DryMass: handling and analyzing quantitative phase microscopy images of spherical, cell-sized objects.

BACKGROUND: Quantitative phase imaging (QPI) is an established tool for the marker-free classification and quantitative characterization of biological samples. For spherical objects, such as cells in suspension, microgel beads, or liquid droplets, a single QPI image is sufficient to extract the radius and the average refractive index. This technique is invaluable, as it allows the characterization of large sample populations at high measurement rates. However, until now, no universal software existed that could perform this type of analysis. Besides the choice of imaging modality and the variety in imaging software, the main difficulty has been to automate the entire analysis pipeline from raw data to ensemble statistics. RESULTS: We present DryMass, a powerful tool for QPI that covers all relevant steps from loading experimental data (multiple file formats supported), computing the phase data (built-in, automated hologram analysis), performing phase background corrections (offset, tilt, second order polynomial) to fitting scattering models (light projection, Rytov approximation, Mie simulations) to spherical phase objects for the extraction of dry mass, radius, and average refractive index. The major contribution of DryMass is a user-convenient, reliable, reproducible, and automated analysis pipeline for an arbitrary number of QPI datasets of arbitrary sizes. CONCLUSION: DryMass is a leap forward for data analysis in QPI, as it not only makes it easier to visualize raw QPI data and reproduce previous results in the field, but it also opens up QPI analysis to users without a background in programming or phase imaging.

Mechanical Mapping of Spinal Cord Growth and Repair in Living Zebrafish Larvae by Brillouin Imaging.

The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time to our knowledge a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and nondestructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal's natural development. The Brillouin shift within the spinal cord remained comparable during development and transiently decreased during the repair processes after spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step toward an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties, and allows us to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.

Accurate evaluation of size and refractive index for spherical objects in quantitative phase imaging.

Measuring the average refractive index (RI) of spherical objects, such as suspended cells, in quantitative phase imaging (QPI) requires a decoupling of RI and size from the QPI data. This has been commonly achieved by determining the object's radius with geometrical approaches, neglecting light-scattering. Here, we present a novel QPI fitting algorithm that reliably uncouples the RI using Mie theory and a semi-analytical, corrected Rytov approach. We assess the range of validity of this algorithm in silico and experimentally investigate various objects (oil and protein droplets, microgel beads, cells) and noise conditions. In addition, we provide important practical cues for the analysis of spherical objects in QPI.

Volume Transitions of Isolated Cell Nuclei Induced by Rapid Temperature Increase.

Understanding the physical mechanisms governing nuclear mechanics is important as it can impact gene expression and development. However, how cell nuclei respond to external cues such as heat is not well understood. Here, we studied the material properties of isolated nuclei in suspension using an optical stretcher. We demonstrate that isolated nuclei regulate their volume in a highly temperature-sensitive manner. At constant temperature, isolated nuclei behaved like passive, elastic and incompressible objects, whose volume depended on the pH and ionic conditions. When the temperature was increased suddenly by even a few degrees Kelvin, nuclei displayed a repeatable and reversible temperature-induced volume transition, whose sign depended on the valency of the solvent. Such phenomenon is not observed for nuclei subjected to slow heating. The transition temperature could be shifted by adiabatic changes of the ambient temperature, and the magnitude of temperature-induced volume transition could be modulated by modifying the chromatin compaction state and remodeling processes. Our findings reveal that the cell nucleus can be viewed as a highly charged polymer gel with intriguing thermoresponsive properties, which might play a role in nuclear volume regulation and thermosensing in living cells.

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.

Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

PNIPAAm microgels with defined network architecture as temperature sensors in optical stretchers.

