High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual “grids” that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm2 in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X ...