The use of optical clearing and multiphoton microscopy for investigation of three-dimensional tissue-engineered...

Recent advances in three-dimensional (3D) tissue engineering have concomitantly generated a need for new methods to visualize and assess the tissue. In particular, methods for imaging intact volumes of whole tissue, rather than a single plane, are required. Herein, we describe the use of multiphoton microscopy, combined with optical clearing, to noninvasively probe decellularized lung extracellular matrix scaffolds and decellularized, tissue-engineered blood vessels. We also evaluate recellularized lung tissue scaffolds. In addition to nondestructive imaging of tissue volumes greater than 4 mm(3), the lung tissue can be visualized using three distinct signals, combined or singly, that allow for simple separation of cells and different components of the extracellular matrix. Because the 3D volumes are not reconstructions, they do not require registration algorithms to generate digital volumes, and maintenance of isotropic resolution is not required when acquiring stacks of images. Once a virtual volume of tissue is generated, structures that have innate 3D features, such as the lumens of vessels and airways, are easily animated and explored in all dimensions. In blood vessels, individual collagen fibers can be visualized at the micron scale and their alignment assessed at various depths through the tissue, potentially providing some nondestructive measure of vessel integrity and mechanics. Finally, both the lungs and vessels assayed here were optically cleared, imaged, and visualized in a matter of hours, such that the added benefits of these techniques can be achieved with little more hassle or processing time than that associated with traditional histological methods.