Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection

Area of Science:

• Optics and Photonics
• Biotechnology
• Molecular Imaging

Background:

• Simultaneous multi-color single-molecule (SM) detection is crucial for advanced biological analysis.
• Existing four-color SM detection systems often require complex optics and may have limitations in throughput.
• Dual-view imaging systems are typically limited to two-color detection.

Purpose of the Study:

• To develop a novel dual-view imaging system for simultaneous four-color SM detection.
• To enhance the throughput and simplicity of multicolor SM detection.
• To provide a versatile platform for next-generation DNA analysis technologies.

Main Methods:

• Developed a dual-view optics system incorporating a wide-range dichroic mirror (WR DM).
• Utilized four species of SM fluorophores (Alexa 488, 555, 647, 680) excited by evanescent-wave illumination.
• Implemented a classification method based on signal ratios from two SM fluorescence spots imaged on an EM-CCD camera at 100 Hz.

Main Results:

• Achieved classification of 597 SM fluorophores into four species with >98% accuracy.
• Demonstrated comparable accuracy to conventional four-color SM detection systems.
• The developed system offers 1.5-fold higher throughput due to more efficient use of the EM-CCD chip.

Conclusions:

• The developed dual-view system with a WR DM enables efficient and accurate four-color SM detection.
• This simple modification of existing dual-view optics significantly increases detection throughput.
• The system is well-suited for applications in next-generation DNA sequencers and microarray-chip analyzers.