In this article, we describe use of Python for large-scale automated server-based bio-image analysis in FARSIGHT, a free and open-source toolkit of image analysis methods for quantitative studies of complex and dynamic tissue microenvironments imaged by modern optical microscopes including confocal, multi-spectral, multi-photon, and time-lapse systems. The core FARSIGHT modules for image segmentation, feature extraction, tracking, and machine learning are written in C++, leveraging widely used libraries including ITK, VTK, Boost, and Qt. For solving complex image analysis task, these modules must be combined into scripts using Python. As a concrete example, we consider the problem of analyzing 3-D multi-spectral brain tissue images surrounding implanted neuroprosthetic devices, acquired using high-throughput multi-spectral spinning disk step-and-repeat confocal microscopy. The resulting images typically contain 5 fluorescent channels, 6,000$\times$10,000$\times$500 voxels with 16 bits/voxel, implying image sizes exceeding 250GB. These images must be mosaicked, pre-processed to overcome imaging artifacts, and segmented to enable cellular-scale feature extraction. The features are used to identify cell types, and perform large-scale analytics for identifying spatial distributions of specific cell types relative to the device. Python was used to build a server-based script (Dell 910 PowerEdge servers with 4 sockets/server with 10 cores each, 2 threads per core and 1TB of RAM running on Red Hat Enterprise Linux linked to a RAID 5 SAN) capable of routinely handling image datasets at this scale and performing all these processing steps in a collaborative multi-user multi-platform environment consisting. Our Python script enables efficient data storage and movement between compute and storage servers, logging all processing steps, and performs full multi-threaded execution of all codes, including open and closed-source third party libraries.