Rapid Autofocus Method Based on LED Oblique Illumination for Metaphase Chromosome Microscopy Imaging System

In clinical practice, microscopes are commonly used for imaging chromosomes to diagnose genetic diseases. Achieving precise and rapid autofocusing is a significant challenge in the advancement of high-throughput chromosome imaging systems. Here, we introduce a rapid autofocus method based on LED oblique illumination for dual-objective configuration in metaphase chromosome imaging system. Our method utilizes a programmable LED array for sample illumination, employing a sequential activation of two LEDs from opposing angles to create oblique illumination. The defocus distance is ascertained through image cross-correlation calculation. Illumination with multiple LEDs in the array is used to acquire bright-field images after completing the focusing. Our method can perform continuous autofocus under a 10× objective and a 100× oil immersion objective, with average focusing errors of 1.29 μm and 0.12 μm, respectively. The total imaging time for a single sample has been significantly reduced from approximately 10 min with conventional methods to just 2 min. This study provides preliminary evidence supporting the viability of developing a next-generation high-throughput chromosome scanner employing a LED array.