Primary assessment of medicines for expected migrastatic potential with holographic incoherent quantitative phase imaging.

Solid tumor metastases cause most cancer-related deaths. The prevention of their occurrence misses suitable anti-metastases medicines newly labeled as migrastatics. The first indication of migrastatics potential is based on an inhibition of in vitro enhanced migration of tumor cell lines. Therefore, we decided to develop a rapid test for qualifying the expected migrastatic potential of some drugs for repurposing. The chosen Q-PHASE holographic microscope provides reliable multifield time-lapse recording and simultaneous analysis of the cell morphology, migration, and growth. The results of the pilot assessment of the migrastatic potential exerted by the chosen medicines on selected cell lines are presented.

Testing anti-cancer drugs with holographic incoherent-light-source quantitative phase imaging.

Quantitative Phase Imaging is becoming an important tool in the objective evaluation of cellular responses to experimental treatment. The technique is based on interferometric measurements of the optical thickness of cells in tissue culture reporting on the distribution of dry mass inside the cells. As the measurement of the optical thickness is interferometric, it is not subjected to the Abbe resolution limit, and the use of an incoherent-light source further increases the accuracy practically achieving 0.93nm in optical path difference corresponding to 4.6 femtograms/µm2. Holographic mode reduces the exposure in comparison to phase-shifting or phase-stepping interference microscopy and allows observation of faster dynamics. An attractive application is in the development of novel anti-cancer drugs and there is an important potential for pretesting chemotherapeutic drugs with biopsy material for personalized cancer treatment. The procedure involves the preparation of live cells in tissue culture, seeding them into suitable observation chambers, and time-lapse recording with an adjusted microscope. Subsequent image processing and statistical analysis are essential last steps producing the results, which include rapid measurements of cell growth in terms of dry-mass increase in individual cells, speed of cell motility and other dynamic morphometric parameters.

Coherence gate manipulation for enhanced imaging through scattering media by non-ballistic light in partially coherent interferometric systems.

Coherence gating is typically exploited for imaging through disordered media by least-scattered (ballistic) light. Ballistic light-based approaches produce clear images only when the proportion of ballistic to multiply scattered (non-ballistic) light is relatively high. To overcome this limitation, we counterintuitively utilize the coherence gate to image by the non-ballistic light, enabling us to retrieve information missing in the ballistic image. We show that non-ballistic images acquired by transversal coherence gate shifting have image quality and spatial resolution comparable to the ballistic image. Combining images for different coherence gate positions, we synthesize an image of quality superior to ballistic light approaches. We experimentally demonstrate our concept on quantitative phase imaging through biological tissue.

Single-Shot Three-Dimensional Orientation Imaging of Nanorods Using Spin to Orbital Angular Momentum Conversion.

The key information about any nanoscale system relates to the orientations and conformations of its parts. Unfortunately, these details are often hidden below the diffraction limit, and elaborate techniques must be used to optically probe them. Here we present imaging of the 3D rotation motion of metal nanorods, restoring the distinct nanorod orientations in the full extent of azimuthal and polar angles. The nanorods imprint their 3D orientation onto the geometric phase and space-variant polarization of the light they scatter. We manipulate the light angular momentum and generate optical vortices that create self-interference images providing the nanorods' angles via digital processing. After calibration by scanning electron microscopy, we demonstrated time-resolved 3D orientation imaging of sub-100 nm nanorods under Brownian motion (frame rate up to 500 fps). We also succeeded in imaging nanorods as nanoprobes in live-cell imaging and reconstructed their 3D rotational movement during interaction with the cell membrane (100 fps).

Phase of white light and its compatibility to the optical path.

The phase of monochromatic light directly relates to the optical path difference (OPD), but finding this connection for spectrally broadband light is challenging. Due to a missing concept of the compatibility between the phase of randomly fluctuating fields and the OPD, demanding scanning is the only proven way for a highly accurate OPD measurement in white light. Here, we use the self-coherence function (SCF) of the spatially incoherent light to reveal the connection between the white-light phase and the OPD. Our method uses an associated field assigned to the SCF to mimic the intensity oscillation of a correlation pattern. The associated field allows restoring a cumulative OPD integrated into the SCF across all spectral constituents. The method is essential for quantitative phase microscopy, in which the SCF is available even in white light, but its processing beyond the quasi-monochromatic approach is still lacking. Improper assessment of the white-light phase may result in a loss of measurement accuracy, as we demonstrate theoretically and experimentally. Deploying our method in coherence-controlled holographic microscopy, we measured the cumulative OPD in the broadband light with a strongly asymmetric spectrum (bandwidth of 150 nm), achieving accuracy better than 5 nm in the measuring depth range of 2 µm.

Automated interpretation of time-lapse quantitative phase image by machine learning to study cellular dynamics during epithelial-mesenchymal transition.

SIGNIFICANCE: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification. AIM: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes. APPROACH: The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images. RESULTS: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes. CONCLUSIONS: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

Geometric-Phase Microscopy for Quantitative Phase Imaging of Isotropic, Birefringent and Space-Variant Polarization Samples.

We present geometric-phase microscopy allowing a multipurpose quantitative phase imaging in which the ground-truth phase is restored by quantifying the phase retardance. The method uses broadband spatially incoherent light that is polarization sensitively controlled through the geometric (Pancharatnam-Berry) phase. The assessed retardance possibly originates either in dynamic or geometric phase and measurements are customized for quantitative mapping of isotropic and birefringent samples or multi-functional geometric-phase elements. The phase restoration is based on the self-interference of polarization distinguished waves carrying sample information and providing pure reference phase, while passing through an inherently stable common-path setup. The experimental configuration allows an instantaneous (single-shot) phase restoration with guaranteed subnanometer precision and excellent ground-truth accuracy (well below 5 nm). The optical performance is demonstrated in advanced yet routinely feasible noninvasive biophotonic imaging executed in the automated manner and predestined for supervised machine learning. The experiments demonstrate measurement of cell dry mass density, cell classification based on the morphological parameters and visualization of dynamic dry mass changes. The multipurpose use of the method was demonstrated by restoring variations in the dynamic phase originating from the electrically induced birefringence of liquid crystals and by mapping the geometric phase of a space-variant polarization directed lens.

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna.

Optical metasurfaces have emerged as a new generation of building blocks for multifunctional optics. Design and realization of metasurface elements place ever-increasing demands on accurate assessment of phase alterations introduced by complex nanoantenna arrays, a process referred to as quantitative phase imaging. Despite considerable effort, the widefield (nonscanning) phase imaging that would approach resolution limits of optical microscopy and indicate the response of a single nanoantenna still remains a challenge. Here, we report on a new strategy in incoherent holographic imaging of metasurfaces, in which unprecedented spatial resolution and light sensitivity are achieved by taking full advantage of the polarization selective control of light through the geometric (Pancharatnam-Berry) phase. The measurement is carried out in an inherently stable common-path setup composed of a standard optical microscope and an add-on imaging module. Phase information is acquired from the mutual coherence function attainable in records created in broadband spatially incoherent light by the self-interference of scattered and leakage light coming from the metasurface. In calibration measurements, the phase was mapped with the precision and spatial background noise better than 0.01 and 0.05 rad, respectively. The imaging excels at the high spatial resolution that was demonstrated experimentally by the precise amplitude and phase restoration of vortex metalenses and a metasurface grating with 833 lines/mm. Thanks to superior light sensitivity of the method, we demonstrated for the first time to our knowledge the widefield measurement of the phase altered by a single nanoantenna while maintaining the precision well below 0.15 rad.

Quantitative phase imaging unravels new insight into dynamics of mesenchymal and amoeboid cancer cell invasion.

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo.

