Low-Numerical Aperture Microscope Objective Boosted by Liquid-Immersed Dielectric Microspheres for Quantum Dot-Based...

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Quantum dot (QD)-based digital immunoassays enable ultrasensitive biomarker detection.
• High numerical aperture (NA) objectives are typically required, limiting their application.
• Developing accessible microscopy techniques is crucial for broader adoption.

Purpose of the Study:

• To develop a high-quality imaging method for single quantum dots (QDs) using a low-NA air objective.
• To enable digital immunoassay analysis with reduced microscopy requirements.
• To assess the performance of this method for prostate-specific antigen (PSA) detection.

Main Methods:

• Utilized a combination of a 20×/0.4 NA air objective and liquid-immersed microspheres (150 μm, n = 2.2) for imaging single QDs.
• Achieved signal-to-noise ratios comparable to high-NA oil objectives (100×/1.4 NA).
• Performed digital analysis of prostate-specific antigen (PSA) in a relevant concentration range.

Main Results:

• High-quality imaging of massive single-QDs was successfully achieved.
• The signal-to-noise ratio was comparable to that obtained with a 100×/1.4 NA oil objective.
• Digital PSA analysis demonstrated a dynamic range of 0-50 ng/mL with a limit of detection of 0.17 ng/mL.
• Serum PSA measurements closely matched hospital-provided values.

Conclusions:

• A low-magnification, low-NA objective combined with microspheres enables high-performance QD imaging for digital immunoassays.
• This approach significantly reduces the barrier to microscopy miniaturization and cost.
• The findings are beneficial for popularizing advanced biomolecular digital analysis techniques.