Optical Histology: A Method to Visualize Microvasculature in Thick Tissue Sections of Mouse Brain

Optical Histology: A Method to Visualize Microvasculature in Thick Tissue Sections of Mouse Brain Austin J. Moy 1,2 , Matthew P. Wiersma 1,2 , Bernard Choi 1,2,3 * 1 Beckman Laser Institute, University of California Irvine, Irvine, California, United States of America, 2 Department of Biomedical Engineering, University of California Irvine, Irvine, California, United States of America, 3 Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, Irvine, California, United States of America Abstract Background: The microvasculature is the network of blood vessels involved in delivering nutrients and gases necessary for tissue survival. Study of the microvasculature often involves immunohistological methods. While useful for visualizing microvasculature at the m m scale in specific regions of interest, immunohistology is not well suited to visualize the global microvascular architecture in an organ. Hence, use of immunohistology precludes visualization of the entire microvasculature of an organ, and thus impedes study of global changes in the microvasculature that occur in concert with changes in tissue due to various disease states. Therefore, there is a critical need for a simple, relatively rapid technique that will facilitate visualization of the microvascular network of an entire tissue. Methodology/Principal Findings: The systemic vasculature of a mouse is stained with the fluorescent lipophilic dye DiI using a method called ‘‘vessel painting’’. The brain, or other organ of interest, is harvested and fixed in 4% paraformaldehyde. The organ is then sliced into 1 mm sections and optically cleared, or made transparent, using FocusClear, a proprietary optical clearing agent. After optical clearing, the DiI-labeled tissue microvasculature is imaged using confocal fluorescence microscopy and adjacent image stacks tiled together to produce a depth-encoded map of the microvasculature in the tissue slice. We demonstrated that the use of optical clearing enhances both the tissue imaging depth and the estimate of the vascular density. Using our ‘‘optical histology’’ technique, we visualized microvasculature in the mouse brain to a depth of 850 m m. Conclusions/Significance: Presented here are maps of the microvasculature in 1 mm thick slices of mouse brain. Using combined optical clearing and optical imaging techniques, we devised a methodology to enhance the visualization of the microvasculature in thick tissues. We believe this technique could potentially be used to generate a three-dimensional map of the microvasculature in an entire organ. Citation: Moy AJ, Wiersma MP, Choi B (2013) Optical Histology: A Method to Visualize Microvasculature in Thick Tissue Sections of Mouse Brain. PLoS ONE 8(1): e53753. doi:10.1371/journal.pone.0053753 Editor: Irene Georgakoudi, Tufts University, United States of America Received February 24, 2012; Accepted December 4, 2012; Published January 23, 2013 Copyright: s 2013 Moy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the Arnold and Mabel Beckman Foundation, the National Institutes of Health grant award R01 HD065536, and the National Institutes of Health Laser Microbeam and Medical Program (LAMMP, a P41 Technology Research Resource, grant number EB015890). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: choib@uci.edu investigated in tumors [1–4], the cardiovascular system [5–7], the brain [8], the eye [9,10], and skin [11,12]. The gold-standard method of visualizing tissue microvascula- ture is the well-established immunohistochemistry technique [13– 15]. Briefly, immunohistochemistry involves the staining of thin tissue slices with fluorescently-labeled antibodies that bind selectively to molecular markers of interest in the tissue, such as the CD31 marker for microvasculature. Once the tissues are stained, a fluorescence microscope is used to visualize and obtain images of the markers that can aid researchers in investigating and understanding the disease state of the tissue. The tissue slices used in immunohistochemistry are typically 1–5 m m in thickness and allow for very detailed, cellular-level interrogation of tissue. Using immunohistochemistry to visualize microvasculature in tissue yields extremely detailed images, but is limited to small spatial regions of interest, typically on the order of hundreds of m m. A single immunohistology slice will allow visualization of several Introduction The survival of mammalian tissue is dependent on the delivery of nutrients and growth factors and the exchange of oxygen and carbon dioxide gases. Nutrient delivery and gas exchange is done through the blood, which travels through the large feeder blood vessels of the vascular system to a network of small blood vessels and finally to the tissue. This network of small blood vessels is known as the microvasculature and its primary functions are twofold: 1) to transport blood to the tissue 2) in doing so, to facilitate delivery of essential nutrients from the blood and exchange gases with the surrounding tissue that are necessary for tissue survival. Thus, the microvasculature plays a pivotal role in the survival and function of tissue and, as a result, the study of the microvasculature in both normal and diseased states is important. The role of the microvasculature has previously been PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e53753