Abstract
Oblique plane imaging, using remote focusing with a tilted mirror, enables direct two-dimensional (2D) imaging of any inclined plane of interest in three-dimensional (3D) specimens. It can image real-time dynamics of a living sample that changes rapidly or evolves its structure along arbitrary orientations. It also allows direct observations of any tilted target plane in an object of which orientational information is inaccessible during sample preparation. In this work, we study the optical resolution of this innovative wide-field imaging method. Using the vectorial diffraction theory, we formulate the vectorial point spread function (PSF) of direct oblique plane imaging. The anisotropic lateral resolving power caused by light clipping from the tilted mirror is theoretically analyzed for all oblique angles. We show that the 2D PSF in oblique plane imaging is conceptually different from the inclined 2D slice of the 3D PSF in conventional lateral imaging. Vectorial optical transfer function (OTF) of oblique plane imaging is also calculated by the fast Fourier transform (FFT) method to study effects of oblique angles on frequency responses.
Highlights
Oblique plane microscopy (OPM) [1,2,3,4,5,6,7] images 2D cross-sections of a specimen that are tilted from the focal plane of a microscope objective lens
To accurately predict the performance of oblique plane imaging for any oblique angle and numerical aperture (NA) regimes, we adopt the vectorial diffraction theory [12] that considers the polarization of electromagnetic waves
It is clear that the tilt of the conventional point spread function (PSF) fails to predict both the minor x-resolution loss and the y-resolution trend over the oblique angles, both of which are attributed to the pupil area loss from the light clipping
Summary
Oblique plane microscopy (OPM) [1,2,3,4,5,6,7] images 2D cross-sections of a specimen that are tilted from the focal plane of a microscope objective lens. Anselmi et al [1] proposed a wide-field oblique plane imaging method by a remote tilting technique, which has simpler configuration than Dunsby’s They explained qualitatively two mechanisms of resolution loss due to the possible light clipping and the inclined detection PSF. Smith et al [2,3] showed point-scanning oblique plane microscopy using a remote scanning technique They studied non-isotropic lateral resolution for all oblique angles in terms of spatial cutoff frequencies deduced from the region of support for the 3D OTF in Fourier space. Their analysis for point-scanning microscopy is not applicable to wide-field oblique plane imaging because their system has no light clipping with a different overall PSF. We calculate vectorial OTF from the FFT of the PSF to examine the effects of oblique angles on the spatial cutoff frequency
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