Live‐Animal Imaging of Renal Function by Multiphoton Microscopy

Abstract

Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. © 2018 by John Wiley & Sons, Inc.

Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs

“…Observation of respiratory rate, color of the mucosa and response to stimulation (ear pinch) are appropriate for determining proper depth of anesthesia and are indirect indicators of blood pressure and heart rate. However, monitoring of blood pressure and heart rate with an intra-arterial catheter or tail vein blood pressure cuff transducer system, and rectal thermometer, to measure core temperature, afford the investigator a more accurate determination of the physiologic state of the animal [1, 5, 9]…”

“…Because of anatomical location of these organs and the fact that many of them have long vascular pedicles that allow considerable mobility, need to two types of incisions are utilized to expose the organs of interest: 1) left flank vertical incision and 2) midline horizontal incision. Many investigators select the inverted microscope as the optimal scope for imaging the intra-abdominal organs that we study [1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29]. …”

“…Prior to use, the animals are inspected for overall health: activity, overall appearance, posture, socialization, and grooming are the most common aspects we observe. Once these criteria have been evaluated and the animals are deemed fit, they may be utilized for imaging [1]. …”

“…The dish is directly mounted into the microscope stage such that the organ can be imaged from below. Oriented in this way, the animal’s weight serves to hold the organ in place in the dish, minimizing respiratory and circulatory motion artifacts [1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29]. This approach also requires surgical procedures that are simpler, faster, and less invasive than those needed to immobilize an organ for imaging on an upright system [1].…”

“…Intravital microscopy has afforded researchers the ability to better understand cellular and subcellular processes within multiple organs in the body [1, 2, 3, 4]. The utilization of the rat and mouse models can be advantageous in that organ structure and function in these species mimics humans.…”