High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis
| Autor: | Xianlin Zheng, Yuhai Zhang, Jie Lu, Dayong Jin, Deming Liu, Jiangbo Zhao, Xiaogang Liu, Robert C. Leif, Wei Ren, James A. Piper, Yiqing Lu |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
business.industry Chemistry Microscope slide Nanotechnology Analytical Chemistry Microsphere 03 medical and health sciences 030104 developmental biology Microscopy Computer vision Artificial intelligence Sample area Luminescence business Encoder Throughput (business) Scanning microscopy |
| Zdroj: | Analytical Chemistry. 88:1312-1319 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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 mm(2) 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 and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|