Acceleration of Neuroscience Research Discovery by Incorporation of Large Area/Volume Microscopic Data Workflows

Anastas Popratiloff, Cheryl Clarkson, Zahra Motahari, Christine A. Brantner
2018 Microscopy and Microanalysis  
Modern microscopes are becoming increasingly complex instruments enabling registration of image sets far beyond a single field of view. This is being achieved by integration of sophisticated scanning stages, capable of moving the field of view in precise synchrony with acquisition, providing reliable meta data encoding time, space and multiple imaging modalities. As a result, increasingly complex multidimensional microscopic data sets are being generated and analyzed. In addition to executing
more » ... tion to executing different imaging modalities, the modern instruments are capable of sample manipulation, which further increases the complexity and size of the data sets that are routinely being analyzed. For all these reasons, multifaceted workflows are required from sample preparation, through imaging to structuring and analyses of the image data. Normal and pathological studies of neuronal networks are a prime example of where complex image data facilitates our understanding of structural organization of neuronal circuits and the specific domains of emerging pathological conditions. The needs of large image data sets is amplified by the fact that in the central nervous system (CNS), cell bodies, axonal terminals and dendritic processes span very large volumes, which ideally should be included in the image sets at appropriate resolution. On the other hand, it is important to identify the pathological process at its emergence, where only rare events, representing foci of the nascent pathological process are present in otherwise normal brain tissue. In this talk, we provide several examples of how modern light, electron and correlative microscopy facilitated our effort to identify the underlying pathology associated with feeding and swallowing deficits present in a mouse model of DiGeorge Syndrome (22q11DS, LgDel). Tissue clearing techniques enabling the imaging of whole organisms have exploded recently and dramatically enhanced our perception of brain architecture and connectivity. Our group uses a 100-yearold technique -BABB [3], [2], to clear whole mouse embryos with the goal of identifying abnormal axonal growth of trigeminal motor and sensory neurons in LgDel mouse. We combine tracing with biocytin/Alexa Fluor and wide dynamic range confocal imaging to produce image sets that support reliable reconstruction of a single neuron and its processes, spanning from the rhombomeres to anterior tip of the mandibular branchial arch. We use hybrid detector -a GaAsP photocathode coupled to avalanche photomultiplier in photon counting regime on Leica SP8. Thus, our experimental setup allows us to register on the same large volume of the embryo, the signal from areas with high photon yield, such as the injection site to very low photon readouts, such as the smallest neuronal processes. Sampling rate was kept at 10% of the excitation rate (80MGHz) of the source to achieve linearity. An oil immersion objective lens matches the refractive index (RI) of BABB and offers large working distance to capture half of the embryo (500 m). As a result of matching RI, the spherical aberrations are minimized leading to the production of robust 3D volumes and segmentation of the axonal trajectories. Unlike BABA, newer clearing approaches such as Clarity do preserve fluorescence from reporter proteins. Additionally, we have also applied passive Clarity clearing, which requires a highly specialized lens (Leica HC Fluotar L 25x/1.0, IMM (n=1.457)), but produces image sets with unparalleled quality. Whereas the light microscopy imaging technology had improved dramatically, the resolution is still limited close to diffraction. Moreover, the amount of cellular and tissue information is restricted to a
doi:10.1017/s1431927618006876 fatcat:o3d76gxghra55kh2ba5nyrex64