Artificial Neural Network (ANN)-Based Determination of Fractional Contributions from Mixed Fluorophores using Fluorescence Lifetime Measurements.

Here we present an artificial neural network (ANN)-approach to determine the fractional contributions Pi from fluorophores to a multi-exponential fluorescence decay in time-resolved lifetime measurements. Conventionally, Pi are determined by extracting two parameters (amplitude and lifetime) for each underlying mono-exponential decay using non-linear fitting. However, in this case parameter estimation is highly sensitive to initial guesses and weighting. In contrast, the ANN-based approach robustly gives the Pi without knowledge of amplitudes and lifetimes. By experimental measurements and Monte-Carlo simulations, we comprehensively show that accuracy and precision of Pi determination with ANNs and hence the number of distinguishable fluorophores depend on the fluorescence lifetimes' differences. For mixtures of up to five fluorophores, we determined the minimum uniform spacing Δτmin between lifetimes to obtain fractional contributions with a standard deviation of 5%. In example, five lifetimes can be distinguished with a respective minimum uniform spacing of approx. 10 ns even when the fluorophores' emission spectra are overlapping. This study underlines the enormous potential of ANN-based analysis for multi-fluorophore applications in fluorescence lifetime measurements.

Advantages and Limitations of Fluorescence Lifetime Measurements Using Single-Photon Avalanche Diode (SPAD) Array Detector: A Comprehensive Theoretical and Experimental Study.

Fast fluorescence lifetime (FL) determination is a major factor for studying dynamic processes. To achieve a required precision and accuracy a certain number of photon counts must be detected. FL methods based on single-photon counting have strongly limited count rates because of the detector's pile-up issue and are suffering from long measurement times in the order of tens of seconds. Here, we present an experimental and Monte Carlo simulation-based study of how this limitation can be overcome using array detectors based on single-photon avalanche diodes (SPADs). We investigated the maximum count rate per pixel to determine FL with a certain precision and accuracy before pile-up occurs. Based on that, we derived an analytical expression to calculate the total measurement time which is proportional to the FL and inversely proportional to the number of pixels. However, a higher number of pixels drastically increases data rate. This can be counteracted by lowering the time resolution. We found that even with a time resolution of four times the FL, an accuracy of 10% can be achieved. Taken all together, FLs between 10 ns and 3 ns can be determined with a 300-pixel SPAD array detector with a measurement time and data rate less than 1 µs and 700 Mbit/s, respectively. This shows the enormous potential of SPAD array detector for high-speed applications requiring continuous data read out.

Combined atomic force microscopy (AFM) and traction force microscopy (TFM) reveals a correlation between viscoelastic material properties and contractile prestress of living cells.

Living cells exhibit a complex mechanical behavior, whose underlying mechanisms are still largely unknown. Emerging from the molecular structure and dynamics of the cytoskeleton, the mechanical behavior comprises "passive" viscoelastic material properties and "active" contractile prestress. To directly investigate the connection between these quantities at the single-cell level, we here present the combination of atomic force microscopy (AFM) with traction force microscopy (TFM). With this combination, we simultaneously measure viscoelastic material parameters (stiffness, fluidity) and contractile prestress of adherent fibroblast and epithelial cells. Although stiffness, fluidity, and contractile prestress greatly vary within a cell population, they are highly correlated: stiffer cells have a lower fluidity and a larger prestress than softer cells. We show that viscoelastic material properties and contractile prestress are both governed by the activity of the actomyosin machinery. Our results underline the connection between a cell's viscoelastic material properties and its contractile prestress and their importance in cell mechanics.

Macro-SICM: A Scanning Ion Conductance Microscope for Large-Range Imaging.

The scanning ion conductance microscope (SICM) is a versatile, high-resolution imaging technique that uses an electrolyte-filled nanopipet as a probe. Its noncontact imaging principle makes the SICM uniquely suited for the investigation of soft and delicate surface structures in a liquid environment. The SICM has found an ever-increasing number of applications in chemistry, physics, and biology. However, a drawback of conventional SICMs is their relatively small scan range (typically 100 µm × 100 µm in the lateral and 10 µm in the vertical direction). We have developed a Macro-SICM with an exceedingly large scan range of 25 mm × 25 mm in the lateral and 0.25 mm in the vertical direction. We demonstrate the high versatility of the Macro-SICM by imaging at different length scales: from centimeters (fingerprint, coin) to millimeters (bovine tongue tissue, insect wing) to micrometers (cellular extensions). We applied the Macro-SICM to the study of collective cell migration in epithelial wound healing.

