The microvasculature serves an imperative function in regulating perfusion and nutrient exchange throughout the body, adaptively altering blood flow to preserve hemodynamic and metabolic homeostasis. Its normal functioning is vital to tissue health, whereas its dysfunction is present in many chronic conditions, including diabetes, heart disease, and cognitive decline. As microvascular dysfunction often appears early in disease progression, its detection can offer early diagnostic information. To detect microvascular dysfunction, one uses imaging to probe the microvasculature’s ability to react to a stimulus, also known as microvascular reactivity (MVR). An assessment of MVR requires an integrated understanding of vascular physiology, techniques for stimulating reactivity, and available imaging methods to capture the dynamic response. Practical considerations, including compatibility between the selected stimulus and imaging approach, likewise require attention. In this review, we provide a comprehensive foundation necessary for informed imaging of MVR, with a particular focus on the challenging endeavor of assessing microvascular function in deep tissues.