Researchers are conducting more genetic experiments that call for use of inverted microscopes so they can view and track the activity of discrete molecules within living cells. An inverted microscope uses a light over the specimen and the objective lens below it. The resulting images reveal the nature of intracellular processes, critical information for tasks that include drug discovery, disease prevention, and biochemistry research. For successful experiments, however, inverted microscopes must stay precisely focused to capture digital time-lapse photographs of fluorescent markers. This means the objective lens on a microscope remains within 100 nm of the focal plane, thereby preventing images from drifting out of focus in time-lapse photography. For such precision, small thermal effects present big challenges. Even an ambient temperature shift of one degree Celsius can alter focus by 200 nm.

Mad City Labs, Madison, Wis., (madcitylabs.com) found it could compensate for thermal expansion and other mechanical tolerances with real-time, high resolution closed-loop motion control. So the company invented an auto-focus correction system, the C-Focus, for microscopes capable of resolving 500-µm features, confocal imaging systems, and others with autofocus mechanisms. The Mercury 3500 programmable encoder from MicroE Systems, Bedford, Mass., (microesys.com) provides the necessary closed-loop feedback.

“The 8.4-mm high encoder has programmable linear resolution in steps from 5 µm to 5 nm for ± 20 nm positioning accuracy and 5 nm of motion-control resolution for the C-Focus system,” says Mad City Labs R&D engineer Jim Mackay

“The encoder must have high resolution to stay focused on a virus cell 50 to 100-nm long,” adds Mackay. “At the same time, the sensor has to be small enough to fit in the cramped confines of an inverted optical microscope. A larger sensor is out of the question because the focal length of a typical inverted microscope is just 7mm.” The low profile encoder sensor makes it possible to retrofit the C-Focus system to almost any inverted microscope.

Mad City Labs' auto-focus design is based on its Nano-F100 piezo-electric stage. It uses the encoder sensor mounted on a low coefficient of thermal expansion (CTE) Invar bracket fixed to the body of the microscope. The encoder's low-CTE glass scale (parallel to the Invar bracket) rides up and down on the aluminum housing to which the objective lens mounts.

To operate the system, a researcher just focuses the microscope on an area of interest and hits the Start button. The C-Focus system then provides automatic correction for thermal expansion and contraction. “The encoder is easy to use because it comes with a separate scale and sensor, individually mounted components,” says Mackay. “The encoder is also easy to setup. It has a relatively broad alignment tolerance, a ±2° sweet spot in the theta Z axis, three times better than competing technologies. Plus, a programmable interpolation in integer steps, programmable output frequency, and circuitry for automatic gain and phase correction make it easy to fine tune system performance.