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Cell-substrate interaction is one of the crucial phenomenon that determines cell behavior and fate. Here, we present the results on the effect of physical properties (i.e., hydrophobicity/hydrophilicity and roughness) of polyethylene oxide (PEO) and 1,4-polyisoprene (PI) thin films on adhesion of two melanoma cell lines, originating from primary tumor site from vertical growth phase (VGP WM115 cells) and from its metastasis to skin (WM266-4 cells). Our findings show that cell–substrate interaction is strongly dependent on the stage of melanoma progression. For both studied cell types, the dependence of cellular adhesive properties (quantified from single cell force spectroscopy) on surface free energy, particularly, on its polar component, was observed. Adhesive forces of VGP WM115 cells to thin polymer films (TPF) with various hydrophobicity and roughness are larger as compared to those observed in the case of metastatic WM266-4 cells. The proliferation of the latter cells was higher as for VGP WM115 cells, what agrees with the invasive phenotype of these melanoma cells. In summary, the metastatic WM266-4 cells adapt better to different substrates properties as compared to VGP WM115 ones.

Understanding the response of cancer cells to ionising radiation is a crucial step in modern radiotherapy. Raman microspectroscopy, together with Partial Least Squares Regression (PLSR) analysis has been shown to be a powerful tool for monitoring biochemical changes of irradiated cells on the subcellular level. However, to date, the majority of Raman studies have been performed using a single spectrum per cell, giving a limited view of the total biochemical response of the cell. In the current study, Raman mapping of the whole cell area was undertaken to ensure a more comprehensive understanding of the changes induced by X-ray radiation. On the basis of the collected Raman spectral maps, PLSR models were constructed to elucidate the time-dependent evolution of chemical changes induced in cells by irradiation, and the performance of PLSR models based on whole cell averages as compared to those based on average Raman spectra of cytoplasm and nuclear region. On the other hand, prediction of X-ray doses for individual cellular components showed that cytoplasmic and nuclear regions should be analysed separately. Finally, the advantage of the mapping technique over single point measurements was verified by a comparison of the corresponding PLSR models.

Keratinocytes are predominant in the uppermost layer of the skin, while fibroblasts dominate in the dermal layer. These cells interact with each other directly when fibroblasts migrate to a region of the wound where they induce keratinocytes proliferation through double paracrine signalling. Since a response from both keratinocytes and fibroblasts dominates during the inflammatory and proliferative phases, the exact knowledge how these two types of cells interact with each other is crucial for deeper understanding of mechanisms involved in the wound healing process. The aim of this study was to quantify alterations in mechanical properties of cells, i.e. fibroblasts and keratinocytes, in conditions mimicking direct cellular interactions observed in wound healing. Single cell elasticity was measured using atomic force microscope. To verify the influence of keratinocyte neighbors on fibroblasts elasticity (and vice versa), the effect of cellular confluency was studied in parallel. Our results enabled us to distinguish cellular density-related effects from intercellular interactions occurring between fibroblasts and keratinocytes. While the presence of keratinocytes affects fibroblasts spreading capability and mechanical properties, the keratinocytes remain unaffected by the fibroblasts. These results highlight the importance of the cellular deformability in understanding of the role of biomechanics in double paracrine signalling as fibroblast-keratinocyte interaction can change the potential of the wound healing.

Atomic force microscopy - infrared (AFM-IR) spectroscopy allows spectroscopic studies in the mid-infrared (mid-IR) spectral region with a spatial resolution better than is allowed by the diffraction limit. We show that the high spatial resolution can be used to perform spectroscopic and imaging studies at the subcellular level in fixed eukaryotic cells. We collect AFM-IR images of subcellular structures that include lipid droplets, vesicles and cytoskeletal filaments, by relying on the intrinsic contrast from IR light absorption. We also obtain AFM-IR absorption spectra of individual subcellular structures. Most spectra show features that are recognizable in the IR absorption spectra of cells and tissue obtained with FTIR technology, including absorption bands characteristic of phospholipids and polypeptides. The quality of the spectra and of the images opens the way to structure and composition studies at the subcellular level using mid-IR absorption spectroscopy.

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C 60 + cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites. Subject category : Mass spectrometry.

Near-field scanning optical microscopy is a technique providing images of structures with spatial resolution better than $\lambda/2$, which is undetectable in far-field where the Abbe law of limiting resolution is critical. In parallel to the optical imaging, topography maps are also acquired. Near-field scanning optical microscopy measurements can be performed both in air and liquid environments. The later makes the technique very useful for biomaterials analysis offering information that could not be obtained with other methods. Our work presents the results of recent studies on application of near-field scanning optical microscopy to imaging of cells in air as well as in physiological buffers. Differences in cell's topography and morphology have been noticed between two cell lines from human bladder non-malignant (HCV29) and malignant (T24) cancers. Presented results are part of the research that characterizes physiological changes of cells depending on stage of cancer.

The aim of this research was to find out whether the passage number effect may influence on the PC-3 cells (the human prostate cancer line derived from bone metastases) response to proton radia- tion. 2 MeV horizontally focused proton microbeam was used as a radiation source. The cells were treated with a counted number of H + ions (50-8000) corresponding to doses of 1.3-209 Gy/cell. For comparison, cell death was also induced by UVC radiation. All cells were stained with Hoechst 33342 and propidium iodide and visualized under a fluorescence microscope. Necrosis was observed at: a) 8000 protonsper cell (corresponding to ~209 Gy/cell) after 2-4 passages, b) 3200 protons per cell (corresponding to ~84 Gy/cell) for cells after 11-14 passages and c) only 800 protons per cell (corresponding to ~2 Gy/cell ) after 47-50 passages. Apoptosis was efficiently induced, by protons, only in cells after 50 passages. The results showed that the laboratory conditions affected cellular response of PC-3 cell line to the proton irradiation. The cellular response to the radiation treatment strongly depends on number of passages.

