Kumar Abhishek

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Kumar
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Abhishek
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  • Preprint
    Visualizing calcium flux in freely moving nematode embryos
    ( 2017-02) Ardiel, Evan L. ; Kumar, Abhishek ; Marbach, Joseph ; Christensen, Ryan ; Gupta, Rishi ; Duncan, William ; Daniels, Jonathan S. ; Stuurman, Nico ; Colón-Ramos, Daniel ; Shroff, Hari
    The lack of physiological recordings from Caenorhabditis elegans embryos stands in stark contrast to the comprehensive anatomical and gene expression datasets already available. Using light-sheet fluorescence microscopy (LSFM) to address the challenges associated with functional imaging at this developmental stage, we recorded calcium dynamics in muscles and neurons and developed analysis strategies to relate activity and movement. In muscles, we found that the initiation of twitching was associated with a spreading calcium wave in a dorsal muscle bundle. Correlated activity in muscle bundles was linked with early twitching and eventual coordinated movement. To identify neuronal correlates of behavior, we monitored brain-wide activity with subcellular resolution and identified a particularly active cell associated with muscle contractions. Finally, imaging neurons of a well-defined adult motor circuit, we found that reversals in the eggshell correlated with calcium transients in AVA interneurons.
  • Article
    Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy
    (Nature Publishing Group, 2017-11-13) Wu, Yicong ; Kumar, Abhishek ; Smith, Corey ; Ardiel, Evan L. ; Chandris, Panagiotis ; Christensen, Ryan ; Rey-Suarez, Ivan ; Guo, Min ; Vishwasrao, Harshad D. ; Chen, Jiji ; Tang, Jianyong ; Upadhyaya, Arpita ; La Riviere, Patrick J. ; Shroff, Hari
    Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.
  • Article
    A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults
    (Frontiers Media, 2022-10-07) Smith, Jayson J. ; Kenny, Isabel W. ; Wolff, Carsten ; Cray, Rachel ; Kumar, Abhishek ; Sherwood, David R. ; Matus, David Q.
    Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adultstages.
  • Article
    Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium
    (American Society for Cell Biology, 2016-05-18) Dundon, Samantha E.R. ; Chang, Shyr-Shea ; Kumar, Abhishek ; Occhipinti, Patricia ; Shroff, Hari ; Roper, Marcus ; Gladfelter, Amy S.
    Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus’ transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale.