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

Area of Science:

• Microscopy and Imaging Technologies
• Biophotonics
• Materials Science

Background:

• Routine microscopy is limited to analyzing few analytes, hindering throughput and quantitative analysis.
• Existing digital microscopy and image analysis techniques for micrometer-sized targets have low throughput and reliability.

Purpose of the Study:

• To develop an advanced on-the-fly stage scanning method for high-precision target location across entire microscope slides.
• To improve the speed, simplicity, and quantitative analysis capabilities of microscopy for micrometer-scale targets.

Main Methods:

• Implemented an orthogonal scanning automated microscopy (OSAM) technique integrating X- and Y-axis linear encoders for real-time positional referencing.
• Utilized a motorized stage for rapid scanning of large sample areas (50 × 24 mm²).
• Incorporated an autofocus unit to compensate for Z-axis slide tilt, improving detection efficiency.

Main Results:

• OSAM scanned a 50 × 24 mm² area in 5.3 minutes, locating 15 μm lanthanide luminescent microspheres with high precision (1.38 μm X, 1.75 μm Y standard deviations).
• Luminescence detection efficiency increased by 35% with an improved coefficient of variation due to the autofocus unit.
• Demonstrated robust quantification of luminescence intensities and lifetimes for various micrometer-scale targets.

Conclusions:

• Advanced OSAM offers significant advantages in speed, throughput, and quantitative analysis for micrometer-scale luminescent targets.
• The technique is capable of analyzing diverse targets including microspheres, microplates, microrods, and nanoparticles in suspension arrays.
• OSAM represents a breakthrough for robust, high-efficiency microscopy of solid-phase samples.