Designing the focal plane spacing for multifocal plane microscopy

Multifocal plane microscopy (MUM) has made it possible to study subcellular dynamics in 3D at high temporal and spatial resolution by simultaneously imaging distinct planes within the specimen. An important question in MUM experiments is how the number of focal planes and their spacings should be chosen to achieve the best possible localization accuracy along the z-axis.

The MUMDesignTool is a new GUI based toolbox, under our previously developed FandPLimitTool, that allows users to calculate the limits of the accuracy with which the 3D location of a single molecules can be estimated when imaged using a MUM setup. The software supports the calculation of these limits along the z-axis for different focal planes in real-time and thereby helps to optimize the design of MUM experiments. The user can calculate limits for a variety of imaging scenarios.

The technical details of the methodology used for calculating the accuracy limits for a MUM setup are given in [1].

MUMDesignTool main window (Rapid mode).

MUMDesignTool configuration window.

MUMDesignTool noise parameter setting and console windows.

Please cite the following papers when appropriate to acknowledge the use of the MUMDesignTool in your work (reverse chronological order):


[1] A. Tahmasbi, S. Ram, J. Chao, A. V. Abraham, F. Tang, E. S. Ward, and R. J. Ober, Designing the focal plane spacing for multifocal plane microscopy. Opt. Express, 22(14):16706-16721, 2014. cite for the software and for MUM

[2] J. Chao, S. Ram, E. S. Ward, and R. J. Ober, Ultrahigh accuracy imaging modality for super-localization microscopy. Nat. Methods, 10:335-338, 2013. cite for EMCCD

[3] J. Chao, E. S.Ward, and R. J. Ober, Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices. Multidim. Sys. Sig. Proc., 23:349-379, 2012. cite for EMCCD

[4] A. V. Abraham, S. Ram, J. Chao, E. S. Ward, and R. J. Ober, Quantitative study of single molecule location estimation techniques. Opt. Express, 17:23352-23373, 2009. cite for the software

[5] J. Chao, S. Ram, A. V. Abraham, E. S. Ward, and R. J. Ober, A resolution measure for three – dimensional microscopy. Opt. Commun., 282:1751-1761, 2009. cite for 3D resolution

[6] S. Ram, E. S. Ward, and R. J. Ober. Beyond Rayleigh’s criterion: A resolution measure with application to single-molecule microscopy. PNAS, 103(12):4457-4462, 2006. cite for 2D resolution

[7] S. Ram, R. J. Ober, and E. S. Ward. How accurately can a single molecule be localized in three dimensions when imaged through a fluorescence microscope? Proc. of SPIE, 5699:426-435, 2005. cite for 3D localization accuracy

[8] R. J. Ober, S. Ram, and E. S.Ward. Localization accuracy in single molecule microscopy.Biophysical Journal, 86:1185-1200, 2004. cite for 2D localization accuracy

Version History

  • V1.0 (June 30, 2014) This is the currently available version.

Minimum System Requirements

  • PC with Core 2 Duo or equivalent processor suggested
  • Windows XP Service Pack 2 *
  • 512 MB RAM
  • 1024 x 768 screen resolution

* Although MATLAB and the MCR are available on the Linux/Unix and MacOS platforms, this executable version of the software can run only on a PC with Windows XP, Windows Server 2008, Windows 7 and Windows 8, and will NOT work in a Linux/Unix or Mac environment.

How to obtain the software

If you are interested in access to one of our software, please contact us at

Download MUMDesignTool User’s Manual pdf-icon

Download MUMDesignTool License Agreement pdf-icon


None available as yet.

Release Notes

This is the first release of the MUMDesignTool which is incorporated with the FandPLimitTool V1.2.

Bug Reporting

We are constantly trying to improve this software and would be very grateful if you could send us a report on any bugs you find. Please send all bug reports to the email with the subject MUMDesignTool Bug. Please include as many details about the bug as you can, including details of all tasks that led up to exposure of the bug. This will help us in finding a solution to the problem. When we have a fix for the problem, we will release an updated version of the software. Please look in the Release Notes section for problems we are already aware of and have solutions for.

MUMDesignTool Development Team

Current Members

Past Members

  • Sripad Ram
  • Felix Tang


The development work for MUMDesignTool was supported by the National Institutes of Health (R01 GM085575).

Contact Address

Raimund J. Ober, Ph.D.

Department of Biomedical Engineering
Texas A&M University
College Station, TX 77843-1114, USA

E. Sally Ward, Ph.D.

Department of Molecular and
Cellular Medicine
Texas A&M Health Science Center
Joe H. Reynolds Medical Sciences Building
College Station, TX 77843-1114, USA