A fast-Fourier-transform method of topography and interferometry is proposed. By computer processing of a noncontour type of fringe pattern, automatic discrimination is achieved between elevation and depression of the object or wave-front form, which has not been possible by the fringe-contour-generation techniques. The method has advantages over moire topography and conventional fringe-contour interferometry in both accuracy and sensitivity. Unlike fringe-scanning techniques, the method is easy to apply because it uses no moving components.

Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry

Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

/pdf/cell-segmentation-methods-for-label-free-contrast-microscopy-4035ft6953.pdf

Cell segmentation methods for label-free contrast microscopy: review and comprehensive comparison.

Tomographic phase microscopy (TPM) is an emerging optical microscopic technique for bioimaging. TPM uses digital holographic measurements of complex scattered fields to reconstruct three-dimensional refractive index (RI) maps of cells with diffraction-limited resolution by solving inverse scattering problems. In this paper, we review the developments of TPM from the fundamental physics to its applications in bioimaging. We first provide a comprehensive description of the tomographic reconstruction physical models used in TPM. The RI map reconstruction algorithms and various regularization methods are discussed. Selected TPM applications for cellular imaging, particularly in hematology, are reviewed. Finally, we examine the limitations of current TPM systems, propose future solutions, and envision promising directions in biomedical research.

Tomographic phase microscopy: principles and applications in bioimaging [Invited]

Coherence-controlled holographic microscope (CCHM) combines off-axis holography and an achromatic grating interferometer allowing for the use of light sources of arbitrary degree of temporal and spatial coherence. This results in coherence gating and strong suppression of coherent noise and parasitic interferences enabling CCHM to reach high phase measurement accuracy and imaging quality. The achievable lateral resolution reaches performance of conventional widefield microscopes, which allows resolving up to twice smaller details when compared to typical off-axis setups. Imaging characteristics can be controlled arbitrarily by coherence between two extremes: fully coherent holography and confocal-like incoherent holography. The basic setup parameters are derived and described in detail and experimental validations of imaging characteristics are demonstrated.

Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope

Quantitative phase imaging systems using white light illumination can exhibit lower noise figures than laser-based systems. However, they can also suffer from object-dependent artifacts, such as halos, which prevent accurate reconstruction of the surface topography. In this work, we show that white light diffraction phase microscopy using a standard halogen lamp can produce accurate height maps of even the most challenging structures provided that there is proper spatial filtering at: 1) the condenser to ensure adequate spatial coherence and 2) the output Fourier plane to produce a uniform reference beam. We explain that these object-dependent artifacts are a high-pass filtering phenomenon, establish design guidelines to reduce the artifacts, and then apply these guidelines to eliminate the halo effect. Since a spatially incoherent source requires significant spatial filtering, the irradiance is lower and proportionally longer exposure times are needed. To circumvent this tradeoff, we demonstrate that a supercontinuum laser, due to its high radiance, can provide accurate measurements with reduced exposure times, allowing for fast dynamic measurements.

/pdf/effects-of-spatial-coherence-in-diffraction-phase-microscopy-gdyltoc7xn.pdf

Effects of spatial coherence in diffraction phase microscopy

In the paper a new off-axis achromatic interferometer configuration of a digital holographic microscope is presented.
The proposed configuration uses a reflective diffraction grating and ensures a high-contrast interference pattern in the
output plane of the microscope using illumination of an arbitrary degree of temporal and spatial coherence. The concept
of this optical system brings a significant improvement of microscope parameters, enables implementation of
conventional observing techniques and is more user-friendly in comparison with the previous generation of
the microscope. The functionality of the microscope has been proved experimentally.

Coherence-controlled holographic microscope

A basic description of the system for optical tomography with diffusive illumination of the phase object is presented. The configuration of the system allows automatic projection scanning of the studied phase object (such as a burner flame or temperature fields around heated elements) in the range of viewing angles from 0 deg to 90 deg. The object’s projections are obtained using multidirectional holographic interferometry and consequently digitized by a CCD camera. A computer then calculates the wavefront deformation for each projection, and the subsequent tomographic reconstruction of each horizontal slice of the object is processed. Finally, all slices are interpolated and displayed. Here we present the experimental setup along with some experimental results.

System for optical tomography

The novel laboratory system for the optical tomography is used to obtain three-dimensional temperature field around a heated element. The Mach-Zehnder holographic interferometers with diffusive illumination of the phase object provide the possibility to scan of multidirectional holographic interferograms in the range of viewing angles from 0 deg to 108 deg. These interferograms form the input data for the computer tomography of the 3D distribution of the refractive index variation, which characterizes the physical state of the studied medium. The configuration of the system allows
automatic projection scanning of the studied phase object. The computer calculates the wavefront deformation for each projection, making use of different methods of Fourier-transform and phase-sampling evaluations. The experimental set-up together with experimental results is presented.

Tomographic system for 3D temperature reconstruction

A three-dimensional configuration of multidirectional Mach-Zehnder holographic interferometer with diffusive illumination
is used in novel phase tomograph. The multidirectional tomography setup with diffusive illumination enables to digitize
many times higher number of projections in comparison with other known configurations. This setup doesn't require to
rotate with the object. The resulting resolution of the reconstructed three-dimensional image depends not only on the choice
of computer tomography algorithm, but first of all on the device ability to digitize sufficient number of complete and
equally spaced projections in given angular range with low noise level. The proposed paper is focused on design and
optimization of the optical tomography setup for the three-dimensional non-contact diagnostic of the physical properties of
stationery placed non-symmetric phase objects. Optimization of geometrical configuration design of multidirectional
holographic interferometr, with respect to significant increase of the signal to noise ratio in interferograms is performed.
Characteristic properties of the multidirectional interferograms of investigated phase objects are discussed and presented.

Martin Antoš

Papers

Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope

Coherence-controlled holographic microscope

System for optical tomography

Tomographic system for 3D temperature reconstruction

New three-dimensional configuration of multidirectional phase tomograph