A Spectral Phasor Perspective in Zebrafish Muscle Development

Hyperspectral imaging provides the potential for assessing biochemical interactions in the zebrafish embryo in a label-free manner that extends beyond conventional morphological and molecular phenotyping. It takes advantage of the intrinsic wavelengths emitted or reflected from a sample without the need for extrinsic staining methods. The specific spectral signature from a sample can arise from chemical interactions, molecular bonds and macro-structural arrangements. A challenge in hyperspectral imaging is the large spectral data sets that result from acquiring a spectrum for every pixel within an image. Spectral Phasor offers an efficient representation of the spectral data as vectors in Fourier space, thereby condensing each spectrum into a single point in a 2-D plot. The Spectral Phasor has been successfully applied to hyperspectral data on protein samples, demonstrating changes in fluorescence signatures. This study proposes an application of Spectral Phasor to the zebrafish muscle development. The skeletal muscle system provides an attractive model for the proof-of-principle experiments in the implementation of Spectral Phasor. Skeletal muscle is a highly organized tissue with myofibrils as the functional unit that contributes to the repetitive segment of the myotome. The modularity of these units provides unique landmarks for anchoring the SP data. Our analysis of muscle suggest that SP can be used for staging the skeletal muscle development

Live 4D Imaging of the Embryonic Vertebrate Heart with Two-Photon Light Sheet Microscopy and Simultaneous Optical Phase Stamping

The developing vertebrate heart is a highly dynamic organ that starts to function early on during embryonic development, even as it continues to undergo dramatic morphological changes and cellular differentiation. Fast and high resolution three-dimensional (3D) imaging is needed to document the intrinsic cellular dynamics of the beating heart, as a critical step in understanding its development. To meet the challenges of obtaining sub-cellular resolution imaging of a dynamic 100-micron length scale 3D structure, which moves quasi-periodically at frequency of a few Hertz, over tens of microns amplitude, we have employed two-photon light sheet microscopy (2p-SPIM) and a novel independent optical phase stamping method to generate well-resolved 3D movies (4D) of the beating heart. Applying this 4D imaging modality to zebrafish embryos, we have found remarkable heterogeneity in cardiomyocyte morphology, gene expression, and behavior both during the cardiac cycle, and over the developmental time. The observed heterogeneity appears to play a key role in the maintenance of tissue geometry and cardiac output as the heart undergoes cycles of contraction and expansion. The variation in cellular morphology and behavior provide new insights into the tight link between cellular dynamics, mechanical environment exerted and felt by the beating heart, and the genetic program that governs not only the differentiation and construction but also the maintenance of this important organ

Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate with molecular sensitivity the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems

Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging

Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos

Pre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations.

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis

Eph-ephrin signaling modulated by polymerization and condensation of receptors

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour