Zbynek Dostal

Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion.

Abstract. Coherence-controlled holographic microscopy (CCHM) in low-coherence mode possesses a pronounced coherence gate effect. This offers an option to investigate the details of cellular events leading to cell death caused by cytopathic turbid emulsions. CCHM capacity was first assessed in model situations that showed clear images obtained with low coherence of illumination but not with high coherence of illumination. Then, the form of death of human cancer cells induced by treatment with biologically active phospholipids (BAPs) preparation was investigated. The observed overall retraction of cell colony was apparently caused by the release of cell-to-substratum contacts. This was followed by the accumulation of granules decorating the nuclear membrane. Then, the occurrence of nuclear membrane indentations signaled the start of damage to the integrity of the cell nucleus. In the final stage, cells shrunk and disintegrated. This indicated that BAPs cause cell death by necrosis and not apoptosis. An intriguing option of checking the fate of cancer cells caused by the anticipated cooperative effect after adding another tested substance sodium dichloroacetate to turbid emulsion is discussed on grounds of pilot experiments. Such observations should reveal the impact and mechanism of action of the interacting drugs on cell behavior and fate that would otherwise remain hidden in turbid milieu.

The Role of Coherence in Image Formation in Holographic Microscopy

Abstract Off-axis digital holographic microscopes (DHM) working with incoherent light have been designed and constructed. Their imaging properties can be changed by variation of the coherence of light. This spans from emulation of classic coherent-light DHM allowing for numerical focusing to incoherent-light DHM characterized by high-quality imaging, no coherence noise, halved limit of lateral resolution, and by coherence-gating effect making imaging in turbid media and optical sectioning possible. We describe theoretically the imaging process of a holographic microscope (HM) and how it is influenced by the coherence of illumination. The 3D coherent transfer function (CTF) reveals the dependence of a spatial frequency passband on the coherence properties of a source. Reduction of coherence leads to the passband broadening i.e. to the resolution enhancement. This effect is obvious also from the form of 3D point spread functions, which allows us to characterize imaging by 3D convolution. Imaging and numerical focusing of planar objects are described by 2D CTF derived from 3D CTF for various defocusing. Results for 2D objects are presented also in a simplified approximate form, which gives deeper insight into the fundaments of imaging. In this approximation, the image formation in a turbid medium by coherence gating is elucidated. In addition, it is shown that the mutual lateral shift of the object and reference beams amplifies higher spatial frequencies of a defocused object and allows an object in a turbid medium to be imaged by diffuse (non-ballistic) light. Important theoretical results are verified experimentally.

Quantitative phase imaging through scattering media by means of coherence-controlled holographic microscope.

Abstract. A coherence-controlled holographic microscope (CCHM) enables quantitative phase imaging with coherent as well as incoherent illumination. The low spatially coherent light induces a coherence gating effect, which makes observation of samples possible also through scattering media. The paper describes theoretically and simulates numerically imaging of a two-dimensional object through a static scattering layer by means of CCHM, with the main focus on the quantitative phase imaging quality. The authors have investigated both strongly and weakly scattering media characterized by different amounts of ballistic and diffuse light. It is demonstrated that the phase information can be revealed also for the case of the static, strongly scattering layer. The dependence of the quality of imaging process on the spatial light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with a model phase object, as well as living carcinoma cells treated in an optically turbid emulsion.

Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior.

Abstract. Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two-dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Quantitative phase imaging through scattering media

Coherence-controlled holographic microscope (CCHM) is an off-axis holographic system. It enables observation of a sample and its quantitative phase imaging with coherent as well as with incoherent illumination. The spatial and temporal coherence can be modified and thus also the quality and type of the image information. The coherent illumination provides numerical refocusing in wide depth range similarly to a classic coherent-light digital holographic microscopy (HM). Incoherent-light HM is characterized by a high quality, coherence-noise-free imaging with up to twice higher resolution compared to coherent illumination. Owing to an independent, free of sample reference arm of the CCHM the low spatial light coherence induces coherence-gating effect. This makes possible to observe specimen also through scattering media. We have described theoretically and simulated numerically imaging of a two dimensional object through a scattering layer by CCHM using the linear systems theory. We have investigated both strongly and weakly scattering media characterized by different amount of ballistic and diffuse light. The influence of a scattering layer on the quality of a phase signal is discussed for both types of the scattering media. A strong dependence of the imaging process on the light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with model samples, as well as real biologic objects particularly then by time-lapse observations of live cells reactions to substances producing optically turbid emulsion.

Automated alignment method for coherence-controlled holographic microscope

Abstract. A coherence-controlled holographic microscope (CCHM) was developed particularly for quantitative phase imaging and measurement of live cell dynamics, which is the proper subject of digital holographic microscopy (DHM). CCHM in low-coherence mode extends DHM in the study of living cells. However, this advantage is compensated by sensitivity of the system to easily become misaligned, which is a serious hindrance to wanted performance. Therefore, it became clear that introduction of a self-correcting system is inevitable. Accordingly, we had to devise a theory of a suitable control and design an automated alignment system for CCHM. The modulus of the reconstructed holographic signal was identified as a significant variable for guiding the alignment procedures. From this, we derived the original basic realignment three-dimensional algorithm, which encompasses a unique set of procedures for automated alignment that contains processes for initial and advanced alignment as well as long-term maintenance of microscope tuning. All of these procedures were applied to a functioning microscope and the tested processes were successfully validated. Finally, in such a way, CCHM is enabled to substantially contribute to study of biology, particularly of cancer cells in vitro.A hyperspectral image projector (HIP) based on liquid crystal on silicon spatial light modulators is explained and demonstrated to generate data cubes. The HIP-constructed data cubes are three-dimensional images of the spatial distribution of spectrally resolved abundances of intracellular light-absorbing oxyhemoglobin molecules in single erythrocytes. Spectrally and spatially resolved image data indistinguishable from the real scene may be used as standard data cubes, so-called digital phantoms, to calibrate image sensors and validate image analysis algorithms for their measurement quality, performance consistency, and interlaboratory comparisons for quantitative biomedical imaging applications.

Coherence-Controlled Holographic Microscopy for Coherence-Gated Quantitative Phase Imaging

We show that the use of incoherent illumination in coherence-controlled holographic microscopy (CCHM) enables coherence-gated quantitative phase imaging of objects through turbid media. Also high lateral resolution and strong suppression of coherence noise is demonstrated.