unwin, R. J., Parker, N. & Duchen, M. R. Multiphoton imaging reveals differences in mitochondrial function between nephron segments. J. Am. Soc. Nephrol. 20, 1293–1302 (2009).
Hato, T. et al. Two-photon intravital fluorescence lifetime imaging of the kidney reveals cell-type specific metabolic signatures. J. Am. Soc. Nephrol. 28, 2420–2430 (2017).
Wang, X. X. et al. FXR/TGR5 dual agonist prevents progression of nephropathy in diabetes and obesity. J. Am. Soc. Nephrol. 29, 118 (2018).
Ranjit, S. et al. Phasor approach to autofluorescence lifetime imaging FLIM can be a quantitative biomarker of chronic renal parenchymal injury. Kidney Int. https://doi.org/10.1016/j.kint.2020.02.019 (2020).
Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv Mikrosk. Anatõmie 9, 413–468 (1873).
Rayleigh. XV. On the theory of optical images, with special reference to the microscope. Lond. Edinb. Dubl. Phil. Mag. 42, 167–195 (1896).
Pawley, J. (Ed.) Handbook of Biological Confocal Microscopy 3rd edn (Springer, 2006).
Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72–84 (2019).
Betzig, E. & Trautman, J. K. Near-field optics: microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science 257, 189–195 (1992).
Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl Acad. Sci. USA 102, 17565–17569 (2005).
Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).
Suleiman, H. Y. et al. Injury-induced actin cytoskeleton reorganization in podocytes revealed by super-resolution microscopy. JCI Insight 2, e94137 (2017).
Suleiman, H. et al. Nanoscale protein architecture of the kidney glomerular basement membrane. eLife 2, e01149 (2013).
Lin, M.-H. et al. Laminin-521 protein therapy for glomerular basement membrane and podocyte abnormalities in a model of pierson syndrome. J. Am. Soc. Nephrol. 29, 1426–1436 (2018).
Motrapu, M. et al. Drug testing for residual progression of diabetic kidney disease in mice beyond therapy with metformin, ramipril, and empagliflozin. J. Am. Soc. Nephrol. 31, 1729–1745 (2020).
Olson, E., Levene, M. J. & Torres, R. Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies. Biomed. Opt. Express 7, 3089–3096 (2016).
Torres, R. et al. Three-dimensional morphology by multiphoton microscopy with clearing in a model of cisplatin-induced CKD. J. Am. Soc. Nephrol. 27, 1102–1112 (2016).
Chozinski, T. J. et al. Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy. Sci. Rep. 8, 10396 (2018).
Zhao, Y. et al. Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat. Biotechnol. 35, 757–764 (2017).
Heine, J. et al. Adaptive-illumination STED nanoscopy. Proc. Natl Acad. Sci. USA 114, 9797–9802 (2017).
Jahr, W., Velicky, P. & Danzl, J. G. Strategies to maximize performance in stimulated emission depletion (STED) nanoscopy of biological specimens. Methods 174, 27–41 (2020).
Lanzanò, L. et al. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nat. Commun. 6, 6701 (2015).
Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).
Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).
Zhao, M. et al. Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy. Biomed. Opt. Express 5, 1296–1308 (2014).
Siegerist, F., Endlich, K. & Endlich, N. Novel microscopic techniques for podocyte research. Front. Endocrinol. 9, 379–379 (2018).
Artelt, N. et al. Comparative analysis of podocyte foot process morphology in three species by 3D super-resolution microscopy. Front. Med. 5, 292–292 (2018).
Pullman, J. M. et al. Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease. Biomed. Opt. Express 7, 302–311 (2016).
Siegerist, F. et al. Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement. Sci. Rep. 7, 11473 (2017).
Korobchevskaya, K., Lagerholm, C. B., Colin-York, H. & Fritzsche, M. Exploring the potential of airyscan microscopy for live cell imaging. Photonics 4, 41 (2017).
