Functional and Structural Imaging with Optical Coherence Microscopy

Fluorescence microscopy techniques are well established research tools and have proven their use in a large variety of biomedical applications. Microscopic molecular contrast is achieved by imaging fluorescent dyes that bind specifically to a molecule of interest and gene expression can be imaged by genetically modifying organisms to express fluorescent proteins. This versatile technique has, however, three limitations: fluorescent labels can cause toxicity or have an unwanted influence on the process under study; the photobleaching effect limits observation time; and the highest acquisition speed is restricted by the fluorophore’s brightness and by the raster scan required for 3-D imaging. For applications where direct molecular contrast is not essential, label-free imaging offers a solution to these issues and can simplify imaging experiments. In this work, we present methods to perform 3-D label-free imaging of the anatomy (structural imaging) and physiology (functional imaging) of living tissue and cells with optical coherence microscopy (OCM). OCM is founded on low-coherence interferometry and acquires 3-D images of the local light back-scattering strength from within highly scattering biological tissue. By measuring in the spectral or Fourier domain, image acquisition is multiplexed over depth and achieves high sensitivity. OCM can therefore offer an important gain in acquisition speed, under the condition that lateral resolution is maintained over the imaging depth. This is realized by extended focus OCM (xfOCM), which uses Bessel beam illumination to create a high lateral resolution over an extended depth of field. The combination of high 3-D resolution and fast acquisition is especially useful for in vivo experiments, where measurement time is limited, and when fast processes are studied. We demonstrate long-term in vivo imaging with xfOCM of amyloid plaque in a mouse model of Alzheimer’s disease (AD), without administration of extrinsic contrast agents. By implementing a 3-D image segmentation algorithm, we further show how xfOCM can be employed to perform a label-free ex vivo transversal study of the development of amyloid plaque in the mouse brain. The high acquisition speed obtained from multiplexing depth acquisition is further exploited to image cerebral blood flow. We first analyze the Doppler frequency spectrum measured by OCM when scatterers such as erythrocytes flow through the system’s focus and show how it relates to the axial and lateral flow velocity components. This then enables quantitative blood flow imaging with xfOCM, with in an unprecedented combination of 3-D resolution and acquisition speed. We apply this technique to label-free in vivo angiography and quantitative blood flow imaging in the murine brain. xfOCM achieves deep tissue penetration due to the use of a near-infrared spectrum. The study of cell cultures, however, does not require such deep penetration, but instead could benefit from higher resolution. For these applications, we introduce visible spectrum OCM (visOCM), enabling label-free and long-term imaging of living cell cultures with 3-D sub-micron resolution. visOCM allows cell morphology to be imaged in thin single layer cultures, but also in thicker three-dimensional cultures and organotypic slices, for which no label-free tomographic microscopy exists to date.