Pharma Focus Europe

A biologist’s guide to planning and performing quantitative bioimaging experiments

Rebecca A. Senft, Barbara Diaz-Rohrer, Pina Colarusso, Lucy Swift, Nasim Jamali, Helena Jambor, Thomas Pengo, Craig Brideau, Paula Montero Llopis, Virginie Uhlmann, Jason Kirk, Kevin Andrew Gonzales, Peter Bankhead, Edward L. Evans III, Kevin W. Eliceiri, Beth A. Cimini


Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.


The optical microscope is a major cornerstone of biological discovery, yet modern-day bioimaging experiments involve much more than looking down the eye-piece and reporting observations. Proper quantification of imaging data requires planning and decision-making at every step of the experimental imaging workflow. In practice, even the most motivated and independent learner cannot master the best practices in microscopy if they do not know what topics they must fully understand and which skills they need to acquire as they work towards proficiency. Ideally, biologists wishing to learn more about microscopy will have convenient local access to training and mentorship as they develop their bioimaging knowledge and skills. Unfortunately, in practice, access to advanced training and support varies widely by site. Thankfully, in addition to a number of hands-on specialized workshops that provide excellent training opportunities, a wealth of high-quality open-source resources have been generated by the bioimaging community.

This Essay serves as a navigational guide for the important considerations needed to best utilize optical microscopy as a quantitative measurement technique. For each major stage of bioimaging experiments (sample preparation, image acquisition, image analysis, and data interpretation; Fig 1), we present key high-level considerations as well as highlighting resources that present further information. We primarily focus on fluorescence microscopy of animal cells and tissues, but many recommendations and resources are generalizable to other kinds of bioimaging (e.g., brightfield microscopy, electron microscopy, histology) and other sample types (plants, prokaryotes, etc.).


The success or failure of a microscopy experiment ultimately rests upon the decisions made in its design and execution; here, we have presented general guidelines and basic concepts that can guide the decision-making process when designing one’s experiments. Designing experiments so that they can be quantitative and reproducible not only benefits the experimental scientist but also the wider community. To that end, we strongly recommend that data, analysis pipelines, and other resources be made open source and freely available to the public. The information presented here is not exhaustive and we further recommend consulting with experts, exploring resources linked here and on the companion website, and reading relevant published literature and protocols. Regional organizations dedicated to bioimaging and/or bioimage analysis are present around the globe and are often in an excellent position to connect beginners with local resources available to them (Table 2).


The authors thank Anne Carpenter, Ellen Dobson, and Pearl Ryder for helpful discussions regarding important resources to highlight.

Citation: Senft RA, Diaz-Rohrer B, Colarusso P, Swift L, Jamali N, Jambor H, et al. (2023) A biologist’s guide to planning and performing quantitative bioimaging experiments. PLoS Biol 21(6): e3002167.

Published: June 27, 2023

Copyright: © 2023 Senft et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Funding was provided by the National Institutes of Health (NIH) (NIH COBA P41 GM135019 to BAC and KWE). This project has been made possible in part by grant number 2020-225720 to BAC from the Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation. VU is supported by European Molecular Biology Laboratory (EMBL) internal funding. ELE is supported by a Morgridge Postdoctoral Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: DIC, differential interference contrast; LB, Luria broth; NA, numerical aperture; PMT, photomultiplier tube; RI, refractive index; ROS, reactive oxygen species; WD, working distance.

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