Sheppard, C. Super-resolution in confocal imaging. Optik 80, 53–54 (1988).
Sheppard, C. J. R., Mehta, S. B. & Heintzmann, R. Superresolution by image scanning microscopy using pixel reassignment. Opt. Lett. 38, 2889–2892 (2013).
Herwig, J. et al. Thrombospondin type 1 domain–containing 7A localizes to the slit diaphragm and stabilizes membrane dynamics of fully differentiated podocytes. J. Am. Soc. Nephrol. 30, 824–839 (2019).
Huff, J. et al. The new 2D superresolution mode for ZEISS Airyscan. Nat. Methods 14, 1223 (2017).
Grgic, I. et al. Imaging of podocyte foot processes by fluorescence microscopy. J. Am. Soc. Nephrol. 23, 785–791 (2012).
Khoury, C. C. et al. Visualizing the mouse podocyte with multiphoton microscopy. Biochem. Biophys. Res. Commun. 427, 525–530 (2012).
Ronconi, E. et al. Regeneration of glomerular podocytes by human renal progenitors. J. Am. Soc. Nephrol. 20, 322–332 (2009).
Höhne, M. et al. Light microscopic visualization of podocyte ultrastructure demonstrates oscillating glomerular contractions. Am. J. Pathol. 182, 332–338 (2013).
Burford, J. L. et al. Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment. Pflugers Arch. 469, 965–974 (2017).
Gyarmati, G. et al. Advances in renal cell imaging. Semin. Nephrol. 38, 52–62 (2018).
Hackl, M. J. et al. Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags. Nat. Med. 19, 1661–1666 (2013).
Peti-Peterdi, J. Multiphoton imaging of renal tissues in vitro. Am. J. Physiol. Renal Physiol. 288, F1079–F1083 (2005).
Peti-Peterdi, J., Burford, J. L. & Hackl, M. J. The first decade of using multiphoton microscopy for high-power kidney imaging. Am. J. Physiol. Renal Physiol. 302, F227–F233 (2012).
Peti-Peterdi, J., Kidokoro, K. & Riquier-Brison, A. Novel in vivo techniques to visualize kidney anatomy and function. Kidney Int. 88, 44–51 (2015).
Peti-Peterdi, J., Kidokoro, K. & Riquier-Brison, A. Intravital imaging in the kidney. Curr. Opin. Nephrol. Hypertens. 25, 168–173 (2016).
Sipos, A., Toma, I., Kang, J. J., Rosivall, L. & Peti-Peterdi, J. Advances in renal (patho)physiology using multiphoton microscopy. Kidney Int. 72, 1188–1191 (2007).
Hato, T., Winfree, S. & Dagher, P. C. Intravital imaging of the kidney. Methods 128, 33–39 (2017).
Molitoris, B. A. & Sandoval, R. M. Intravital multiphoton microscopy of dynamic renal processes. Am. J. Physiol. Renal Physiol. 288, F1084–F1089 (2005).
Polesel, M. & Hall, A. M. Axial differences in endocytosis along the kidney proximal tubule. Am. J. Physiol. Renal Physiol. 317, F1526–F1530 (2019).
Dunn, K. W. et al. Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am. J. Physiol. Cell Physiol. 283, C905–C916 (2002).
Endres, B. T. et al. Intravital imaging of the kidney in a rat model of salt-sensitive hypertension. Am. J. Physiol. Renal Physiol. 313, F163–F173 (2017).
Kaverina, N. V. et al. Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging. PLoS One 12, e0173891 (2017).
Schießl, I. M., Hammer, A., Riquier-Brison, A. & Peti-Peterdi, J. Just look! Intravital microscopy as the best means to study kidney cell death dynamics. Semin. Nephrol. 36, 220–236 (2016).
Schuh, C. D. et al. Long wavelength multiphoton excitation is advantageous for intravital kidney imaging. Kidney Int. 89, 712–719 (2016).
