KEYWORDS: Optical imaging, Confocal microscopy, Stereoscopy, 3D image processing, In vivo imaging, Signal to noise ratio, Scattering, Microscopy, Microscopes, In vitro testing
Fluorescent genetically encoded voltage indicators can be combined with optical imaging to provide high-throughput electrophysiologic recordings with single-spike resolution and subthreshold sensitivity. Such voltage imaging is highly demanding in terms of signal collection; thus, most experiments have been performed with widefield one-photon microscopy. Unfortunately, widefield techniques are susceptible to out-of-focus background and scattering, which degrades SNR, especially in high-density slice or in vivo experiments. In this work, we describe a multi-plane near-kHz-rate confocal microscope that effectively suppresses undesired background. This technique enables more densely labeled in vitro and in vivo imaging experiments, critical for the dissection of neural circuit dynamics.
In this presentation we will describe a new method for non-contact in vivo corneal and lenticular microscopy. The technique is based on fundus retroillumination, i.e. anterior segment back-illumination via reflection from the posterior fundus. As such, the retroillumination microscope provides a unique transmission imaging configuration sensitive to forward-scattered light. To enhance intrinsic phase-gradient contrast, we apply asymmetric illumination. The technique produces micron-scale lateral resolution images across a large 1 mm diagonal field of view in the central cornea. We will show representative images of the epithelium, the subbasal nerve plexus, large stromal nerves, dendritic immune cells, endothelial nuclei, and the anterior crystalline lens fibers and nuclei. Finally, we will discuss potential clinical applications and extension to three-dimensional imaging.
Widefield ocular fundus imaging is conventionally performed in a reflection geometry. In this configuration, back-reflections from inner retinal layers, such as the nerve fiber layer, the inner limiting membrane, or even the anterior walls of large blood vessels, are often encountered, and may obscure the visibility of deeper features. Moreover, spectroscopic quantification of endogenous chromophores is complicated since the final image is a summation of reflections from several fundus layers (i.e. no single absorption pathlength can safely be assumed). Researchers have sought to model the fundus reflections, however the models are sensitive to the populations used and particular imaging platform. In theory, unwanted superficial reflections could be avoided and light path modeling could be simplified by adopting a transmission imaging geometry. We present an alternative transillumination fundus imaging strategy based on deeply penetrating near-infrared (NIR) light delivered transcranial near the subject’s temple. A portion of this light diffuses through bone and illuminates the posterior eye not from the front, as with conventional methods, but rather mostly from behind. As such, we image light transmitted through the fundus rather than back-reflected off multiple fundus layers. This single-pass measurement geometry simplifies absorption pathlength considerations and provides complementary information to fundus reflectometry. The use of NIR light enables imaging as deep as the choroid. Importantly, the technique is compatible with reflection-based techniques and we have shown that it works well with a commercial non-mydriatic fundus camera. Combining information from these two illumination approaches should improve spectroscopic analysis of the fundus.
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