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50-60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells.

Biomechanical and biomolecular characterization of extracellular matrix structures in human colon carcinomas.

The extracellular matrix (ECM) is extensively remodeled in tumor tissues. Overproduction of collagens, pathological collagen crosslinking and alignment of fibers are major processes that ultimately result in an increased tissue stiffness. Although it is known that glycosaminoglycans (GAGs) play an important role in tumor signaling, their contribution to the biomechanical properties of tumor ECM is unknown. In this study, ECM structures of human colon carcinoma and normal (control) colon tissues were histologically identified. Using atomic force microscopy (AFM) nanoindentation, we show that the collagen-rich regions within the ECM of colon carcinoma tissues were significantly stiffer than the submucosal collagen-rich layer of control tissues. Screening of these regions with Raman microspectroscopy revealed significantly different molecular fingerprints for collagen fibers in colon carcinoma tissues compared to control tissues. We further showed an increased alignment of collagen fibers and elevated levels of GAG immuno-reactivity within the collagen network of colon carcinoma tissues. GAGs such as heparan sulfate and chondroitin sulfate were detected in significantly elevated levels in collagen fibers of carcinoma tissues. Moreover, immunodetection of the collagen-associated proteoglycan decorin was significantly decreased in carcinomas tissues of individual patients when compared with the corresponding control tissues. Overall a strong patient-to-patient variability was evident in the ECM composition, structure and biomechanics of individual colon carcinoma tissues. Although, biomechanical characteristics of tumor ECM were not directly impacted by GAG content, GAGs might play an important role during the mechanical and structural remodeling of pathological tumor ECM. To manipulate GAG expression and deposition in tumor microenvironments could represent a novel potential therapeutic strategy.

Viscoelastic properties of normal and cancerous human breast cells are affected differently by contact to adjacent cells.

Malignant transformation drastically alters the mechanical properties of the cell and its response to the surrounding cellular environment. We studied the influence of the physical contact between adjacent cells in an epithelial monolayer on the viscoelastic behavior of normal MCF10A, non-invasive cancerous MCF7, and invasive cancerous MDA-MB-231 human breast cells. Using an atomic force microscopy (AFM) imaging technique termed force clamp force mapping (FCFM) to record images of the viscoelastic material properties, we found that normal MCF10A cells are stiffer and have a lower fluidity at confluent than at sparse density. Contrarily, cancerous MCF7 and MDA-MB-231 cells do not stiffen and do not decrease their fluidity when progressing from sparse to confluent density. The behavior of normal MCF10A cells appears to be governed by the formation of stable cell-cell contacts, because their disruption with a calcium-chelator (EGTA) causes the stiffness and fluidity values to return to those at sparse density. In contrast, EGTA-treatment of MCF7 and MDA-MB-231 cells does not change their viscoelastic properties. Confocal fluorescence microscopy showed that the change of the viscoelastic behavior in MCF10A cells when going from sparse to confluent density is accompanied by a remodeling of the actin cytoskeleton into thick stress fiber bundles, while in MCF7 and MDA-MB-231 cells the actin cytoskeleton is only composed of thin and short fibers, regardless of cell density. While the observed behavior of normal MCF10A cells might be crucial for providing mechanical stability and thus in turn integrity of the epithelial monolayer, the dysregulation of this behavior in cancerous MCF7 and MDA-MB-231 cells is possibly a central aspect of cancer progression in the epithelium. STATEMENT OF SIGNIFICANCE: We measured the viscoelastic properties of normal and cancerous human breast epithelial cells in different states of confluency using atomic force microscopy. We found that confluent normal cells are stiffer and have lower fluidity than sparse normal cells, which appears to be governed by the formation of cell-cell contacts. Contrarily, confluent cancer cells do not stiffen and not have a decreased fluidity compared to sparse cancer cells and their viscoelastic properties are independent of cell-cell contact formation. While the observed behavior of normal cells appears to be crucial for providing the mechanical stability and therefore the integrity of the epithelial monolayer, the dysregulation of this behavior in cancer cells might be a central aspect of early stage cancer progression and metastasis in the epithelium.

LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells.

LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage.