Single-exposure 3D label-free microscopy based on color-multiplexed intensity diffraction tomography

Abstract

We present a 3D label-free refractive index (RI) imaging technique based on single-exposure intensity diffraction tomography (sIDT) using a color-multiplexed illumination scheme. In our method, the chromatic light-emitting diodes (LEDs) corresponding R/G/B channels in an annular programmable ring provide oblique illumination geometry that precisely matches the objective’s numerical aperture. A color intensity image encoding the scattering field of the specimen from different directions is captured, and monochromatic intensity images concerning three color channels are separated and then used to recover the 3D RI distribution of the object following the process of IDT. In addition, the axial chromatic dispersion of focal lengths at different wavelengths introduced by the chromatic aberration of the objective lens and the spatial position misalignment of the ring LED source in the imaging system’s transfer functions modeling are both corrected to significantly reduce the artifacts in the slice-based deconvolution procedure for the reconstruction of 3D RI distribution. Experimental results on MCF-7, Spirulina algae, and living Caenorhabditis elegans samples demonstrate the reliable performance of the sIDT method in label-free, high-throughput, and real-time (∼24 fps) 3D volumetric biological imaging applications.

Quantitative Measurements of Red Blood Cell Indices Using Spectroscopic Differential Phase-Contrast Microscopy

“…Advanced forms of ODT demonstrated a tomographic RI imaging capability from a single image acquisition. Zhou et al, for example, introduced color-multiplexed diffraction tomography capable of producing an RI tomogram in a single shot . This method, however, exhibits nonisotropic spatial resolution and image artifacts due to the significant amount of missing information in the object 3D spectrum that can be obtained with a single measurement.…”

Live-cell analysis framework for quantitative phase imaging with slightly off-axis digital holographic microscopy

“…This requires measurements must be performed at the single cell level with high precision and high throughput, thus bringing challenges to microscopy. Quantitative Phase Imaging (QPI) techniques are rapidly gaining momentum and popularity as new imaging tools in cellular biology (Zuo et al, 2017;Fan et al, 2019;Li et al, 2019;Zuo et al, 2020;Lu et al, 2022;Zhou et al, 2022), where Digital holographic microscopy (DHM) (Cuche et al, 1999;Mann et al, 2005;Huang et al, 2022) combines interferometric technique, modern CCD sensor and image processing systems, opening up a new perspective for the accurate characterization of physical properties of living cells.…”