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.
Highlights
The development of multifocal plane microscopy (MUM) in the recent past has made it possible to track the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging different focal planes within the sample [1,2,3,4,5,6,7]
As a first step we investigate the influence of different plane spacings on the axial-PLAMMUM assuming that the number of focal planes is two
The behavior of the axial-practical localization accuracy measure (PLAM) of a 2-plane MUM setup for different plane spacings is illustrated in the left-hand side plots of Fig. 2, where it is shown that altering the plane spacing changes the flatness of the curve
Summary
The development of multifocal plane microscopy (MUM) in the recent past has made it possible to track the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging different focal planes within the sample [1,2,3,4,5,6,7]. In [13] a 4-plane MUM setup providing a 10 μm viewing and tracking depth is used to study the 3D single molecule dynamics in live epithelial cells. The second requirement is to allow for a relatively large viewing and tracking depth across the sample in order to cover the z-range over which the cellular process of interest occurs Taking into account these design considerations, we provide guidelines to set up appropriate MUM experiments for different applications. We introduce a new software module called MUMDesignTool that calculates and plots the PLAM along the z-axis and provides a user-friendly framework for finding the appropriate number of planes and plane spacings for a MUM setup. The results of the paper can be reproduced using the MUMDesignTool
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