Intravital Imaging of the Kidney Using Multiparameter Multiphoton Microscopy

Abstract

Intravital optical microscopy provides a powerful means of studying the cell biology in the most physiologically relevant setting. The ability of multiphoton microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of multiple organs. Presented here are examples of how two-photon microscopy can be applied to intravital studies of kidney physiology and the study of disease processes. These include studies of cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters. Two-photon microscopy can now provide a level of investigation previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. Therefore, application of this technology will provide direct visualization of organ-specific and cell-specific responses to an array of stimuli and therapeutic approaches, enhancing our understanding and treatment of disease processes.

Quantitative intravital microscopy of hepatic transport

“…Results presented extend previous studies of cultured cells, demonstrating that rifampin inhibits uptake of organic anions by hepatocytes in vivo and provide support for an iterative model of hepatic transport. Although used here to evaluate the effects of chronic rifampin treatment, the ability to analyze processes occurring in the timescale of seconds to minutes also provides intravital microscopy with the unique ability to detect acute, or transient drug effects which, when combined with additional readouts of injury, 43,60,[62][63][64][65][66] provides the potential to characterize the chronology of cholestatic liver injury. Since is both properly sedated and physiologically supported.…”

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

“…Multiphoton excitation imaging was employed to quantitatively reveal microvascular function (Dunn et al, 2003;Molitoris & Sandoval, 2005) as well as glomerular filtration and permeability within kidney with living animals (Kang et al, 2006;Sipos et al, 2007). In a further study, they showed how this nonlinear optical imaging can be used to observe and quantify drug delivery in an intact, living and functioning kidney.…”

Improving signal levels in intravital multiphoton microscopy using an objective correction collar

“…While the surface of the kidney is easily imaged by conventional optical microscopy, imaging into the kidney is best approached by MPM. [2,3] However, we have found that MPM is capable of collecting images from only the first two hundred microns into the kidney. Here we evaluate the role of spherical aberration in signal attenuation with depth into fixed and living kidney tissues, and evaluate a simple solution to improve deep-tissue MPM.…”

“…[2][3][4][5][6][7][8][9][10] Additionally, because multiphoton microscopy is a fluorescence technique, it has the ability to localize multiple specific molecules simultaneously. Multiphoton microscopy has also been shown to allow extended observation of even highly sensitive processes without detectable damage.…”