Small, D. M., Sanchez, W. Y. & Gobe, G. C. Intravital multiphoton imaging of the kidney: tubular structure and metabolism. Methods Mol. Biol. 1397, 155–172 (2016).
Ashworth, S. L., Sandoval, R. M., Tanner, G. A. & Molitoris, B. A. Two-photon microscopy: visualization of kidney dynamics. Kidney Int. 72, 416–421 (2007).
Hall, A. M., Rhodes, G. J., Sandoval, R. M., Corridon, P. R. & Molitoris, B. A. In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury. Kidney Int. 83, 72–83 (2013).
Dickson, L. E., Wagner, M. C., Sandoval, R. M. & Molitoris, B. A. The proximal tubule and albuminuria: really! J. Am. Soc. Nephrol. 25, 443–453 (2014).
Molitoris, B. A. et al. siRNA Targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury. J. Am. Soc. Nephrol. 20, 1754–1764 (2009).
Sandoval, R. M., Kennedy, M. D., Low, P. S. & Molitoris, B. A. Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy. Am. J. Physiol. Cell Physiol. 287, C517–C526 (2004).
Schiessl, I., Fremter, K., Burford, J., Castrop, H. & Peti-Peterdi, J. Long-term cell fate tracking of individual renal cells using serial intravital microscopy. Methods Mol. Biol. 2150, 25–44 (2020).
Schuh, C. D. et al. Combined structural and functional imaging of the kidney reveals major axial differences in proximal tubule endocytosis. J. Am. Soc. Nephrol. 29, 2696 (2018).
Peti-Peterdi, J., Morishima, S., Bell, P. D. & Okada, Y. Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus. Am. J. Physiol. Renal Physiol. 283, F197–F201 (2002).
Burford, J. L. et al. Intravital imaging of podocyte calcium in glomerular injury and disease. J. Clin. Invest. 124, 2050–2058 (2014).
Salmon, A. H. J. et al. Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction. J. Am. Soc. Nephrol. 23, 1339–1350 (2012).
Nakano, D. et al. Multiphoton imaging of the glomerular permeability of angiotensinogen. J. Am. Soc. Nephrol. 23, 1847–1856 (2012).
Endlich, N. et al. Two-photon microscopy reveals stationary podocytes in living zebrafish larvae. J. Am. Soc. Nephrol. 25, 681–686 (2014).
Brähler, S. et al. Intravital and kidney slice imaging of podocyte membrane dynamics. J. Am. Soc. Nephrol. 27, 3285–3290 (2016).
Wang, C. & Ji, N. Characterization and improvement of three-dimensional imaging performance of GRIN-lens-based two-photon fluorescence endomicroscopes with adaptive optics. Opt. Express 21, 27142–27154 (2013).
Bao, H., Allen, J., Pattie, R., Vance, R. & Gu, M. Fast handheld two-photon fluorescence microendoscope with a 475 μm × 475 μm field of view for in vivo imaging. Opt. Lett. 33, 1333–1335 (2008).
Akhoundi, F., Qin, Y., Peyghambarian, N., Barton, J. K. & Kieu, K. Compact fiber-based multi-photon endoscope working at 1700 nm. Biomed. Opt. Express 9, 2326–2335 (2018).
Liang, W., Hall, G., Messerschmidt, B., Li, M.-J. & Li, X. Nonlinear optical endomicroscopy for label-free functional histology in vivo. Light Sci. Appl. 6, e17082 (2017).
Sinefeld, D., Paudel, H. P., Ouzounov, D. G., Bifano, T. G. & Xu, C. Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence. Opt. Express 23, 31472–31483 (2015).
Zheng, W. et al. Adaptive optics improves multiphoton super-resolution imaging. Nat. Methods 14, 869–872 (2017).
Digman, M. A. & Gratton, E. Analysis of diffusion and binding in cells using the RICS approach. Microsc. Res. Tech. 72, 323–332 (2009).
