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Publisher Summary This chapter describes a new cytometry technique based on the combination of Two-photon microscopy (TPM) and high-speed imaging techniques. TPM is a high-resolution fluorescence microscopy technique that provides 3D images of cells and tissues. It is well suited for imaging tissues. By incorporating high-speed image capabilities, it provides images of large populations of cells within their native tissue environment using TPM and extends standard 2D image cytometry into 3D. The native environment of cells is within the 3D environment of their host tissue but standard preparation for both flow and image cytometry involves the dissociation of cells from their tissue matrix. The chapter provides an overview of some of the early cytometry techniques such as flow, image, and tissue-based cytometry. Flow cytometry (FCM) is conducted with cells in suspension, whereas image cytometry studies are typically conducted in 2D culture. The biochemical, mechanical, and intracellular inputs present in a tissue affect the behavior of a cell. The underlying principles and instrumentation of two-photon tissue cytometry and some emerging applications of this technology are also described.