Location: Room E212A Anatomy/Zoology Building. Tim Stasevich, PI

This custom-built fluorescence microscope is based on an ASI RAMM frame and has been tailored for advanced live-cell imaging, photomanipulation, and automated single-cell picking. The system is equipped with a high-sensitivity Andor iXon Ultra 888 EMCCD camera and a high-NA Olympus objective set, including 60x oil (NA 1.5), 20x air (NA 0.80), and 10x air (NA 0.40). A 300 mm achromatic tube lens is used in place of the standard 180 mm Olympus lens, resulting in a 1.67× increase in effective magnification.

Four laser lines are available for excitation: 405 nm (250 mW, Stradus), 488 nm (60 mW, OBIS LS), 561 nm (50 mW, OBIS LS), and 637 nm (140 mW, OBIS LX). The excitation path is split into two independent arms. One arm, used for imaging, incorporates a 2D galvo scanner for ring-TIRF and HILO illumination, which provides uniform excitation and enhances signal-to-noise ratio. The other arm is dedicated to photomanipulation and also includes a 2D galvo, allowing spatially flexible illumination patterns for applications such as photobleaching and photoactivation.

TTL triggering coordinates all major components, including the camera, lasers, Z piezo, and filter wheel, enabling precise synchronization during time-sensitive experiments. A near-infrared LED-based focus lock maintains stable focus throughout long-term imaging sessions, including overnight time-lapses. Environmental conditions are controlled with an Okolab UNO chamber (5% CO₂ and temperature control).

The microscope integrates a CellSorter micropipette-based system for automated single-cell collection. Cells can be selectively retrieved from chambered coverslip dishes and transferred to multi-well plates for downstream analysis. The system supports pre- and post-pick imaging to verify collection and is well-suited for workflows requiring isolation of individual cells or small groups under visual guidance.

Control and automation are implemented through Micro-Manager, PycroManager, and Arduino with custom software modules developed in-house. Real-time image analysis, including spot detection and targeted photobleaching/photoactivation, can be implemented to support adaptive experimental workflows.

Training is not provided, and access is currently limited to fee-for-service or research collaboration though grant funding mechanisms. For further information contact: Tim.stasevich@colostate.edu