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All researchers recording images using basic widefield microscopy.


A few simple questions to help ensure your images faithfully reveal what’s happening at the microscale.


Use objective measures provided by your imaging software—such as the histogram display—to avoid misinterpretations caused by limitations of the display and human visual perception.1-3

Before you begin

01. What is the purpose of your imaging—creating an attention-getting journal cover, saving a reference for your notebook, or capturing the changes in dynamic processes over time?

Is your sample optimized for imaging?

02. Have you ensured that background signal is minimized?

03. Is the specificity of your label sufficient to be seen above background?

04. Is your label at a sufficient concentration to be seen, but not high enough to be toxic?

Are your camera, optics, and filters optimized for your experiment?

05. Are the wavelengths of your filters consistent with your fluorophores?

06. Are your objective and media of similar refractive index?

07. Is your coverslip thickness the correct size for your experiment and setup?

08. Is the magnification high enough to resolve the features of interest?

09. Have you removed all unnecessary filters and optics from the light path (i.e., polarizers, phase rings, etc.)?

10. Do you have even illumination across the entire field?

11. Have you eliminated all stray light, including filtering out IR?

12. Are you using ND filters (neutral density filters), if possible, to minimize sample bleaching or phototoxicity?

13. Does your camera have adequate QE at the necessary wavelengths?

14. Is your camera fast enough to capture the dynamics of your system?

Are your image acquisition parameters optimized?

15. Have you set up an informative file naming system?

16. If your camera has gain control, is it set appropriately?

17. Is your exposure time optimized?

18. Have you set the histogram to visualize the object(s) of interest (i.e., cell body, dendrite, etc.)?

To learn more about these questions, read our User Guide, “Can You Trust What You See?


  1. Adelson, E. H. in Cogn. Neurosci. (Gazzaniga, M.) (MIT Press, 1999).
  2. Capo-Aponte, J. E. et al. Visual perception and cognitive performance. at <http://www.usaarl.army.mil/publications/HMD_Book09/files/Section%2018%20-%20Chapter%2010%20Visual%20Perception%20and%20Cognitive%20Performance.pdf>
  3. What optical illusions show us about visual perception. Brain Top Bottom at <http://thebrain.mcgill.ca/flash/a/a_02/a_02_p/a_02_p_vis/a_02_p_vis.html>
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