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.

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.

Holographic tomography: techniques and biomedical applications [Invited].

Holographic tomography (HT) is an advanced label-free optical microscopic imaging method used for biological studies. HT uses digital holographic microscopy to record the complex amplitudes of a biological sample as digital holograms and then numerically reconstruct the sample's refractive index (RI) distribution in three dimensions. The RI values are a key parameter for label-free bio-examination, which correlate with metabolic activities and spatiotemporal distribution of biophysical parameters of cells and their internal organelles, tissues, and small-scale biological objects. This article provides insight on this rapidly growing HT field of research and its applications in biology. We present a review summary of the HT principle and highlight recent technical advancement in HT and its applications.

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.

Reconstruction method for extended depth-of-field optical diffraction tomography.

In the paper we present a novel method of extended depth-of-field limited-angle optical diffraction tomography, in which the change of a focal plane position is performed with a liquid focus-tunable lens. One sinogram is acquired for each state of a focus-tunable lens. After acquisition process is complete, all sinograms are independently reconstructed and stitched to form the final tomographic reconstruction. The presented solution effectively extends the applicability of the Rytov approximation to relatively thick samples and provides uniform resolution of 3D tomographic reconstructions. The method is also combined with Generalized Total Variation Iterative Constraint algorithm, which minimizes distortion of the results due to the limited angular range of acquired projections. The combined solution is dedicated to investigation of transparent and semi-transparent biological micro-structures, like cells and tissue slices.

Illumination-related errors in limited-angle optical diffraction tomography.

In the paper, the design and tolerances of optical systems and scanning components used in limited-angle optical diffraction tomography are analyzed in order to improve the performance of the measurement systems and to encourage the application of tomography as a standard method for quantitative analysis of 3D refractive index distribution in biological microstructures. The first part of the presented analysis consists of component selection for the scanning device and optical system in the illumination part of the setup and the influence of the illumination wavefront on reconstruction quality. In the second part, the sensitivity of the tomographic reconstruction quality to three representative measurement-related errors based on synthetic data is demonstrated. Finally, a configuration of the system, selected to minimize reconstruction errors, is proposed and alignment tolerances simulated using the Monte Carlo method are provided.

Holographic tomography with scanning of illumination: space-domain reconstruction for spatially invariant accuracy.

The paper presents two novel, space-domain reconstruction algorithms for holographic tomography utilizing scanning of illumination and a fixed detector that is highly suitable for imaging of living biomedical specimens. The first proposed algorithm is an adaptation of the filtered backpropagation to the scanning illumination tomography. Its space-domain implementation enables avoiding the error-prone interpolation in the Fourier domain, which is a significant problem of the state-of-the-art tomographic algorithm. The second proposed algorithm is a modified version of the former, which ensures the spatially invariant reconstruction accuracy. The utility of the proposed algorithms is demonstrated with numerical simulations and experimental measurement of a cancer cell.

Generalized total variation iterative constraint strategy in limited angle optical diffraction tomography.

Due to incompleteness of input data inherent to Limited Angle Tomography (LAT), specific additional constraints are usually employed to suppress image artifacts. In this work we demonstrate a new two-stage regularization strategy, named Generalized Total Variation Iterative Constraint (GTVIC), dedicated to semi-piecewise-constant objects. It has been successfully applied as a supplementary module for two different reconstruction algorithms: an X-ray type solver and a diffraction-wise solver. Numerical tests performed on a detailed phantom of a biological cell under conical illumination pattern show significant reduction of axial blurring in the reconstructed refractive index distribution after GTVIC is added. Analogous results were obtained with experimental data.

Active limited-angle tomographic phase microscope.

We demonstrate an active, holographic tomography system, working with limited angle of projections, realized by optical-only, diffraction-based beam steering. The system created for this purpose is a Mach­Zehnder interferometer modified to serve as a digital holographic microscope with a high numerical aperture illumination module and a spatial light modulator (SLM). Such a solution is fast and robust. Apart from providing an elegant solution to viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different illumination scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied here to reconstruct nonpiecewise constant samples. Finally, the accuracy of full measurement and the proposed processing path is tested for a calibrated three-dimensional micro-object as well as a biological object--C2C12 myoblast cell.

Accurate approach to capillary-supported optical diffraction tomography.

A new holographic data processing path for accurate quantitative tomographic reconstruction of 3D samples placed in a cylindrical capillary is proposed. The method considers strong unintentional focusing effects induced by the inner cylindrical boundary of the vessel: 1) introduction of cylindrical wave illumination of a sample, and 2) object wave deformation. The first issue is addressed by developing an arbitrary illumination tomographic reconstruction algorithm based on filtered backpropagation, while the second by a novel correction algorithm utilizing the optical rays analysis. Moreover, the processing path includes a novel holographic method for correction of spherical aberration related to refraction at a planar surface. Utility of the developed data processing path is proven with numerical simulations and experimental measurement of a specially prepared test sample.

Tomographic phase microscopy of living three-dimensional cell cultures.

A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a "3-D cluster" as well as subcellular organelles like the nucleoli and local internal refractive index changes.