Standardizing image assessment in optical diffraction tomography.

Optical diffraction tomography (ODT) has gradually become a popular label-free imaging technique that offers diffraction-limited resolution by mapping an object's three-dimensional (3D) refractive index (RI) distribution. However, there is a lack of comprehensive quantitative image assessment metrics in ODT for studying how various experimental conditions influence image quality, and subsequently optimizing the experimental conditions. In this Letter, we propose to standardize the image assessment in ODT by proposing a set of metrics, including signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and structural distinguishability (SD). To test the feasibility of the metrics, we performed experiments on angle-scanning ODT by varying the number of illumination angles, RI contrast of samples, sample feature sizes, and sample types (e.g., standard polystyrene beads and 3D printed structures) and evaluated the RI tomograms with SNR, CNR, and SD. We further quantitatively studied how image quality can be improved, and tested the image assessment metrics on subcellular structures of living cells. We envision the proposed image assessment metrics may greatly benefit end-users for assessing the RI tomograms, as well as experimentalists for optimizing ODT instruments.

3D scattering microphantom sample to assess quantitative accuracy in tomographic phase microscopy techniques.

In this paper we present a structurally-complex biomimetic scattering structure, fabricated with two-photon polymerization, and utilize this object in order to benchmark a computational imaging system. The phantom allows to tailor the scattering by modifying its degrees of freedom i.e. refractive index contrast and scattering layer dimensions and incorporates a 3D imaging quality test, representing a single cell within tissue. While the sample may be used with multiple 3D microscopy techniques, we demonstrate the impact of scattering on three tomographic phase microscopy (TPM) reconstruction methods. One of these methods assumes the sample to be weak-scattering, while the other two take multiple scattering into account. The study is performed at two wavelengths (visible and near-infrared), which serve as a scaling factor for the scattering phenomenon. We find that changing the wavelength from visible into near-infrared impacts the applicability of TPM reconstruction methods. As a result of reduced scattering in near-infrared region, the multiple-scattering-oriented techniques perform in fact worse than a method aimed for weak-scattering samples. This implies a necessity of selecting proper approach depending on sample's scattering characteristics even in case of subtle changes in the object-light interaction.

Near-infrared, wavelength, and illumination scanning holographic tomography.

We present a holographic tomography technique in which the projections are acquired using both wavelength and illumination scanning in the near-infrared region. We show how to process the acquired data to obtain correct values of three-dimensional refractive index distributions in both single-wavelength and multi-wavelength data acquisition schemes and how to properly account for the dispersion of the sample. We perform numerical and experimental comparisons of different illumination scenarios to determine the most efficient measurement protocol. We show that the multi-wavelength protocol is advantageous in terms of signal-to-noise ratio and contrast-to-noise ratio over single-wavelength protocols, even for the same number of projections used for reconstructions. Finally, we show that this approach is suitable for providing high-quality refractive index distributions of relatively thick colon cancer samples.

Numerical refractive index correction for the stitching procedure in tomographic quantitative phase imaging.

Tomographic quantitative phase imaging (QPI) lacks an absolute refractive index value baseline, which poses a problem when large dense objects extending over multiple fields of view are measured volume by volume and stitched together. Some of the measurements lack the natural baseline value that is provided by the mounting medium with a known refractive index. In this work, we discuss the problem of the refractive index (RI) baseline of individual reconstructed volumes that are deprived of access to mounting medium due to the extent of the object. The solution of this problem is provided by establishing the RI offsets based on the overlapping regions. We have proven that the process of finding the offset RI values may be justifiably reduced to the analogous procedure in the 2D baseline correction (2D-BC). Finally, we proposed the enhancement of the state-of-the-art 2D-BC procedure previously introduced in the context of 2D QPI. The processing is validated at the examples of a synthetic dataset and a liver organoid.

Roadmap on Digital Holography-Based Quantitative Phase Imaging.

Quantitative Phase Imaging (QPI) provides unique means for the imaging of biological or technical microstructures, merging beneficial features identified with microscopy, interferometry, holography, and numerical computations. This roadmap article reviews several digital holography-based QPI approaches developed by prominent research groups. It also briefly discusses the present and future perspectives of 2D and 3D QPI research based on digital holographic microscopy, holographic tomography, and their applications.

Common-path intrinsically achromatic optical diffraction tomography.

In this work we propose an open-top like common-path intrinsically achromatic optical diffraction tomography system. It operates as a total-shear interferometer and employs Ronchi-type amplitude diffraction grating, positioned in between the camera and the tube lens without an additional 4f system, generating three-beam interferograms with achromatic second harmonic. Such configuration makes the proposed system low cost, compact and immune to vibrations. We present the results of the measurements of 3D-printed cell phantom using laser diode (coherent) and superluminescent diode (partially coherent) light sources. Broadband light sources can be naturally employed without the need for any cumbersome compensation because of the intrinsic achromaticity of the interferometric recording (holograms generated by -1st and +1st conjugated diffraction orders are not affected by the illumination wavelength). The results show that the decreased coherence offers much reduced coherent noise and higher fidelity tomographic reconstruction especially when applied nonnegativity constraint regularization procedure.

Multi-incidence digital holographic profilometry with high axial resolution and enlarged measurement range.

In this work, multi-incident digital holographic profilometry for microscale measurements is presented. This technique assembles the set of object fields from captured holograms for generation of the longitudinal scanning function (LSF). Numerical propagation is used for refocusing, and thus, the LSF can be determined at any given plane along the optical axis. The LSF takes maximum value for in focus object points, which are used to obtain full-field height distribution of the sample. This principle is the base of proposed measurement technique. Three capturing holograms strategies, which give control over the shape of the LSF, unambiguous measurement range, axial resolution, and noise immunity, are discussed. The conclusions of this work are supported by numerical and experimental results.

3D-printed biological cell phantom for testing 3D quantitative phase imaging systems.

As the 3D quantitative phase imaging (QPI) methods mature, their further development calls for reliable tools and methods to characterize and compare their metrological parameters. We use refractive index engineering during two-photon laser photolithography to fabricate a life-scale phantom of a biological cell with internal structures that mimic optical and structural properties of mammalian cells. After verification with a number of reference techniques, the phantom is used to characterize the performance of a limited-angle holographic tomography microscope.

Projection extrapolation routine for tight-frame limited-angle optical diffraction tomography.

We propose a data-replenishment-type expansion of the modified Gerchberg-Papoulis (GP) algorithm for limited-angle optical diffraction tomography (LAODT), which prevents artifact buildup in the GP reconstructions of confined bulk objects tightly fitting the active field of view (FoV) of the LAODT microscope. Objects crossing the FoV borders are not considered. The method relies on a Fourier-based forward projector complementary to the GP solver with no additional constraints. Fourier space regridding errors are minimized by means of one-dimensional oversampling in the axial direction, which is demonstrated to be more efficient than standard projection padding. Verification of both synthetic and experimental sinograms confirms the ability of the procedure to deduce missing projection parts necessary for the correct reconstruction.