Allen, J. R., Ross, S. T. & Davidson, M. W. Single molecule localization microscopy for superresolution. J. Opt. 15, 094001 (2013).
Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat. Methods 16, 1045–1053 (2019).
Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).
Hebisch, E., Wagner, E., Westphal, V., Sieber, J. J. & Lehnart, S. E. A protocol for registration and correction of multicolour STED superresolution images. J. Microsc. 267, 160–175 (2017).
Wegner, W. et al. In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins. Sci. Rep. 7, 11781 (2017).
Wassie, A. T., Zhao, Y. & Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nat. Methods 16, 33–41 (2019).
Huff, J. The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat. Methods 12, i–ii (2015).
Unnersjö-Jess, D. et al. Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion. Kidney Int. 93, 1008–1013 (2018).
L.L. was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) under MFAG 2018-ID. 21931 project. E.G. and S.R. were supported by National Institutes of Health (NIH) P50 GM076516 and NIH P41GM103540 to E.G. A.E.L. and M.L. were supported by NIH 5R01DK116567 (NIDDK), and National Institute on Ageing (NIA) 5R01AG049493 to M.L.
The authors declare no competing interests.
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- Two-photon excitation microscopy
A microscopy technique in which fluorophores can be excited at wavelengths that are double the characteristic wavelengths of excitation, as a consequence of the simultaneous absorption of two photons.
- Deep imaging via emission recovery
(DIVER). A detector assembly that improves imaging in deep tissues by maximizing the amount of fluorescence emission collected by a large area (6.45 cm2) photomultiplier tube in the forward direction of the excitation propagation.
- Fluorescence recovery after photobleaching
(FRAP). A microscopy method whereby a small area of the sample is photobleached and as the molecules repopulate that area, intensity is recovered. This increase in intensity enables calculation of diffusion properties.
- Fluorescence correlation spectroscopy
(FCS). A technique that analyses the intensity fluctuations resulting from the movement of fluorescent molecules in and out of an optically restricted volume (~1 µl), which enables calculations of the diffusion parameters, concentrations and photophysical properties of the molecules.
- Förster resonance energy transfer
(FRET). An event that results from the transfer of excitation energy from one fluorescent molecule (the donor) to another (the acceptor) when they are in close proximity (<10 nm). FRET decreases the donor intensity and fluorescence lifetime and increases the acceptor fluorescence.
- Nanoscale precise imaging by rapid beam oscillation
(nSPIRO). A microscopy technique based on the scanning of a laser beam around the surface of apical microvilli or other cellular protrusions. The detected signal and the position of the scanner are used to perform high-resolution imaging of the protrusions.
- Confocal microscopy
An optical imaging technique that provides rejection of out-of-focus signal by combining laser scanning with a pinhole in the detection path. The typical resolution of confocal fluorescence microscopy is ~200 nm in the focal plane and ~600 nm along the axial direction owing to its efficient rejection of out-of-focus signals.
An environment-sensitive fluorophore used to perform biophysical studies on membranes. The spectral properties of Laurdan are sensitive to the penetration of water into membranes.
- Generalized polarization domains
Portions of a membrane characterized by the same value of generalized polarization, a parameter that quantifies the spectral shift of Laurdan in different lipid environments.
- Giant unilamellar vesicles
(GUVs). Simplified model membrane systems, 1–100 μm in size, in which components of the cell membrane, including lipids and transmembrane proteins, are combined with cell components of the membrane to create a lipid bilayer.
- Total internal reflection microscopy
(TIRF). A microscopy approach in which total internal reflection of the excitation light limits fluorescence detection to a sub-diffraction, ~100 nm region above the glass, called the evanescent field. Only the fluorophores that are in this small area are excited and give light.
- Fluorescence lifetime imaging microscopy
(FLIM). A microscopy approach that measures the lifetime (average time the fluorophore stays in the excited state) in each of the pixels of an image. FLIM can be used to distinguish signals from different species even if they have similar emission spectra.
