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DURIP Success: Confocal Microscope System

In 2021, Prof. Diane Hoffman-Kim's research group at Brown University was awarded a DURIP to purchase a Nikon Confocal Microscope for imaging cortical spheroids

Neuroinflammation, oxidative stress, and apoptotic and necrotic cell death are some of the biological responses that occurs in the minutes to days after the initial injury from TBI. The details about injury threshold progression are not well understood; therefore, it is critical that the brain injury progression is explored in order to fully understand the pathological dynamics of TBI. Our lab is working to study the TBI progression from multiple modalities of TBI in our 3D neurospheroids.

The neurospheroids are cultured for 10-14 days in vitro and then subjected to, for example, rotational acceleration or blunt impact TBI. To assess the degree of brain injury, neurospheroids are stained with calcein AM (CaAM) to label live cells and ethidium homodimer-1 (EthD1) to label dead cells and evaluated hours to days post injury using confocal microscopy imaging. Confocal microscopy is used to functionally characterize the neurospheroids through calcium imaging, and for immunohistochemistry staining of structural biomarkers including neurite networks and glial cells.

Experimental end-point analysis relies on confocal microscopy and is performed in the minutes to days following injury; therefore, it is critical that we have access to a confocal to examine the neurospheroids at multiple post injury timepoints in order to understand the pathological progression of TBI. Utilizing confocal microscopy as our end-point injury analysis provides many advantages including; reduction of background interference, reproducibility, and the ability to collect serial optical sections from our

neurospheroids. Therefore, having access to the confocal for precisely timed and longitudinal experiments optimizes our ability to carry out multiple experiments with complex injury modalities and outputs, including our repeat TBI experiments and our TBI biomarker experiments



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