Increasing the acquisition speed in oblique plane microscopy via aliasing
Authors/Creators
- 1. UT Southwestern Medical Center
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2.
Southwestern Medical Center
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3.
MRC Laboratory of Molecular Biology
Contributors
Data collector:
Project leader:
Researcher (2):
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1.
Southwestern Medical Center
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2.
MRC Laboratory of Molecular Biology
- 3. UT Southwestern Medical Center
Description
Oblique plane microcopy (OPM), a variant of light-sheet fluorescence microscopy (LSFM), enables rapid volumetric imaging without mechanically scanning the sample or an objective. In an OPM, the sample space is mapped to a distortion free image space via remote focusing, and the oblique light-sheet plane is mapped via a tilted tertiary imaging system onto a camera. As a result, the 3D point-spread function and optical transfer function (OTF) are tilted to the optical axis of the tertiary imaging system. To satisfy Nyquist sampling, small scanning steps are required to encompass the tilted 3D OTF, slowing down acquisition and increasing sample exposure. Here we show that a judicious amount of under-sampling can lead to a form of aliasing in OPM that can be recovered without a loss of spatial resolution while minimizing artifacts. The resulting speed gains depend on the optical parameters of the system and reach 2-4-fold in our demonstrations. We leverage this method for rapid subcellular 3D imaging of mitochondria and the endoplasmic reticulum.
Technical info
The high resolution OPM data (Figure 6A-C and in Figure 7) has a px’ and py’ pixel size of 147 nm. We acquired 3D data with 207 nm scan step size, which resulted in isotropic pixels (i.e. pz’=207nm*sin(45) ). The data was then digitially downsampled 4-fold. Figure 8 was directly acquired at 828nm step size, or we digitally down-sampled an existing stack that was acquired with 207nm step size.
The mesoscopic OPM (Figure 6D-F) has a px’ and py’ pixel size of 1.15 microns, and the scanning step was set to 1.6 microns to achieve critical Nyquist sampling.
In all cases, the light-sheet was tilted by 45 degrees to the optical axis of the primary objective.
Files
Figure6.zip
Additional details
Funding
- National Institutes of Health
- Transformative microscopes to image across spatiotemporal scales R35 GM133522-
- National Institutes of Health
- Omni Oblique Plane Microscope to spread light-sheet based imaging in biomedical research R01EB035538