Sampling efficiency of a single-cell capillary electrophoresis system

Area of Science:

• Analytical Chemistry
• Biotechnology
• Microscopy and Imaging

Background:

• Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is vital for single-cell analysis.
• Pulsed laser lysis systems are used with CE-LIF for high temporal resolution, but sampling efficiency concerns exist.

Purpose of the Study:

• To develop and validate a novel method for estimating cell sampling efficiency (SE) in single-cell CE-LIF.
• To evaluate the impact of laser pulse energy and focal position on SE during laser-induced cell lysis.

Main Methods:

• Assembled a single-cell CE-LIF system integrated with a microscope and a nanosecond pulsed laser for cell lysis.
• Developed a novel SE estimation method using fluorescent calibration microspheres and flow cytometry, independent of cell volume.
• Analyzed green fluorescent protein (GFP) in single mammalian cells, varying laser pulse energy (2-9 microJ) and focal position.

Main Results:

• The novel method successfully measured SE without prior knowledge of cell volume.
• Maximum SE was achieved at the lowest laser pulse energy (2 microJ) with the pulse focused directly on the cell.
• Different laser pulse energies and focal positions significantly affected cell sampling efficiency.

Conclusions:

• The developed method provides a robust way to quantify SE in single-cell CE systems.
• Optimizing nanosecond pulsed laser parameters (energy and focus) is essential for maximizing cell content sampling.
• Careful optimization is required for efficient single-cell analysis using laser-induced lysis in CE-LIF.