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain and process these data. However, the majority of experimental data, especially from individual studies of regular-sized laboratories never reach wider research community. A graphical user interface (GUI) engine called Neurovascular Network Explorer 2.0 (NNE 2.0) has been created as a tool for simple and low-cost sharing and exploring of vascular imaging data. NNE 2.0 interacts with a database containing optogenetically-evoked dilation/constriction time-courses of individual vessels measured in mice somatosensory cortex in vivo by 2-photon microscopy. NNE 2.0 enables selection and display of the time-courses based on different criteria (subject, branching order, cortical depth, vessel diameter, arteriolar tree) as well as simple mathematical manipulation (e.g. averaging, peak-normalization) and data export. It supports visualization of the vascular network in 3D and enables localization of the individual functional vessel diameter measurements within vascular trees. NNE 2.0, its source code, and the corresponding database are freely downloadable from UCSD Neurovascular Imaging Laboratory website1. The source code can be utilized by the users to explore the associated database or as a template for databasing and sharing their own experimental results provided the appropriate format.

Dual-polarization interference microscopy for advanced quantification of phase associated with the image field.

A new concept of dual-polarization spatial light interference microscopy (DPSLIM) is proposed and demonstrated experimentally. The method works with two orthogonally polarized modes in which signal and reference waves are combined to realize the polarization-sensitive phase-shifting, thus allowing advanced reconstruction of the phase associated with the image field. The image phase is reconstructed directly from four polarization encoded interference records by a single step processing. This is a progress compared with common methods, in which the phase of the image field is reconstructed using the optical path difference and the amplitudes of interfering waves, which are calculated in multiple-step processing of the records. The DPSLIM is implemented in a common-path configuration using a spatial light modulator, which is connected to a commercial microscope Nikon E200. The optical performance of the method is demonstrated in experiments using both polystyrene microspheres and live LW13K2 cells.

Automated classification of cell morphology by coherence-controlled holographic microscopy.

In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity.

Distinctive behaviour of live biopsy-derived carcinoma cells unveiled using coherence-controlled holographic microscopy.

Head and neck squamous cell carcinoma is one of the most aggressive tumours and is typically diagnosed too late. Late diagnosis requires an urgent decision on an effective therapy. An individualized test of chemosensitivity should quickly indicate the suitability of chemotherapy and radiotherapy. No ex vivo chemosensitivity assessment developed thus far has become a part of general clinical practice. Therefore, we attempted to explore the new technique of coherence-controlled holographic microscopy to investigate the motility and growth of live cells from a head and neck squamous cell carcinoma biopsy. We expected to reveal behavioural patterns characteristic for malignant cells that can be used to imrove future predictive evaluation of chemotherapy. We managed to cultivate primary SACR2 carcinoma cells from head and neck squamous cell carcinoma biopsy verified through histopathology. The cells grew as a cohesive sheet of suspected carcinoma origin, and western blots showed positivity for the tumour marker p63 confirming cancerous origin. Unlike the roundish colonies of the established FaDu carcinoma cell line, the SACR2 cells formed irregularly shaped colonies, eliciting the impression of the collective invasion of carcinoma cells. Time-lapse recordings of the cohesive sheet activity revealed the rapid migration and high plasticity of these epithelial-like cells. Individual cells frequently abandoned the swiftly migrating crowd by moving aside and crawling faster. The increasing mass of fast migrating epithelial-like cells before and after mitosis confirmed the continuation of the cell cycle. In immunofluorescence, analogously shaped cells expressed the p63 tumour marker, considered proof of their origin from a carcinoma. These behavioural traits indicate the feasible identification of carcinoma cells in culture according to the proposed concept of the carcinoma cell dynamic phenotype. If further developed, this approach could later serve in a new functional online analysis of reactions of carcinoma cells to therapy. Such efforts conform to current trends in precision medicine.

Multimode fibre: Light-sheet microscopy at the tip of a needle.

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

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

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.

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

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.

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

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.

Multimodal holographic microscopy: distinction between apoptosis and oncosis.

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - "Multimodal holographic microscopy (MHM)". The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI- phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.

Automated alignment method for coherence-controlled holographic microscope

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.