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Assuring Comparability in Wide-Field Fluorescence Microscopy Imaging

NIST developed a procedure for characterizing the performance of a fluorescence microscope by benchmarking the detection threshold, saturation, and linear dynamic range to a physical artifact, such as a fluorescent material. This method has already been implemented by several universities and companies, and is proven to improve the reproducibility of wide-field fluorescence imaging. Our strategy adds value to image data repositories by benchmarking the conditions under which the images were acquired.


Step-by-Step Guide
Follow BBD's procedure by downloading this guide.

MicroManager Script
Review this script developed to facilitate the implementation of our procedure in other laboratories.

Excel Spreadsheet
Chart the benchmarked parameters using the attached Excel spreadsheet to ensure instrument performance over time. 

Watch us implement this procedure in a NIST laboratory.

Technical Note
Review this procedure as published in Cytometry Part A.

Get Involved:

Researchers and product developers are now able to implement our new procedure in quantitative wide-field fluorescence microscopy. NIST invites users to provide feedback on the usability and effectiveness of this analytical tool. We are also interested in knowing where you need benchmarking protocols in your quantitative imaging pipeline. Contact michael.halter [at] (Michael Halter) to learn more about this project and ways to collaborate with NIST.


Created March 24, 2015, Updated November 15, 2019