Designing the Focal Plane Spacing for Multifocal Plane Microscopy

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Designing the Focal Plane Spacing for Multifocal Plane Microscopy

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Title: Designing the Focal Plane Spacing for Multifocal Plane Microscopy
Author(s):
Tahmasbi, Amir;
Ram, Sripad;
Chao, Jerry;
Abraham, Anish V.;
Tang, Felix W.;
Ward, E. S.;
Ober, Raimund J.
Item Type: article
Keywords: Multifocal plane microscopy
Practical localization accuracy measure
Focal planes
Abstract: 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. MUM allows high accuracy localization of a point source along the z-axis since it overcomes the depth discrimination problem of conventional single plane microscopy. 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. Here, we propose approaches based on the Fisher information matrix and report spacing scenarios called strong coupling and weak coupling which yield an appropriate 3D localization accuracy. We examine the effect of numerical aperture, magnification, photon count, emission wavelength and extraneous noise on the spacing scenarios. In addition, we investigate the effect of changing the number of focal planes on the 3D localization accuracy. We also introduce a new software package that provides a user-friendly framework to find appropriate plane spacings for a MUM setup. These developments should assist in optimizing MUM experiments.
Publisher: Optical Society of American (OSA)
ISSN: 1094-4087
Persistent Link: http://dx.doi.org/10.1364/OE.22.016706
http://hdl.handle.net/10735.1/4173
Terms of Use: ©2014 Optical Society of America
Sponsors: US National Institutes of Health (R01 GM085575)

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