Abstract
A major challenge in three-dimensional (3D) microscopy is to obtain accurate spatial information while simultaneously keeping the microscopic samples in their native states. In conventional 3D microscopy, axial resolution is inferior to spatial resolution due to the inaccessibility to side scattering signals. In this study, we demonstrate the isotropic microtomography of free-floating samples by optically rotating a sample. Contrary to previous approaches using optical tweezers with multiple foci which are only applicable to simple shapes, we exploited 3D structured light traps that can stably rotate freestanding complex-shaped microscopic specimens, and side scattering information is measured at various sample orientations to achieve isotropic resolution. The proposed method yields an isotropic resolution of 230 nm and captures structural details of colloidal multimers and live red blood cells, which are inaccessible using conventional tomographic microscopy. We envision that the proposed approach can be deployed for solving diverse imaging problems that are beyond the examples shown here.
Highlights
Improving the three-dimensional (3D) spatial resolution is a fundamental challenge in modern microscopy
The missing cone problem impedes the accurate evaluation of axially thin 3D samples, such as rod-like microcrystals[4], red blood cells (RBCs)[5,6], and bacteria[7]
The analysis showed that the isotropic reconstruction of the poly(methyl acrylate) (PMA) dimer exhibited the maximum improved axial resolution (Fig. 5a, b)
Summary
Improving the three-dimensional (3D) spatial resolution is a fundamental challenge in modern microscopy. Most imaging modalities record 3D information with sub-micrometre resolution via axial[1] or illumination scanning[2], their axial resolution is inferior to the lateral resolution due to the finite numerical aperture (NA) of a condenser and an objective lens. This long-standing challenge is known as the missing cone problem because the Fourier spectrum of a reconstructed tomogram contains no information in a conical region along the optical axis[3]. A straightforward approach is the rotation of a sample loaded in a microcapillary[8,9] or a rotating tip[10,11]
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