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

Optical microscopy encompasses a wide array of instruments and applications, each designed with the goal of obtaining the best possible image. However, achieving that goal depends on multiple factors, including, but not limited to, choice of microscope sample and calibration standards. Alignment or calibration standards are mandatory to obtain the best image possible. Currently, standards on the market are limited by such factors as fluorescence, sample volume and sample-to-sample interaction, and aberrations. Reported here is the testing of pollen as an imaging phantom, a designed material or device that mimics human tissue for the purpose of reliable and accurate calibration of an imaging system, and the benchmarking of a recently built multimodal adaptive optics multiphoton fluorescence microscope system. The developed imaging phantom was used to compare intensity values to those of an industry standard (TetraSpeck^™). Comparisons were made using deconvolutions of the captured images and their resultant point-spread functions in addition to measurements taken of surface characteristics. All comparisons between imaging media, imaging conditions per photomultiplier tube (PMT), and the interaction of media and imaging conditions showed significant p-values. Results showed that medium selection had a substantial impact on intensity output and was thus influenced by the choice of PMT (signal output from a sample). Results showed the sample itself impacted the intensity readings, as indicated by the comparison of two-dimensional (TetraSpeck^™) and three-dimensional (pollen grains) samples. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for multimodal adaptive optics multiphoton fluorescence microscope (mAO-MPFM) analysis Support Protocol: Pollen collection Basic Protocol 2: Observing autofluorescence and surface structures of pollen grains using mAO-MPFM Basic Protocol 3: Sample preparation for cold field emission scanning electron microscope (cFE-SEM) imaging Basic Protocol 4: Observing surface characteristics of pollen grains using cFE-SEM Basic Protocol 5: Pollen grain structure manual and mask measurement for fluorescent and SEM images with comparisons Basic Protocol 6: Intensity analysis of fluorescence images with Image-Pro and Prism.