neurovascular-network-explorer-20-simple-tool-for-exploring-sharing

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 ...

Brno University of Technology

Malignant cells appear to possess a special aptitude for survival. We attempted to prove this in vitro by acute nutritional and energy deprivation as a survival threat. A phosphate-buffered saline (PBS) survival test in cell culture allowed static observations. These were supplemented by classic and quantitative phase-contrast time-lapse microscopy. From one normal and four neoplastic cell populations, no cells survived 77 hours of exposure to PBS. Only G3S2 derived from a human breast carcinoma survived 60 hours. Cells in sparse culture were more vulnerable than those in dense. Epithelial cells were more vigorous than mesenchymal cells. Cells of greater malignancy resisted longer. Evaluation in culture as detailed by digital holographic microscopy (DHM) revealed an increase in the compactness of the intracellular mass motility from normal to metastasizing mesenchymal cells, thus reaching the level of epithelial G3S2 cells. Studying the PBS survival test with DHM opens a new approach to investigations of the structural integrity of neoplastic cells.

Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy.

The paper deals with quantitative phase imaging of two-height-level surface reliefs. The imaging is considered to be a linear system and, consequently, the Fourier transform of the image is the product of the Fourier transform of a 2D function characterizing the surface and a specific 2D coherent transfer function. The Fourier transform of functions specifying periodic surface reliefs is factorized into two functions similar to lattice and structure amplitudes in crystal structure analysis. The approach to the imaging process described in the paper enables us to examine the dependence of the phase image on the surface geometry. Theoretical results are verified experimentally by means of a digital holographic microscope.

Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process.

Transmitted-light coherence-controlled holographic microscope (CCHM) based on an off-axis achromatic interferometer allows us to use light sources of arbitrary degree of temporal and spatial coherence. Besides the conventional DHM modes such as quantitative phase contrast imaging and numerical 3D holographic reconstruction it provides high quality (speckle-free) imaging, improved lateral resolution and optical sectioning by coherence gating. Optical setup parameters and their limits for a technical realization are derived and described in detail. To demonstrate the optical sectioning property of the microscope a model sample uncovered and then covered with a diffuser was observed using a low-coherence light source.

Coherence-controlled holographic microscope.

Fresnel Incoherent Correlation Holography (FINCH) allows digital reconstruction of incoherently illuminated objects from intensity records acquired by a Spatial Light Modulator (SLM). The article presents wave optics model of FINCH, which allows analytical calculation of the Point Spread Function (PSF) for both the optical and digital part of imaging and takes into account Gaussian aperture for a spatial bounding of light waves. The 3D PSF is used to determine diffraction limits of the lateral and longitudinal size of a point image created in the FINCH set-up. Lateral and longitudinal resolution is investigated both theoretically and experimentally using quantitative measures introduced for two-point imaging. Dependence of the resolving power on the system parameters is studied and optimal geometry of the set-up is designed with regard to the best lateral and longitudinal resolution. Theoretical results are confirmed by experiments in which the light emitting diode (LED) is used as a spatially incoherent source to create object holograms using the SLM.

Point spread function and two-point resolution in Fresnel incoherent correlation holography.

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.

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. 

Multimodal holographic microscopy: distinction between apoptosis and oncosis.

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.

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

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.

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

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.

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

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.

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

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.

Automated classification of cell morphology by 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.

Article	Year
Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy. Anticancer research\| PubMed ID: 19528500	2009
Quantitative-phase-contrast imaging of a two-level surface described as a 2D linear filtering process. Optics express\| PubMed ID: 20940953	2010
Coherence-controlled holographic microscope. Optics express\| PubMed ID: 20941100	2010
Point spread function and two-point resolution in Fresnel incoherent correlation holography. Optics express\| PubMed ID: 21934923	2011
Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope. Optics express\| PubMed ID: 23787662	2013
Multimodal holographic microscopy: distinction between apoptosis and oncosis. PloS one\| PubMed ID: 25803711	2015
Quantitative phase imaging through scattering media by means of coherence-controlled holographic microscope. Journal of biomedical optics\| PubMed ID: 26244853	2015
Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion. Journal of biomedical optics\| PubMed ID: 26334859	2015
Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior. Journal of biomedical optics\| PubMed ID: 26340954	2015
Multimode fibre: Light-sheet microscopy at the tip of a needle. Scientific reports\| PubMed ID: 26657400	2015
Automated classification of cell morphology by coherence-controlled holographic microscopy. Journal of biomedical optics\| PubMed ID: 28836416	2017
Distinctive behaviour of live biopsy-derived carcinoma cells unveiled using coherence-controlled holographic microscopy. PloS one\| PubMed ID: 28846747	2017

Radim Chmelik

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