Quantitative microbiology with widefield microscopy: navigating optical artefacts for accurate interpretations

Area of Science:

• Microbiology
• Microscopy
• Biophysics

Background:

• Time-resolved live-cell imaging is vital for quantitative microbiology.
• Microbial cell dimensions near the microscope's depth of field and diffraction effects complicate image interpretation.
• 2D widefield images capture projected 3D information, blurred by the point spread function.

Purpose of the Study:

• Investigate the impact of diffraction and projection on microbial cell size and content quantification.
• Identify and characterize novel artifacts arising from these optical phenomena.
• Develop strategies for accurate microbial cell analysis in microscopy.

Main Methods:

• Utilized simulations and targeted experiments to study optical effects.
• Analyzed the interplay of projection and diffraction in 2D widefield microscopy.
• Developed machine learning-based analysis methods to correct for identified artifacts.

Main Results:

• Discovered new artifacts from projection and diffraction, significantly biasing size and fluorescence quantification.
• Demonstrated substantial errors in single-molecule counting due to these optical effects.
• Quantification errors were found to be complex and challenging to eliminate.

Conclusions:

• Awareness of projection and diffraction artifacts is critical for accurate microbial imaging.
• New experimental designs and machine learning approaches can mitigate these artifacts.
• Accurate quantification of microbiological processes is achievable with advanced analysis methods.