Stretching individual living cells with light is a standard method to assess their mechanical properties. Yet, heat introduced by the laser light of optical stretchers may unwittingly change the mechanical properties of cells therein. To estimate the temperature induced by an optical trap, we introduce cell-sized, elastic poly(N-isopropylacrylamide) (PNIPAAm) microgels that relate temperature changes to hydrogel swelling. For their usage as a standardized calibration tool, we analyze the effect of free-radical chain-growth gelation (FCG) and polymer-analogous photogelation (PAG) on hydrogel network heterogeneity, micromechanics, and temperature response by Brillouin microscopy and optical diffraction tomography. Using a combination of tailor-made PNIPAAm macromers, PAG, and microfluidic processing, we obtain microgels with homogeneous network architecture. With that, we expand the capability of standardized microgels in calibrating and validating cell mechanics analysis, not only considering cell and microgel elasticity but also providing stimuli-responsiveness to consider dynamic changes that cells may undergo during characterization.

Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.

Three-dimensional correlative single-cell imaging utilizing fluorescence and refractive index tomography.

Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.

Droplet-Assisted Microfluidic Fabrication and Characterization of Multifunctional Polysaccharide Microgels Formed by Multicomponent Reactions.

Polysaccharide-based microgels have broad applications in multi-parametric cell cultures, cell-free biotechnology, and drug delivery. Multicomponent reactions like the Passerini three-component and the Ugi four-component reaction are shown in here to be versatile platforms for fabricating these polysaccharide microgels by droplet microfluidics with a narrow size distribution. While conventional microgel formation requires pre-modification of hydrogel building blocks to introduce certain functionality, in multicomponent reactions one building block can be simply exchanged by another to introduce and extend functionality in a library-like fashion. Beyond synthesizing a range of polysaccharide-based microgels utilizing hyaluronic acid, alginate and chitosan, exemplary in-depth analysis of hyaluronic acid-based Ugi four-component gels is conducted by colloidal probe atomic force microscopy, confocal Brillouin microscopy, quantitative phase imaging, and fluorescence correlation spectroscopy to elucidate the capability of microfluidic multicomponent reactions for forming defined polysaccharide microgel networks. Particularly, the impact of crosslinker amount and length is studied. A higher network density leads to higher Young's moduli accompanied by smaller pore sizes with lower diffusion coefficients of tracer molecules in the highly homogeneous network, and vice versa. Moreover, tailored building blocks allow for crosslinking the microgels and incorporating functional groups at the same time as demonstrated for biotin-functionalized, chitosan-based microgels formed by Ugi four-component reaction. To these microgels, streptavidin-labeled enzymes are easily conjugated as shown for horseradish peroxidase (HRP), which retains its activity inside the microgels.

Mechanical deformation induces depolarization of neutrophils.

The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.

Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.

Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore.

Accurate chromosome segregation depends on proper kinetochore-microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore-centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores.

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term 'bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

Real-time imaging of DNA damage in yeast cells using ultra-short near-infrared pulsed laser irradiation.

Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200-1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.

Selective on site separation and detection of molecules in diluted solutions with super-hydrophobic clusters of plasmonic nanoparticles.

Super-hydrophobic surfaces are bio-inspired interfaces with a superficial texture that, in its most common evolution, is formed by a periodic lattice of silicon micro-pillars. Similar surfaces reveal superior properties compared to conventional flat surfaces, including very low friction coefficients. In this work, we modified meso-porous silicon micro-pillars to incorporate networks of metal nano-particles into the porous matrix. In doing so, we obtained a multifunctional-hierarchical system in which (i) at a larger micrometric scale, the super-hydrophobic pillars bring the molecules dissolved in an ultralow-concentration droplet to the active sites of the device, (ii) at an intermediate meso-scale, the meso-porous silicon film adsorbs the low molecular weight content of the solution and, (iii) at a smaller nanometric scale, the aggregates of silver nano-particles would measure the target molecules with unprecedented sensitivity. In the results, we demonstrated how this scheme can be utilized to isolate and detect small molecules in a diluted solution in very low abundance ranges. The presented platform, coupled to Raman or other spectroscopy techniques, is a realistic candidate for the protein expression profiling of biological fluids.

Mechanical stress downregulates MHC class I expression on human cancer cell membrane.

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1), indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.