Targeted spectroscopy in the eye fundus.

Significance: The assessment of biomarkers in the eye is rapidly gaining traction for the screening, diagnosis, and monitoring of ocular and neurological diseases. Targeted ocular spectroscopy is a technology that enables concurrent imaging of the eye fundus and analysis of high-quality spectra from a targeted region within the imaged area. This provides structural, compositional, and functional information of specific regions of the eye fundus from a non-invasive approach to ocular biomarker detection. Aim: The aim of our study was to demonstrate the multimodal functionality and validation of targeted ocular spectroscopy. This was done in vitro, using a reference target and a model eye, and in vivo. Approach: Images and spectra from different regions of a reference target and a model eye were acquired and analyzed to validate the system. Targeted ocular fluorescence spectroscopy was also demonstrated with the same model. Subsequently, in vivo imaging and diffuse reflectance spectra were acquired to assess blood oxygen saturation in the optic nerve head and the parafovea of healthy subjects. Results: Tests conducted with the reference target showed accurate spectral analysis within specific areas of the imaging space. In the model eye, distinct spectral signatures were observed for the optic disc, blood vessels, the retina, and the macula, consistent with the variations in tissue composition and functions between these regions. An ocular oximetry algorithm was applied to in vivo spectra from the optic nerve head and parafovea of healthy patients, showing significant differences in blood oxygen saturation. Finally, targeted fluorescence spectral analysis was performed in vitro. Conclusions: Diffuse reflectance and fluorescence spectroscopy in specific regions of the eye fundus open the door to a whole new range of monitoring and diagnostic capabilities, from assessment of oxygenation in glaucoma and diabetic retinopathy to photo-oxidation and photodegradation in age-related macular degeneration.

Sensitivity of visible range multi-wavelength algorithms for retinal tissue oximetry to acquisition parameters.

This study examined the sensitivity of broadband spectroscopy algorithms for retinal tissue oximetry to spectral acquisition parameters. Monte Carlo simulations were conducted on a 4-layer retinal model to assess the impact of various parameters. The optimal spectral range for accurate measurements was determined to be 530 nm to 585 nm. Decreased spectral resolution below 4 nm significantly reduced accuracy. Using an acquisition area larger than the blood vessel resulted in an underestimation of oxygen saturation, especially for high values. A threshold was observed where increased light intensity had no significant impact on measurement variability. The study highlights the importance of informed parameter selection for accurately assessing retinal microcapillary oxygenation and studying local hemodynamics.

Millimetric scale two-photon Bessel-Gauss beam light sheet microscopy with three-axis isotropic resolution using an axicon lens.

Significance: Typical light sheet microscopes suffer from artifacts related to the geometry of the light sheet. One main inconvenience is the non-uniform thickness of the light sheet obtained with a Gaussian laser beam. Aim: We developed a two-photon light sheet microscope that takes advantage of a thin and long Bessel-Gauss beam illumination to increase the sheet extent without compromising the resolution. Approach: We use an axicon lens placed directly at the output of an amplified femtosecond laser to produce a long Bessel-Gauss beam on the sample. We studied the dopaminergic system and its projections in a whole cleared mouse brain. Results: Our light sheet microscope allows an isotropic resolution of 2.4 µm in all three axes of the scanned volume while keeping a millimetric-sized field of view, and a fast acquisition rate of up to 34 mm2/s. With slight modifications to the optical setup, the sheet extent can be increased to 6 mm. Conclusion: The proposed system's sheet extent and resolution surpass currently available systems, enabling the fast imaging of large specimens.

Monte-Carlo simulation and tissue-phantom model for validation of ocular oximetry.

Ocular oximetry, in which blood oxygen saturation is evaluated in retinal tissues, is a promising technique for the prevention, diagnosis and management of many diseases and conditions. However, the development of new tools for evaluating oxygen saturation in the eye fundus has often been limited by the lack of reference tools or techniques for such measurements. In this study, we describe a two-step validation method. The impact of scattering, blood volume fraction and lens yellowing on the oximetry model is investigated using a tissue phantom, while a Monte Carlo model of the light propagation in the eye fundus is used to study the effect of the fundus layered-structure. With this method, we were able to assess the performance of an ocular oximetry technique in the presence of confounding factors and to quantify the impact of the choroidal circulation on the accuracy of the measurements. The presented strategy will be useful to anyone involved in studies based on the eye fundus diffuse reflectance.

Transcriptional repression of Plxnc1 by Lmx1a and Lmx1b directs topographic dopaminergic circuit formation.

Mesodiencephalic dopamine neurons play central roles in the regulation of a wide range of brain functions, including voluntary movement and behavioral processes. These functions are served by distinct subtypes of mesodiencephalic dopamine neurons located in the substantia nigra pars compacta and the ventral tegmental area, which form the nigrostriatal, mesolimbic, and mesocortical pathways. Until now, mechanisms involved in dopaminergic circuit formation remained largely unknown. Here, we show that Lmx1a, Lmx1b, and Otx2 transcription factors control subtype-specific mesodiencephalic dopamine neurons and their appropriate axon innervation. Our results revealed that the expression of Plxnc1, an axon guidance receptor, is repressed by Lmx1a/b and enhanced by Otx2. We also found that Sema7a/Plxnc1 interactions are responsible for the segregation of nigrostriatal and mesolimbic dopaminergic pathways. These findings identify Lmx1a/b, Otx2, and Plxnc1 as determinants of dopaminergic circuit formation and should assist in engineering mesodiencephalic dopamine neurons capable of regenerating appropriate connections for cell therapy.Midbrain dopaminergic neurons (mDAs) in the VTA and SNpc project to different regions and form distinct circuits. Here the authors show that transcription factors Lmx1a, Lmx1b, and Otx2 control the axon guidance of mDAs and the segregation of mesolimbic and nigrostriatal dopaminergic pathways.