Benchmarking Assessment and Implementation of an Imaging Phantom in Nonlinear Optical Microscopy

Area of Science:

• Optical Microscopy
• Biophotonics
• Materials Science

Background:

• Accurate calibration of optical microscopy systems is crucial for reliable imaging.
• Existing calibration standards have limitations, including fluorescence, sample volume, and interactions.
• Novel imaging phantoms are needed to overcome these limitations and improve calibration accuracy.

Purpose of the Study:

• To test pollen grains as a novel imaging phantom for optical microscopy calibration.
• To benchmark a newly developed multimodal adaptive optics multiphoton fluorescence microscope system (mAO-MPFM).
• To compare pollen as a phantom against industry-standard calibration materials like TetraSpeck™.

Main Methods:

• Pollen grains were prepared and analyzed using the mAO-MPFM system.
• Image intensity values were compared with TetraSpeck™ using deconvolutions and surface characteristic measurements.
• Analyses included comparisons across different imaging media, conditions, and photomultiplier tubes (PMTs).

Main Results:

• Pollen grains demonstrated significant differences in intensity output compared to TetraSpeck™.
• Medium selection and PMT choice substantially influenced intensity readings.
• The three-dimensional nature of pollen grains impacted intensity measurements differently than two-dimensional standards.

Conclusions:

• Pollen grains are a viable and effective imaging phantom for optical microscopy calibration.
• The choice of imaging medium and detector significantly affects image intensity.
• This study provides a new approach for calibrating advanced multiphoton fluorescence microscopy systems.