Evaluation of Focus Measures for Hyperspectral Imaging Microscopy Using Principal Component Analysis

An automatic focusing system is a crucial component of automated microscopes, adjusting the lens-to-object distance to find the optimal focus by maximizing the focus measure (FM) value. This study develops reliable autofocus methods for hyperspectral imaging microscope systems, essential for extracting accurate chemical and spatial information from hyperspectral datacubes. Since FMs are domain- and application-specific, commonly, their performance is evaluated using verified focus positions. For example, in optical microscopy, the sharpness/contrast of visual peculiarities of a sample under testing typically guides as an anchor to determine the best focus position, but this approach is challenging in hyperspectral imaging systems (HSISs), where instant two-dimensional hyperspectral images do not always possess human-comprehensible visual information. To address this, a principal component analysis (PCA) was used to define the optimal ("ideal") optical focus position in HSIS, providing a benchmark for assessing 22 FMs commonly used in other imaging fields. Evaluations utilized hyperspectral images from visible (400-1100 nm) and near-infrared (900-1700 nm) bands across four different HSIS setups with varying magnifications. Results indicate that gradient-based FMs are the fastest and most reliable operators in this context.