Multicolor plate reader fluorescence calibration.

Area of Science:

• Biotechnology
• Microbiology
• Analytical Chemistry

Background:

• Plate readers are essential for measuring cell growth and fluorescence.
• Current plate reader data lacks standardization, reported in arbitrary units, limiting reproducibility.
• Previous work established calibration for absorbance and green fluorescence.

Purpose of the Study:

• To extend calibration protocols for red and blue fluorescence measurements.
• To establish standardized units for red fluorescence using sulforhodamine-101.
• To establish standardized units for blue fluorescence using Cascade Blue.

Main Methods:

• Developed serial dilution protocols for red and blue fluorescence calibration.
• Utilized sulforhodamine-101 as a calibrant for red fluorescence.
• Utilized Cascade Blue as a calibrant for blue fluorescence.
• Conducted an interlaboratory study to evaluate calibration efficacy.

Main Results:

• The new calibration protocols demonstrate comparable precision to existing methods.
• Red and blue fluorescence measurements show improved cross-laboratory comparability.
• Standardized calibration enables more reliable bacterial fluorescence quantification.

Conclusions:

• The developed calibration methods enhance the utility and reproducibility of plate reader fluorescence data.
• Standardized red and blue fluorescence measurements facilitate robust interlaboratory comparisons.
• These advancements support more accurate quantification of protein expression in bacterial cells.