An automated protocol for performance benchmarking a widefield fluorescence microscope.

Area of Science:

• Biomedical Imaging
• Optical Microscopy
• Analytical Chemistry

Background:

• Widefield fluorescence microscopy is crucial for biological sample assessment and cell response quantification.
• Limited standardized methods exist for evaluating and benchmarking fluorescence microscope performance.
• Accessible benchmarking tools are needed for quantitative research and instrumental method validation.

Purpose of the Study:

• To develop and evaluate an automated method for characterizing fluorescence imaging system performance.
• To benchmark key analytical parameters including detection threshold, saturation, and linear dynamic range.
• To establish reliable reference materials for microscope calibration and validation.

Main Methods:

• An automated method was developed to characterize fluorescence imaging systems.
• Benchmarking involved assessing detection threshold, saturation, and linear dynamic range.
• Uranyl-ion-doped glass and Schott 475 GG filter glass were used as reference materials.

Main Results:

• The automated method effectively characterized fluorescence imaging system performance.
• Both uranyl-ion-doped glass and Schott 475 GG filter glass proved suitable as reference materials.
• The reference materials enabled accurate day-to-day intensity calibration.

Conclusions:

• The developed automated method provides a robust approach for benchmarking fluorescence microscopes.
• Schott 475 GG filter glass is a viable, commercially available reference material for quantitative fluorescence imaging.
• This method enhances the reliability of fluorescence microscopy as a quantitative analytical tool.