Imaging Oral Presentations

Cell motility requires the orchestration of many dynamic cellular systems. To understand their interactions, we are manipulating protein activity with seconds and micron resolution in living cells and animals. This talk will describe new tools to identify and control long range allosteric interactions, using inserted domains to confer response to either small molecules or light. A new optogenetic building block (Zdark) that binds selectively to the dark state of the LOV domain has been used in approaches for versatile control of protein activity, regulation of nuclear translocation, and control of endogenous proteins with light. These tools have been designed to be easy to use, and applicable to a broad range of proteins.

Imaging in living biological specimens is limited by the amount of illumination exposure the sample can survive, the environmental conditions in the imaging chamber and the light scattering properties of the sample itself. Selective Plane Illumination Microscopy (SPIM and a host of other like modality monikers) addresses these concerns like no other optical imaging system. It does so by using a sheet of illumination at only the imaged plane thus reducing photo damage dramatically, especially in combination with high-resolution, greater than video rate sCMOS cameras as a detector. Sample rotation can enable complete coverage of large, opaque specimens. These advances allow relatively large biological organisms to be imaged in 3D at single cell resolution over increasingly long periods of time. These developing technologies of light sheet will ultimately still require complimentary imaging modalities to continue into the meso scale of vertebrate model systems. µPET/CT/SPECT/MR instrumentation, while utilizing a different range of the electromagnetic spectrum are using similar technology advances and approaches to increase the temporal and spacial resolution. For example, µCT is capable of <40µm resolution and functional MR data can be collected in 100 ms increments. In core facilities like the University Imaging Centers at the University of Minnesota (http://uic/umn/edu), these powerful technologies routinely produces massive, terabyte size datasets that have computationally challenging and time-consuming data set requirements, especially in the pre-processing visualization prior to analysis. Ultimately the data must be further mined and correlated. Existing software solutions implemented in Fiji, µManager, or in commercial softwares like Nikon Elements, are performing chained processing steps on a single computer where user input is required even for digital steps that are routinely needed. As these multi-modal data collection machines increase their output, such approaches become problematic since processing can take days. We are developing a workflow of the data management and image processing in an automated and traceable manner of a subset of the data so that effective decisions can be made on the validity of the collection while in process. We hope to develop an automated workflow with minimum user interaction that is easily scalable to multiple datasets or time points on a digital pipeline that would include options ranging from “low-cost” cluster options to High Performance Computing through our Minnesota Super Computing Institute (https://www.msi.umn.edu/research). Solutions with “cloud” providers like Google are also being explored. All with a goal that the visualization of the data is faster than the total acquisition time required for collecting the images.

Light sheet microscopy is rapidly becoming a main stream technology for fluorescence imaging in 3D. The whole plane illumination of light sheet allows for rapid 3D imaging with relatively high resolution. The development and continual refinement of sample clearing technologies has allowed light sheet to become the technology of choice for multi-colour 3D imaging of entire organs. The selective plane illumination and lack of out of focus fluorescence excitation and photo-damage has also made light sheet technology ideal for imaging of small relatively transparent living samples. This has allowed for tracking of cell movement and organ, tissue or organism development with high spatial and temporal resolution. Given the ever increasing utility of light sheet the available instrumentation has grown considerable but much of the equipment is developed with certain applications in mind. Therefore, core facility implementations of light sheet can be challenging. There is choice of equipment, the need to match the instrument to the needs of the core facility research community, challenges with sample preparation often including the use of highly toxic reagents and the challenges of working with massive 3D and 4D data sets and performing image analysis on these complex image sets. This round table will start with a brief overview of the current technologies available both commercially and as “home-built” systems. Then there will be an open discussion with experts in the field with advice on how to tackle the many challenges of implementing light sheet microscopy in a core facility setting.

The histological assessment of the central nervous system in three dimensions has previously been very time consuming and prone to errors of interpretation. Advances in optical systems, especially light sheet fluorescence microscopy, and new tissue clearing methods have dramatically improved visualization of fluorescently labeled axons and support cells. Investigating axonal regeneration requires stringent axonal tracing methods as well as the use of animal models in which transgenic axonal labeling is not available. Using rodent models of optic nerve and spinal cord injury, we labeled axon tracts of interest using Adeno Associated Virus and chemical tracers and performed chemical-based tissue clearing to image multiple axon types using light sheet and confocal microscopy. We also investigated the relationships between axons and scar-forming cells at the injury site as well as connections between sensory axons and motor pools in the spinal cord. Finally, we used these methods to trace axons in non-human primates. This reproducible and adaptable virus-based approach can be combined with transgenic mice or with chemical-based tract-tracing methods, providing scientists with flexibility in obtaining axonal trajectory information from transparent tissue.