subcellular imaging of liquid silicone coated-intestinal epithelial cells
Surface contamination at the nano-contact between the tip of the atomic force microscope and the surface of the cell and the formation of the water bridge limit the maximum spatial resolution that the cell can achieve under environmental conditions. The structural information of the fixed intestinal epithelial cell membrane is enhanced by the manufacture of the silicone liquid membrane, which prevents the accumulation of environmental pollutants and water at the interface between the cell membrane and the tip of the atomic force microscope. A clean and stable experimental platform allows for visualization of the structure and direction of microfluff on the tip cell membrane under standard laboratory conditions and for registration of topographic features in microfluff. The methods developed here can be used to preserve and imaging contaminants The free morphology of fixed cells is the center of basic research in cell biology and the emerging field of digital pathology. Surface contamination and formation of nano-water bridges can reduce the amount of spatial information obtained through atomic force microscopy (AFM) Measurement under environmental conditions. Specifically, the water bridge tends to be formed in a hydrophilic nano-contact, such as the interface between the AFM tip and the solid surface. Carry High Resolution AFM imaging under environmental conditions (in air) , Limiting surface contamination and controlling the parameters formed by a water bridge driven by tip capillary condensation is critical Surface interface. Thermodynamics of water bridge formation and rupture is well known, including humidity, probe radius, distance between probe and sample, influence of pulling force The force and friction on the structure of the Water Bridge. Supported by a numerical model based on the Kelvin equation, density functional theory and Monte Carlo simulations suggest that water bridges fluctuate with changes in the local physical and chemical environment, thus limiting the maximum spatial resolution that an AFM tool that operates in a water medium can achieve However, maintain and study sub- Structure and Dynamics of proteins and cells in natural state. Previously, AFM studies have shown that it is possible to track protein movement and capture cell surface receptor interactions in the room Compared with the low temperature AFM measurements performed in the ultra-cold and thermal stability experimental platform, the temperature in the water medium is an environment with high experimental requirements. Although it is necessary to study Biomolecular Dynamics under physiological conditions, it has been shown that the complex structure of fixed cells (in a dry state) An electron microscope operated in a vacuum can be distinguished better than using AFM in water. In electron microscopy studies of fixed cells, samples were not contaminated by the environment, but at the expense of losing soluble cell content. Therefore, an alternative approach is needed to utilize the latest advances in instruments and meters, retain cell morphology and simultaneously detect subcell topology features, without the need for multi-step sample preparation procedures like AFM-based cell imaging frozen slices. This simple and reliable method will have a direct impact on the clinical level screening of pathological diseases characterized by cell structure failure. . Red blood cell diseases such as sickle-cell anaemia. As proof of the principle of high resolution imaging of cell structure under environmental conditions, we present a solution to fix the details of microfluff structure in the tip domain of intestinal epithelial cells. When cultured to melt on a petri dish or a permeable membrane insert, the epithelial cells become polarized. The most characteristic structure of intestinal epithelial cells is the closely arranged tip microfluff, also known as the brush edge. Microfluff is an important structure to increase the surface of intestinal cells and is the main place for nutrient absorption, participating in mechanical conduction. Here we encapsulate the cell surface in a silicone liquid film prepared by controlled spray deposition and use an AFM probe ( Not completely immersed in liquid silica gel) Take silicone liquid- Surface of cells coated. Silicone liquids minimize probe drift by protecting cell surface topology from environmental contaminants, thus providing a suitable platform for scanning probe microscope measurement, thus achieving in standard laboratory conditions Fusion Caco- 2 single layer of intestinal epithelial cells with well-differentiated Brush edge ( See supplementary map and) Is a hydrophilic surface (Fig. ) Contact angle with water (Θ)of (23u2009±u20093) An average of more than ten locations on the cell layer. The morphology of cells can be degraded over time by interaction with surrounding hydrocarbon contaminants. To solve this problem, we spray Cover the cell surface with liquid silica gel and form a thin liquid membrane on the top of the epithelial cell layer. Three key parameters have been optimized for the manufacture of silicone liquid film by spray The deposition shown in the schematic diagram (Fig. ) The working distance between the nozzle and the sample, the pressure of the compressed gas and the volume of the sprayed silicone liquid. A mean Θ of (63u2009±u20095) Epithelial cells coated with liquid silica gel were measured (Fig. ) Use the same protocol as the uncoated epithelial cell sample. A substantial increase in the water contact angle from 23 ° to 63 ° indicates that the silicone liquid coating significantly increases the hydrophobic properties of the sample. Molecular dynamics (MD) The simulation confirms the increase in the large measurement of the contact angle, showing the calculated RSI (80u2009±u20095) On a silicone