Abstract:We describe a new ultrahigh speed Spectral OCT instrument making use of a CMOS camera and demonstrate high quality in vivo imaging of the anterior segment of the human eye. The high flexibility of the designed imaging system allows a wide range of imaging protocols. Two-and three-dimensional high quality OCT images of the cornea, the anterior chamber and the crystalline lens are presented. A high acquisition rate, up to 135,000 A-scans/second enables three-dimensional reconstruction of the anterior segment during lenticular accommodation, blinking and pupillary reaction to light stimulus. We demonstrate OCT tomographic real time imaging of the lens dynamics during accommodation and high quality OCT cross-sectional images of the entire anterior segment of the eye from the cornea up to posterior part of the crystalline lens. 2009 Optical Society of America

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Speckle pattern, which is inherent in coherence imaging, influences significantly axial and transversal resolution of Optical Coherence Tomography (OCT) instruments. The well known speckle removal techniques are either sensitive to sample motion, require sophisticated and expensive sample tracking systems, or involve sophisticated numerical procedures. As a result, their applicability to in vivo real-time imaging is limited. In this work, we propose to average multiple A-scans collected in a fully controlled way to reduce the speckle contrast. This procedure involves non-coherent averaging of OCT A-scans acquired from adjacent locations on the sample. The technique exploits scanning protocol with fast beam deflection in the direction perpendicular to lateral dimension of the cross-sectional image. Such scanning protocol reduces the time interval between A-scans to be averaged to the repetition time of the acquisition system. Consequently, the averaging algorithm is immune to bulk motion of an investigated sample, does not require any sophisticated data processing to align cross-sectional images, and allows for precise control of lateral shift of the scanning beam on the object. The technique is tested with standard Spectral OCT system with an extra resonant scanner used for rapid beam deflection in the lateral direction. Ultrahigh speed CMOS camera serves as a detector and acquires 200,000 spectra per second. A dedicated A-scan generation algorithm allows for real-time display of images with reduced speckle contrast at 6 frames/second. This technique is applied to in vivo imaging of anterior and posterior segments of the human eye and human skin.

We introduce a new type of scanning protocols, called segmented protocols, which enable extracting multi-range flow velocity information from a single Spectral OCT data set. The protocols are evaluated using a well defined flow in a glass capillary. As an example of in vivo studies, we demonstrate two- and three-dimensional imaging of the retinal vascular system in the eyes of healthy volunteers. The flow velocity detection is performed using a method of Joint Spectral and Time domain OCT. Velocity ranging is demonstrated in imaging of retinal vasculature in the macular region and in the optic disk area characterized by different flow velocity values. Additionally, an enhanced visualization of retinal capillary network is presented in the close proximity to macula.

Abstract:We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 µm axial and 0.4 µm lateral resolution maintained over a depth of 40 µm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.