KEYWORDS: In vivo imaging, Light sources and illumination, Confocal microscopy, Signal to noise ratio, Optical imaging, Neuroimaging, Crosstalk, Brain, Tissues, Tissue optics
Advancements in genetically encoded voltage indicators (GEVIs) have made it possible to measure cellular membrane potential changes optically. But performing GEVI imaging in vivo remains highly challenging due to factors such as low GEVI concentrations, modest signal dynamic range, tissue scattering, out-of-focus fluorescence. To address these challenges, we developed a microscopy technique that take advatanges from both widefield targeted illumination and confocal background rejection, enabling high SNR low crosstalk GEVI imaging across millimeter fields-of-view, at supra-kilohertz frame rates, over extended durations, and at high penetration depths. We demonstrate our technique under a variety of imaging conditions across multiple brain regions and with different classes of GEVIs.
Recent improvements in genetically encoded voltage indicators have enabled optical imaging of action potentials and subthreshold membrane voltage from single neurons in the mammalian brain. However, most current voltage imaging techniques can only simultaneously sample a few cell, limited either by strong background or small field-of-view. We show that, both theoretically and experimentally, targeted illumination with a widefield microscopy can significantly improve voltage imaging performance by improving signal contrast and reducing background cross-contamination. With such improvements, we demonstrated large-scale voltage imaging with fully genetically encoded voltage indicator SomArchon from tens of neurons over a large anatomical area, while maintaining signal contrast over a prolonged recording duration of several continuous minutes.
High-speed laser scanning microscopy is crucial for monitoring fast dynamical events. We here present a novel strategy that enables ultrafast lateral optical scanning for high-speed high-throughput laser scanning microscopes. Our technique is flexible that it can be adapted to one-dimensional line scan or two-dimensional frame scan, with pixel rate up to tens of megahertz. We integrate this strategy into a two-photon microscope for kilohertz frame-rate imaging.
Significance: Digital holographic microscopy is widely used to get the quantitative phase information of transparent cells.
Aim: However, the sample phase is superimposed with aberrations. To quantify the phase information, aberrations need to be fully compensated.
Approach: We propose a technique to obtain aberration-free phase imaging, using the derivative-based principal component analysis (dPCA).
Results: With dPCA, almost all aberrations can be extracted and compensated without requirements on background segmentation, making it efficient and convenient.
Conclusions: It solves the problem that the conventional principal component analysis (PCA) algorithm cannot compensate the common but intricate higher order cross-term aberrations, such as astigmatism and coma. Moreover, the dPCA strategy proposed here is not only suitable for aberration compensation but also applicable for other cases where there exist cross-terms that cannot be analyzed with the PCA algorithm.
We present a single-shot multiplane widefield imaging strategy using a z-splitter prism, which can be assembled from off-the-self components and only requires a single camera. We further introduce a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging, making it a versatile tool for a wide range of biological or biomedical imaging applications.
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