Violet laser diodes in flow cytometry: an update.

Area of Science:

• Flow cytometry
• Laser technology
• Biophotonics

Background:

• Violet laser diodes (VLDs) are effective replacements for krypton-ion lasers in stream-in-air cytometers.
• Previous VLDs (<25 mW) were sufficient for some fluorochromes but potentially inadequate for low-expression applications like FRET.
• Higher power VLDs are needed to improve photon saturation for sensitive assays.

Purpose of the Study:

• To evaluate the performance of higher-power violet laser diodes (55 mW and 100 mW) for flow cytometry.
• To assess their ability to excite various violet-excited fluorochromes, including Cyan Fluorescent Protein (CFP).
• To determine if increased laser power improves instrument sensitivity and precision.

Main Methods:

• A dual module 404 nm VLD (55 mW or 100 mW) was integrated into a stream-in-air flow cytometer.
• Peripheral blood mononuclear cells (PBMCs) labeled with violet-excited probes were analyzed.
• Violet-excited fluorescent microspheres were used to assess sensitivity thresholds.
• Cuvette flow cytometry served as a baseline for sensitivity.

Main Results:

• The 100 mW dual module VLD achieved sensitivity and precision comparable to cuvette-based systems.
• Single 55 mW VLD modules showed slightly reduced sensitivity for microspheres and fluorochromes compared to the 100 mW source.
• Increased laser power led to a detectable improvement in instrument sensitivity.

Conclusions:

• Higher power VLDs (up to 100 mW) offer improved performance in stream-in-air flow cytometry.
• The 100 mW dual module VLD significantly enhances instrument sensitivity, approaching that of cuvette systems.
• These findings support the use of more powerful VLDs for sensitive flow cytometry applications.