RESUMEN
We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low-photodamage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime, enhancing its penetration depth. Our present study uses a conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.
RESUMEN
Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients - one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.
Asunto(s)
Células Madre Pluripotentes Inducidas , Succionato-Semialdehído Deshidrogenasa , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Femenino , Succionato-Semialdehído Deshidrogenasa/deficiencia , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/patología , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas , Discapacidades del DesarrolloRESUMEN
We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally, we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.