The major characteristics of cancer metastasis is the ability of the primary tumor cells to migrate by way of the blood or lymph vessels and to form tumors at multiple, distant sites. There are evidences that cancer progression is characterized by disruption and/or reorganization of cytoskeleton (i.e. cellular scaffold). This is accompanied by various molecular alterations influencing the overall mechanical resistance of cells. Current approach in diagnosis focuses mainly on microbiological, immunological, and pathological aspects rather than on the biomechanics of diseases. The determination of mechanical properties of an individual living cell has became possible with the development of local measurement techniques, such as atomic force microscopy, magnetic or optical tweezers. The advantage of them lies in the capability to measure living cells at a single cell level and in liquid conditions, close to natural environment. Here, we present the studies on mechanical properties of single cells originating from various cancers. The results show that, independently of the cancer type (bladder, melanoma, prostate, breast and colon), single cells are characterized by the lower Young's modulus, denoting higher deformability of cancerous cells. However, the obtained Young's modulus values were dependent on various factors, like the properties of substrates used for cell growth, force loading rate, or indentation depth. Their influence on elastic properties of cells was considered. Based on these findings, the identification of cancerous cells based on their elastic properties was performed. These results proved the AFM capability in recognition of a single, mechanically altered cell, also in cases when morphological changes are not visible. The quantitative analysis of cell deformability carried out using normal (reference) and cancerous cells and, more precisely, their characterization (qualitative and quantitative) can have a significant impact on the development of methodological approaches toward precise identification of pathological cells and would allow for more effective detection of cancer-related changes.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young’s modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.

Most methods developed to study protein binding to distinct surfaces can only determine the average amount of adsorbed protein or merely provide (qualitative) information on its spatial distribution. Both these features can be characterized rigorously by integral geometry analysis of fluorescence micrographs. This approach is introduced here to compare the relative protein adsorption onto various polymer surfaces: polystyrene (PS), poly(methyl methacrylate) (PMMA), poly( n-butyl methacrylate) (PnBMA), poly( tert-butyl methacrylate) (PtBMA), and PS(PETA) and cross-linked poly(ethylene oxide) (PEO*(PETA)), admixed with pentaerythritol triacrylate (PETA). The polymeric surfaces were incubated for 15 min in phosphate-buffered saline (pH 7.4) containing 125 mug/mL fluorescently labeled lectins, either lentil lectin (LcH) or concanavalin A (ConA). Fluorescence images were recorded at identical conditions (physiological buffer, same exposure time, magnification, gain). For each image, taken a few times for each polymer, the distribution and average value of the normalized intensity were determined. The results show that the binding of LcH to PS(PETA), PtBMA, PS, PnBMA, PMMA, and PEO*(PETA) can be expressed by the ratio of the following values (mean +/- 95% confidence interval): 0.356 +/- 0.022, 0.298 +/- 0.030, 0.241 +/- 0.014, 0.083 +/- 0.008, 0.039 +/- 0.008, and 0.010 +/- 0.006, respectively. In turn, the relative adsorption of ConA is described by the values 0.252 +/- 0.016, 0.217 +/- 0.014, 0.222 +/- 0.016, 0.046 +/- 0.006, 0.116 +/- 0.008, and 0.006 +/- 0.002, respectively. Low dispersions of fluorescence intensity around average values indicate homogeneous distribution of adsorbed proteins. The introduced approach enables a fast and easy way not only to quantify the relative amount of bound proteins but also to characterize quantitatively the organization of their surface distribution, as demonstrated for patchlike protein adsorption onto the polymer blend surface.

The atomic force microscopy (AFM) is a technique that is widely used to study the mechanical properties of biological samples in conditions close to natural. In particular, the technique can deliver the information about both the cell stiffness and the unbinding force of individual molecular complexes. Cell stiffness seems to be a global parameter describing the overall changes occurring in the cell structure, particularly in the cellular scaffold called cytoskeleton. The determination of the unbinding forces, acting between a single pair of molecules, brings more specific information about the interaction forces. Both types of AFM measurements can be successfully applied for the detection and quantification of the alterations of mechanical properties in cancerous cells with the special emphasis on the development of a new technique for the cancer detection on a single cell level.

Data included in this article are associated with the research article entitled ‘Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry’ (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin. Keywords: ToF SIMS, Single cells preparation, AFM imaging

Nowadays, it is well established that changes of cell stiffness observed by atomic force microscopy are linked with the cell cytoskeleton. Its structural and functional alterations are underlying major diseases such as cancer, inflammation or neurodegenerative disorders. So far, the use of atomic force microscopy is mostly focused on the determination of the Young modulus using the modified Hertz model. It can quantitatively describe the elastic properties of living cells, however, its value is burdened by the fact that cells are neither isotropic nor homogeneous material. Often, during the atomic force microscopy measurements, the hysteresis between the loading and unloading curves are observed which indicates the dissipation of an energy. In our studies, the index of plasticity was introduced to enumerate such effect during a single loading–unloading cycle. As the results show, such approach delivers an additional parameter describing the mechanical state of cell cytoskeleton. The analysis was performed on test samples where the mechanical properties of the melanoma cells were changed by glutaraldehyde and cytochalasin D treatments. The non-treated cells were compared with fibroblasts.