Quantitative cerebral blood flow imaging with extended-focus optical coherence microscopy

Quantitative three-dimensional blood flow imaging is a valuable technique to investigate the physiology of the brain. Two-photon microscopy (2PM) allows quantification of the local blood flow velocity with micrometric resolution by performing repeated line scans, but prohibitively long measurement times would be required to apply this technique to full three-dimensional volumes. By multiplexing the image acquisition over depth, Fourier domain optical coherence tomography (FDOCT) enables quantification of blood flow velocities with a high volume acquisition rate, albeit at a relatively low spatial resolution. Extended-focus optical coherence microscopy (xfOCM) increases the lateral resolution without sacrificing depth of field and therefore combines the high volume acquisition rate of FDOCT with a resolution comparable to 2PM. Here, we demonstrate high-resolution quantitative imaging of the blood flow velocity vector's magnitude in the adult murine brain with xfOCM.

Functional and Structural Imaging with Optical Coherence Microscopy

Arno Pino Bouwens

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.

EPFL2013

Structural and functional brain imaging using extended-focus optical coherence tomography and microscopy

Paul James Marchand

Neuroimaging techniques aim at revealing the anatomy and functional organisation of cerebral structures. Over the past decades, functional magnetic resonance imaging (fMRI) has revolutionized our understanding of human cerebral physiology through its ability to probe neural activity throughout the entire brain in a non-invasive fashion. Nevertheless, despite recent technological improvements, the spatial resolution of fMRI remains limited to a few hundreds of microns, restricting its use to macroscopic studies. Microscopic imaging solutions have been proposed to circumvent this limitation and have enabled revealing the existence of various cerebral structures, such as neuronal and vascular networks and their contribution to information processing and blood flow regulation within the brain. Optical imaging has proven, through its increased resolution and available contrast mechanisms, to be a valuable complement to fMRI for cellular-scale imaging. In this context, we demonstrate here the capabilities of an extension of optical coherence tomography, termed extended-focus optical coherence tomography (xf-OCT), in imaging cerebral structure and function at high resolution and very high acquisitions rates. Optical coherence tomography is an interferometric imaging technique using a low-coherence illumination source to provide fast, three-dimensional imaging of the back-scattering of tissue and cells. By multiplexing the interferometric ranging over several spectral channels, Fourier-domain OCT performs depth-resolved imaging at very high acquisition rates and high sensitivity. Increasing the lateral resolution of optical systems typically reduces the available depth-of-field and thus hampers this depth multiplexing advantage of OCT. Extended-focus systems aim at alleviating this trade-off between imaging depth and lateral resolution through the use of diffraction-less beams such as Bessel beams, providing high resolution imaging over large depths. The xf-OCT system therefore combines fast acquisition rates and high resolution, both characteristics required to image and study the structure and function of microscopic constituents of cerebral tissue. In this work, we performed functional brain imaging using the ability of xf-OCT to obtain quantita- tive measurements of blood flow in the brain. Changes in blood velocity evoked by neuronal activation were monitored and maps of hemodynamic activity were generated by adapting tools routinely used in fMRI to xf-OCT imaging. Additionally, three novel xf-OCT instruments are presented, wherein the advantages of different spectral ranges are exploited to reach specific imaging parameters. The increased contrast and resolution afforded by an illumination in the visible spectral range was used in two extended-focus optical coherence microscopy (xf-OCM) implementations for subcellular imaging of ex-vivo brain slices and cellular imaging of neurons, capillaries and myelinated axons in the superficial cortex in-vivo. Subsequently, an xf-OCT system is presented, operating in the infrared spectral range, wherein the reduced scattering enabled imaging the smallest capillaries deep in the murine cortex in-vivo.

EPFL2018

In vivo imaging of pancreatic endocrine islets

Michael Friedrich, Joan Goulley, Anne Grapin-Botton, Theo Lasser, Rainer Leitgeb, Christophe Pache, Martin Villiger

Extended focus optical coherence microscope (xfOCM) circumvents the compromise between lateral resolution and depth of field by us of a Bessel-like illumination beam. The high sensitivity and parallel depth profiling of Fourier domain optical coherence tomography are preserved, and combined with high isotropic resolution of 1.5 - 2 μm. To comply with the requirements for in vivo measurements, beam scanning had to be implemented. We then performed measurements, first of excised pancreas, validated by standard immunohistochemistry, to investigate the structures that can be observed. For a quantitative analysis, a semi-automatic islet detection algorithm evaluated the islet size, position, contrast and homogeneity. The influence of streptozotocin on the signature of the islets was investigated in a next step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo, visualizing pancreatic lobules, ducts, blood vessels and individual islets of Langerhans.