- Diffusion coefficients
A parameter that describes how fast a molecule undergoes Brownian motion in a given medium.
- Single-particle tracking
(SPT). A class of microscopy methods for tracking the movement of molecules or cell components. It involves continuous updating of the centre of a scanned orbit based on feedback provided by the recorded fluorescence intensity, which keeps the component of interest at the orbital centre with nanometre precision.
- Fluorescence cross-correlation spectroscopy
An extension of fluorescence correlation spectroscopy (FCS) that allows analysis of intensity fluctuations in two different colours from two different molecules. Correlated fluctuations in both channels results from two interacting molecules diffusing together whereas a lack of correlated intensity fluctuations shows the absence of interaction.
- Scanning FCS
An extension of fluorescence correlation spectroscopy (FCS) that allows analysis of intensity fluctuations at multiple locations of a sample as a result of fast laser scanning.
- Raster image correlation spectroscopy
(RICS). A spatiotemporal fluorescence correlation technique that measures the diffusion of a molecule of interest in images and determines the diffusion coefficients by fitting the resulting correlation function to a diffusion model.
- Number and brightness
(N&B). A fluorescence correlation technique that measures the temporal fluctuations of intensity at each pixel of an image and provides maps of the concentration and the degree of oligomerization of a molecule or proteins.
- Pair correlation functions
A fluorescence correlation technique that analyses the fluctuations detected at two separate locations of a sample. It can be used to determine the presence of barriers to diffusion.
- Orbital tracking
A single-particle tracking technique that involves continuous updating of the centre of a scanned orbit based on feedback provided by the recorded fluorescence intensity, which keeps the component of interest at the orbital centre with nanometre precision.
- Second harmonic generation
(SHG). A non-linear optical process where two photons of the same wavelengths are combined to create a photon of half of the initial wavelength and double the initial photon energy. In biological systems, collagen I and II and myelin fibres lack a centre of symmetry and generate an SHG signal.
- Third harmonic generation
(THG). A non-linear optical process where three photons of the same wavelengths are combined to create a photon of one-third of the initial wavelength and three times the initial photon energy. This is generated because of changes in the refractive index and is especially useful for identifying tissue boundaries and lipid droplets.
- Photomultiplier tube
A detection device that converts the incident light into an electrical signal.
A digital device for detection of fluorescence lifetime imaging microscopy (FLIM) data in the frequency domain. The device measures the demodulation and phase shift of the fluorescence intensity with respect to a modulated excitation source.
- Phasor plot
A method of describing fluorescence lifetime imaging microscopy (FLIM) data by converting the intensity decay (time domain) and modulation and phase shifts (frequency domain) to the polar plot to separate pixels of FLIM images into different phasor points for a fast fit-free analysis of lifetime data.
- π–π interactions
Non-covalent interactions that can occur between the aromatic groups of different molecules.
A graphical fit-free way of analysing the fluorescence lifetime data from an image. Reciprocity between the phasor plot and the fluorescence lifetime imaging microscopy (FLIM) image means that selection of a particular area on an image can show the lifetime signature of those areas whereas selection of an area on the phasor plot can show areas of the image that have similar lifetime signatures.
- Optical elements
Components of an optical setup (for example, a lens, a mirror, a filter).
- Optical clearing
The treatment of biological tissues with exogenous agents capable of reducing scattering and increasing imaging penetration depth.
- Expansion microscopy
A technique whereby the tissue is put in a polyelectrolyte hydrogel that can isotropically expand the biological specimen.
- Moiré patterns
Interference patterns generated when two identical periodic patterns are rotated one with respect to the other.
- Bessel beam excitation
The excitation of fluorescence by a special type of laser beam whose shape is described by a Bessel function.
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Ranjit, S., Lanzanò, L., Libby, A.E. et al. Advances in fluorescence microscopy techniques to study kidney function. Nat Rev Nephrol 17, 128–144 (2021). https://doi.org/10.1038/s41581-020-00337-8