The cryo-imaging system (Fig. 1 ) consists of four major subsystems—the cryomicrotome, the microscope imaging system, the robotic xyz positioner, and the computer control system. The cryomicrotome is a motorized, large section, whole body cryo-microtome (Model 8250, Vibratome, St. Louis, MO) with section thickness adjustable between 2 and 40 µm and a maximum specimen dimension of 250 mm × 110 mm × 5 mm. For an automated cryo-imaging setup, we interfaced appropriate control signals within the cryo-microtome to communicate with the control computer over a single Ethernet cable. A computer/manual selector switch was added to the operator pushbutton box to allow manual override and control. The cryomicrotome frame was mechanically modified to connect an XYZ robotic positioner carrying the imaging system. The imaging subsystem comprises of a stereo microscope (SZX12, Olympus, Japan), coaxial fluorescent attachment with multiple filter cubes, low light digital camera (Retiga Exi, QImaging, Canada), and brightfield and fluorescent light sources. The long working distance of the stereo microscope allows for optional tissue collection. Using multiple microscope objectives and zoom settings, the field-of-view can be varied to cover an entire mouse or down to a small organ and image at in-plane resolution of ≈3 µm. To enable very high-resolution imaging over a mouse-sized FOV, we designed a 3-axis robotic positioner. The computer control system automatically pans the positioner over the specimen for a high-resolution tiled image acquisition.
The computer control system controls the sectioning and image acquisition sequence through the custom developed Programmable Sectioning and Cryo-Imaging (ProSCI) software. Through the Graphical User Interface (GUI), the operator enters specimen information, sets up the illumination sources, chooses imaging modality (brightfield, fluorescence or both), and defines the imaging regions-of-interest (ROI). In case a ROI needs to be imaged at a high-resolution, tiled image acquisition can be setup by defining the area and the software calculates the minimum number of tiles necessary to image the ROI. The Image Processing and Visualization system is a quad-core Windows 64-bit PC with 32GB of RAM (Dell Inc, TX) capable of handling large cryo-image volumes. A typical whole mouse, if imaged at ≈15 µm in-plane resolution and sectioned at 40 µm, generates typically >50 GB of raw data. A suite of MATLAB (Math-works, Natick, MA) custom programs and custom AMIRA (Mercury Computer Systems, San Diego, CA) scripts are used for image preprocessing tasks and 3D visualization.
The computer control system controls the sectioning and image acquisition sequence through the custom developed Programmable Sectioning and Cryo-Imaging (ProSCI) software. Through the Graphical User Interface (GUI), the operator enters specimen information, sets up the illumination sources, chooses imaging modality (brightfield, fluorescence or both), and defines the imaging regions-of-interest (ROI). In case a ROI needs to be imaged at a high-resolution, tiled image acquisition can be setup by defining the area and the software calculates the minimum number of tiles necessary to image the ROI. The Image Processing and Visualization system is a quad-core Windows 64-bit PC with 32GB of RAM (Dell Inc, TX) capable of handling large cryo-image volumes. A typical whole mouse, if imaged at ≈15 µm in-plane resolution and sectioned at 40 µm, generates typically >50 GB of raw data. A suite of MATLAB (Math-works, Natick, MA) custom programs and custom AMIRA (Mercury Computer Systems, San Diego, CA) scripts are used for image preprocessing tasks and 3D visualization.
