Optical coherence tomography (OCT) is a non-contact method for imaging the topological and internal microstructure of samples in three dimensions. OCT can be configured as a conventional microscope, as an ophthalmic scanner, or using endoscopes and small diameter catheters for accessing internal biological organs. In this Primer, we describe the principles underpinning the different instrument configurations that are tailored to distinct imaging applications and explain the origin of signal, based on light scattering and propagation. Although OCT has been used for imaging inanimate objects, we focus our discussion on biological and medical imaging. We examine the signal processing methods and algorithms that make OCT exquisitely sensitive to reflections as weak as just a few photons and that reveal functional information in addition to structure. Image processing, display and interpretation, which are all critical for effective biomedical imaging, are discussed in the context of specific applications. Finally, we consider image artifacts and limitations that commonly arise and reflect on future advances and opportunities. ! hundreds of images per second while preserving the resolution and overall quality of previous time-domain technology. [17][18][19] With the development of essential component technologies, fd-OCT technology became a better fit to the requirements of endoscopic applications and imaging in highly scattering tissues; sd-OCT more naturally met the needs in ophthalmology and retinal imaging in particular. Although the underlying factors have shifted somewhat in later years, sd-OCT remains most prevalent in ophthalmic imaging and fd-OCT more common in endoscopic applications. Other specific attributes of the Fourier-domain approaches determine which is optimal for angiographic, polarimetric and elastographic imaging.In ophthalmology, OCT has become a standard of care for retinal imaging since 2003[20] and it is routinely used to guide refractive and cataract surgeries. In cardiology, intravascular OCT is internationally available for guiding coronary stenting and is routinely used in research related to coronary artery disease and acute myocardial infarction. [21] In gastroenterology, endoscopic OCT provides the ability to comprehensively interrogate the esophagus for neoplastic change and intramucosal cancer. [22] Although endobronchial OCT is not yet commercially available, research studies have shown its ability to diagnose and monitor pulmonary disease. [23] In preclinical research, OCT is routinely used to image small animal tumor models to investigate features such as angiogenesis and the response to novel anti-angiogenic therapies. [24] The field of OCT continues to evolve, expanding into new applications, and capitalizing on new instrument capabilities. In this Primer, we present the principles that underpin reflectance, polarimetric, angiographic, and elastographic imaging with OCT and describe special requirements in the most common biomedical imaging applications. In addition, we provid...