Thinning of tissue in vitreous ice so that macromolecular complexes may be viewed in their native environment for cryo-tomography may be accomplished by cryo-FIB milling (Focused Ion Beam Milling) or vitreous cryo-sectioning. Both techniques require expensive equipment and are technically challenging. This forum will focus on the practical aspects of these techniques so the attendee will have an understanding of the basic process for each technique as well as each techniques strengths and limitations. Vitreous cryo-sections have been the method of choice for thinning cells to 500nm or less but cryo-focused ion beam milling has become popular because samples are free of sectioning artifacts like compression and crevasses. Cryo-FIB samples may be milled into wedges, lamellas or the lift-out technique may be used to place the lamella on a TEM grid. The techniques may be combined to trim down bulk samples in the cryo-microtome before cryo-FIB milling a lamella. Recent developments in micromanipulator assisted cryo-sectioning have improved the quality of vitreous sections for tomography.
This symposium, focused on Atomic Force Microscopy (AFM), is intended to be a forum for the exchange of ideas and knowledge on the characterization of materials/biomaterials. Invited papers will include those utilizing AFM instrumentation and force spectroscopy techniques for characterization of structure-function relationships and materials properties of surfaces, films and interfaces. Specifically, topics including force spectroscopy, fast scanning, conductive tip, or new imaging methodologies are of interest. Target attendees will have a variety of backgrounds from engineering and/or scientific disciplines at all levels of analytical expertise.
Light sheet microscopy, also known as Selective Plane Illumination Microscopy (SPIM), has emerged in recent years as the technique of choice to capture dynamic biological processes over multiple spatial and temporal scales. The unique parallelized "sheet" illumination strategy of SPIM enables high imaging speed, high signal to noise ratio, and reduced photo-induced damage. I will review recent work in our own lab in implementing SPIM with nonlinear excitation, and in applying SPIM to a variety of in vivo imaging applications. Our current work will be described in extending and combining SPIM with other imaging approaches to achieve synchronous volumetric imaging, at cellular resolution, over mesoscopic-scaled volume regions, to observe dynamic processes such as the blood flow in embryonic beating hearts and brain-wide neuronal activity in small live animals.