Robotic Preparation of Tissue Specimens for TEM and Volume EM

Specimen preparation procedures for high-contrast transmission electron microscopy (TEM) and Volume EM (vEM) include multiple contrast enhancing steps making them some of the longest and most complex tissue processing protocols performed for EM. Additionally, 4 of the steps for vEM need heating or cooling. These specimen preparation workflows call for use of noxious, hazardous, and often carcinogenic chemicals including uranium salts, arsenic, heavy metals, cyanide, lead and osmium tetroxide. These long and complex workflows provide ample opportunity for human error and are currently performed manually in most cases. The PrepmasterTM 5100 is a new specimen preparation robot which automatically prepared optimally sized 1 liver tissue specimens for TEM and vEM. Operationally, the technician simply placed the 1 mm diameter x 1-5 mm length specimens in the specimen plate with 50 microliters of buffer, then placed the specimen plate on the temperature-controlled specimen dock and pressed “Start”. The Prepmaster then completed the hundreds of individual liquid handling steps required to prepare the specimen for resin embedding and imaging. A protocol2 was designed by Thomas J. Deerinck, Eric A. Bushong, Andrea Thor and Mark H. Ellisman in the Ellisman Lab at USCD to enhance signal for backscatter electron imaging of epoxy embedded mammalian tissue at low accelerating voltages (1-3 keV)”. This protocol was programmed into the Prepmaster 5100 specimen preparation robot and liver specimens were processed and imaged. The Prepmaster reliably and consistently prepares tissue samples, cells seeded on coverslips, and most other samples procured for life science research or diagnosis, such as organoids or retinas. Cardiac muscle image from UW-Madison. Prepared on Prepmaster 5100. Ellisman rOTO Protocol. Microtomy and imaging by Benjamin K. August on a FEI CM120 STEM at 40,000X. Note the well-defined intercalated disc line running horizontally. Tissue specimen before (left) and after (right) processing.