film (Fig. ) Compared with the calculation of CTR (17u2009±u20093) On an uncoated cell membrane ( Supplementary Map). The ability to detect structural deviations in microfluff geometry is essential to determine their function during nutrient absorption and release and to coordinate immune responses. To this end, we used AFM probes in intermittent contact mode to analyze the microfluff structure on epithelial cells. First of all, we are concerned about the imaging stability of AFM probes, where only the tip- Apex is immersed in silica gel liquid (Fig. ). The figure shows the drift rate of AFM probe in silicone liquid (blue curve)and water (red curve) Scan for more than 2 hours Calibrate the 5 µm area on the grid sample (Fig. inset) The temperature in the laboratory is (23u2009±u20091) C in intermittent contact mode. The near- Constant probe drift rates measured in silica gel liquids with a scan rate of 2 hz ≤ 1 nm per minute highlight imaging stability, with increasing probe drift measured in water using the same scan parameters The low vapor pressure, high viscosity and low surface tension of silicone liquids bypass the need to deliver additional liquids through flow Cells, thereby improving imaging stability, which is necessary to extract complex structure information from cells. The picture is big- Region AFM images recorded in phase Contrast patterns on the surface of epithelial cells during intermittent contact pattern measurement. Capture physical chemical surface properties, such as bullet dissipation, adhesion, and friction, through a phase image that detects the phase lag between the cantilever oscillation relative to the cantilever response. The phase signal can record minor changes in the surface topology that are difficult to resolve directly from the height signal ( Supplementary Map). The phase-Contrast images (Fig. ) Display vertical (marked as I), parallel ( Marked as II, as shown in the figure. )and random ( Supplementary Map) Orientation of microfluff relative to epithelial cell membrane. These different packaging patterns are separated by sharp regional boundaries ( It is depicted as a white line in the picture. ). We calculate an average root. mean-square (RMS) Surface roughness value (25u2009±u20095) Vertical nm (region I)and (60u2009±u20098) Nm for parallel connection (region II) Structure, reflecting the differences in packaging order and orientation within the microfluff structure. Reposition the AFM probe ( Within Region 2) Reduced scan area shows an array of fingers Microfluff ( AFM height image, fig. ). Resolution of data shown in Figure 1 Comparable to previous AFM studies, these studies were microfluff structures on the top epithelial cell membrane of the unfixed cells exposed to imaging and peakforce tapping patterns in water Media. More detailed spatial information of a single microfluff filament (Fig. ) Can be obtained by optimizing the Scan parameters ( See the method section). For reference, figureshows close- Scanning electron microscopy analysis of the stacking structure of microfluff (Fig. ). AFM height image (Fig. ) And the corresponding crossover Profile of section (Fig. Display representative line profiles) Reveal Close-packed finger- Similar to microfluff structures with significant changes in local height and diameter. By averaging through multiple cross-section analyses, we calculate the mean microfluff diameter of 102nm with a lower confidence interval (CIL) Upper limit of 98 nm and confidence interval (CIU)of 104u2009nm (Fig. ). We calculate confidence intervals (CI) Statistical distribution as shown in figure 95%is non-gaussian. We report the mean microfluff diameter with a groundbreaking highLow temperature resolution It is known that the diameter of a single actin filament varies from 6-10 nm along the microfluff length by electron microscopy. The phase- The contrast image reveals more details about the configuration of a single microfluff unit (Fig. ). In particular, we observe lateral stripes along the entire length of the microfluff unit ( Used to only use high- Resolution electron microscope at low temperature) , Visible after amplification-In the phase Image ( Mark with a red arrow in the figure. ) In the overlay image (Fig. ). As far as we know, this level of detail about cell structure under environmental conditions was not previously available with atomic energy microscopy. The AFM phase data show that it is based on a non-uniform stripe spacing of 46 nm with: 42 nm and CIU: 50 nm Gaussian statistical distribution shown in Figure 1( Supplementary Map For fringe periodicity measured along microfluff). Previous high- Resolution electron microscopy studies of a single microfluff structure report a similar transverse stripe with a periodicity of 30-40 nm, comparable to the one measured here. These structural features along the microfluff length are attributed to cross Depending on the chemical environment in which cells are fixed, there may be different strands of the bridge periodically. High spatial resolution of fine structure of microfluff obtained in liquid silica gel with atomic force microscope at room temperature Temperature presents a method for maintaining fixed cell features and visualizing cellular substructures. This method allows for direct imaging of nano-features on the cell surface and along the length of a single microfluff. This approach is compatible with commercially available independent AFM tools and tools used in conjunction with optical microscope technology, which makes it suitable for field deployment screening of diseases associated with cell failure. We expect that this microscope-based approach will have an impact in emerging areas of digital pathology, and it is important to register information on diseased cells in a fixed state without environmental contaminants and high enough Spatial resolution of data analysis based on algorithm.