RESUMEN
A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creaHng designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light-sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.
RESUMEN
Dispersion remains an enduring challenge for the characterization of wavelength-dependent transmission through optical multimode fiber (MMF). Beyond a small spectral correlation width, a change in wavelength elicits a seemingly independent distribution of the transmitted field. Here we report on a parametric dispersion model that describes mode mixing in MMF as an exponential map and extends the concept of principal modes to describe the fiber's spectrally resolved transmission matrix (TM). We present computational methods to fit the model to measurements at only a few, judiciously selected, discrete wavelengths. We validate the model in various MMF and demonstrate an accurate estimation of the full TM across a broad spectral bandwidth, approaching the bandwidth of the best-performing principal modes, and exceeding the original spectral correlation width by more than two orders of magnitude. The model allows us to conveniently study the spectral behavior of principal modes, and obviates the need for dense spectral measurements, enabling highly efficient reconstruction of the multispectral TM of MMF.
RESUMEN
Imaging through optical multimode fiber (MMF) has the potential to enable hair-thin endoscopes that reduce the invasiveness of imaging deep inside tissues and organs. Active wavefront shaping and fluorescent labeling have recently been exploited to overcome modal scrambling and enable MMF imaging. Here, we present a computational approach that circumvents the need for active wavefront control and exogenous fluorophores. We demonstrate the reconstruction of depth-gated confocal images through MMF using a raster-scanned, focused input illumination at the fiber proximal end. We show the compatibility of this approach with quantitative phase, dark-field, and polarimetric imaging. Computational imaging through MMF opens a new pathway for minimally invasive imaging in medical diagnosis and biological investigations.
RESUMEN
Reciprocity is a fundamental principle of wave physics and directly relates to the symmetry in the transmission through a system when interchanging the input and output. The coherent transmission matrix (TM) is a convenient method to characterize wave transmission through general media. Here, we demonstrate the optical reciprocal nature of complex media by exploring their TM properties. We measured phase-corrected TMs of forward and round-trip propagation in a single polarization state through a looped 1 m-long step-index optical multimode fiber (MMF) to experimentally verify a transpose relationship between the forward and backward transmission. This symmetry impedes straightforward MMF calibration from proximal measurements of the round-trip TM. Furthermore, we show how focusing through the MMF with digital optical phase conjugation is compromised by system loss since time reversibility relies on power conservation. These insights may inform the development of new imaging techniques through complex media and coherent control of waves in photonic systems.
RESUMEN
Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices.SIGNIFICANCE STATEMENT A new technique for simultaneous optogenetic stimulation and calcium imaging across wide areas of brain slice enables high-throughput mapping of neuronal excitability and synaptic transmission.
