We wish to thank the Allen Institute founders, Paul G. Allen and Jody Allen, for their vision, encouragement, and support. An integrated imaging and staining platform for cubic millimeter scale array tomography Forrest Collman, Robert Seraﬁn, Sarah Davis, Olga Gliko, Tom M Keenan, Kristy Parker, Linnaea E Ostroﬀ, Stephen J Smith Synapse Biology Department, Allen Institute for Brain Science, Seattle WA 98103 Array tomography (AT) involves reconstruction of images acquired from arrays of serial ultrathin sections, which can be imaged by fluorescence microscopy (FM-AT) for proteometric analysis, or by scanning electron microscopy (EM-AT) for higher resolution. Axial resolution is defined by the thickness of physical sections (50-200 nm); the lateral resolution of FM imaging is optimal given the direct adhesion of sections to an optical coverslip. However, the throughput of data acquisition is slowed by the image acquisition time, and the need for human intervention to stain and set up samples on the microscope. The first AT systems acquired data at a throughput of ~16 seconds per 4 channel image (Micheva 2007). Recent work incorporated a hardware-based autofocus technology that improved throughput to 5-12 sec per 4 channel image (Rah 2013), achieving 0.1 mm3 in 878 imaging hours with 200 nm-thick sections. Data acquisition throughput is further impaired by the overhead involved in staining and setting the sample up for imaging. As part of the Open Synaptome Project (http://opensynapto.me) effort to improve all aspects of ATomo based synaptic analysis, we are developing a next-generation system to achieve imaging throughput on the order of 0.2mm3 per day per microscope. We will describe the design of the imaging system, which include hardware-based autofocus, large format sCMOS sensors, motionless high intensity wide-field laser illumination, and a custom open-source software solution. We will also describe our efforts to construct a robofluidic staining solution fully integrated with the microscope. Our goal is to enable continual staining and imaging without human intervention, thus increasing both throughput and consistency of results ultrathin section onto coverglass IF stain IF imaging poststain FESEM elution ABSTRACT ARRAY TOMOGRAPHY IMAGING brain-map.org alleninstitute.org y (mm) 0 ul 50 ul 100 ul 150 ul 200 ul 250 ul 300ul Fluorescein Intensity (AU) 0 500 100 200 300 400 Wash In x (mm) y (mm) Wash Out 0 ul 50 ul 100 ul 150 ul 200 ul 250 ul 300ul Fluorescein Intensity (AU) 0 500 100 200 300 400 x (mm) 300ul 0 10 x (mm) >95% washin with 200-250ul >99% washout with 300 ul Non-saturating/quenching concentration of fluoroscein 10x air objective, median intensity of field, 50μl increments Fully automated pipetting and image acquisition across x,y grid Pipette In Pipette Out 1.5 mm 3.25 mm pipetting robot inverted microscope 96-well chiller tips 45 mm 3D printed mold PDMS (Sylgard) poured mold Passive pumping design allows simple unpowered flow-cell compatible with vertical pipetting robot (Walker 2002) PASSIVE PUMPING FLOW CELL PROTOTYPE MOSAIC PLANNER - OPEN SOURCE ARRAY TOMOGRAPHY SOFTWARE REFERENCES AND GRANT SUPPORT COVERSLIP FLATNESS LASER ILLUMINATION time per 4 channel frame (sec) auto-focus filter-change exposure stage move Standard Upright Automated Microscope Zeiss Observer Z.1, 4 filter cubes, x-cite mercury halide liquid light guide IMAGING SPEED IMPROVEMENTS Improvement 16.1 sec per 4 images 892 images/hour hardware auto-focus (Rah et al. 2015) 100-400ms focus time ~2000 images/hour Estimated Speed Increase ~10-50 ms change time ~4000 images/hour quad band dichroic illumination switching sCMOS field size 4.2 vs 1.4 megapixels effectively 2.9x faster laser illumination ~5x power density ~12,000 images/hour NET IMPROVEMENT ~40x faster net imaging 500 nm X Y XY PSF 500 nm Z Y YZ PSF 50 100 1 nm/μm across 200 μm 500μm μmanager Core API hardware agnostic microscope commands images/status Camera Illumination Stage Microsc