When choosing an autofocus system for automated microscopy, key factors include the required sampling rate, sample surface characteristics, working distance, autofocus accuracy and repeatability, optical configuration, and integration with the system’s motion and control architecture.
Sampling Rate and Tracking Speed
For automated microscopy systems that scan or move continuously, the autofocus sensor needs to provide sufficiently fast feedback to maintain focus during motion. A higher sampling rate can be particularly important when the system operates at high speed or when rapid changes in sample height need to be tracked.
Sample Surface Characteristics
The type and complexity of the sample surface can have a significant impact on autofocus performance. Patterned, multilayer, or otherwise challenging surfaces may require an autofocus technology that can distinguish between different surface features and maintain focus on the intended surface.
For this reason, it is important to consider not only how fast an autofocus system operates, but also how its sensing method performs on the specific samples being imaged.
Working Distance
The required distance between the objective and the sample is another important consideration. The autofocus sensor should provide a suitable standoff distance for the microscope configuration and the physical requirements of the application.
A longer available standoff can be particularly useful when the system needs additional space between the optical assembly and the sample.
Accuracy and Repeatability
In automated imaging and inspection, maintaining a consistent focus position is essential for repeatable imaging. When selecting an autofocus system, consider both its static autofocus accuracy and its performance while tracking a moving or changing surface.
It is also important to consider the depth of field of the objective, since autofocus performance is often specified relative to objective DOF.
Optical and System Integration
An autofocus sensor needs to work as part of the complete microscopy system. Factors such as laser wavelength, optical configuration, compatibility with the objective, and the relationship between the autofocus focal plane and the imaging plane should be considered.
Integration with the Z-axis motion system and control architecture is equally important. Configurable outputs and communication interfaces can make it easier to integrate the autofocus sensor with different actuators, stages, and system controllers.
Two Approaches: Line and Dot Autofocus
Different autofocus architectures can be better suited to different applications.
WDI’s PFA-DT uses a single-dot structured-light pattern and provides a sampling rate of up to 8 kHz. It is designed for applications where high-speed tracking is a priority, including biomedical imaging, high-content screening, genotyping, and DNA sequencing.
PFA-LN uses a line structured-light pattern and Advanced Multi-Segment Processing. It is designed for challenging, patterned, and multi-surface samples, including semiconductor wafers and OLED displays, and provides sampling rates up to 3 kHz, or up to 5 kHz in SWIFT mode.
Both sensors provide autofocus repeatability of ±0.25 objective DOF or better in static operation and ±0.33 objective DOF or better during fast tracking.
Choosing the Right Autofocus Solution
The right autofocus system depends on the complete application: how fast the system moves, what type of surface is being imaged, how much working distance is required, what level of focus accuracy is needed, and how the sensor will integrate with the rest of the microscope.
PFA-DT and PFA-LN from WDI provide two different approaches to automated focus tracking, allowing system designers to select a configuration according to their specific imaging requirements.