Fig. 1. Setup for acoustic manipulation and…
Fig. 1. Setup for acoustic manipulation and micro-indentation.
a , b Side and front view…
Fig. 1. Setup for acoustic manipulation and micro-indentation.a, b Side and front view schematics describing the 3D characterization of a pollen grain. Acoustic streaming, generated by exciting trapped microbubbles, allows the trapping of pollen grains and their re-orientation with respect to the force sensor. This enables the 3D mechanical characterization of a sample. H denotes the height of the microbubble, θ the rotational angle of the specimen, and x, y, z define the coordinate system where the force F applied by the sensor in z-direction leads to a displacement Δz. c A schematic showing the manipulation and indentation of a single C. elegans nematode. By exciting multiple parallel microbubbles, the same design used for pollen grains can be applied to manipulate a nematode. d A photograph of the setup with the manipulation device, consisting of a PDMS structure and a piezoelectric transducer, as well as the force sensor used for mechanical characterization of the specimens. e A slowly rotating auto-fluorescent pollen grain. Stable rotation of the pollen grain is controlled via acoustic excitation. A denotes a specific location on the pollen grain’s surface which is tracked during the rotation of the specimen. Scale bar: 50 µm.
Fig. 2. 3D indentation of lily pollen…
Fig. 2. 3D indentation of lily pollen grains.
a A scanning electron microscopy (SEM) image…
Fig. 2. 3D indentation of lily pollen grains.a A scanning electron microscopy (SEM) image of dehydrated lily pollen grains; note that the intine is not accessible. b An SEM image of a fully hydrated lily pollen grain, exposing the intine at the colpus. c A schematic of a lily pollen grain highlighting the different surface areas, i.e., the soft colpus and the stiff exine with the lumina. d A graphic showing the structure of a pollen grain cell wall in cross-section. At the colpus, the exine is absent and the intine is exposed to the surroundings. e Two violin plots containing all measurements (m = 300) for n = 30 biologically independent lily pollen grains in deionized water. The data is divided into intine (brown) and exine (green) stiffness values. The box represents the interquartile range, the center line represents the median, and the whiskers represent the 5th and 95th percentiles. The maxima and minima are denoted by the start and end of the violin plots. The inset shows the independent measurements (m = 10) for a single pollen grain, where intine and exine are separated. The individual measurements are represented by dots. f A graph showing two indentation curves from different regions of the same pollen grain. The stiffness values ki and ke are obtained from the slopes. g Two boxplots to compare the stiffness ratios ki/ke of lily pollen grains in deionized water (violet) to the ratios of lily pollen grains in CaCl2 solution (red). The boxes represent the interquartile ranges, the center lines represent medians, and the whiskers denote the ranges of minima and maxima. The stiffness ratio of the pollen grains in a CaCl2 solution significantly increased, indicating specific stiffening of the intine due to Ca2+-mediated crosslinking of pectin. Each stiffness ratio was derived from m = 10 independent measurements on a single specimen. Each stiffness ratio distribution was calculated from n = 30 biologically independent samples. h The apparent stiffness distribution for the exine structure (green) of non-hydrated pollen grains. The data was collected through (m = 50) single indentations of 50 biologically independent samples. The box represents the interquartile range, the center line represents the median, and the whiskers denote the range of minima and maxima. i The pressurized (turgor pressure P = 0.2 MPa) simulation of an average lily pollen grain. The two cell wall layers, i.e., the intine and exine, are clearly visible with the intine being exposed at the colpus. The color bar shows Cauchy stress in the cell wall in MPa. The side view shows the slight variation in pollen grain curvature between different regions of the exine as well as at the colpus and has been taken into account for the simulated indentations. j Simulated indentations of the exine and intine resulting in apparent stiffness values ke and ki of 12.7 N/m and 7.9 N/m, respectively, for a material stiffness ratio Ee/Ei of 10. The turgor pressure is P = 0.2 MPa and the Poisson’s ratio is 0.3. The dashed line denotes the maximum force applied during experimental characterization. Scale bars: a = 25 µm, b = 10 µm, i = 25 µm. Source Data is available as a source data file for e–h, j.
Fig. 3. 3D indentation of C. elegans…
Fig. 3. 3D indentation of C. elegans .
a A fluorescence image of a C. elegans…
Fig. 3. 3D indentation of C. elegans.a A fluorescence image of a C. elegans nematode trapped in the vicinity of microbubbles using acoustic radiation forces. The region of interest (ROI), where the mechanical characterization was performed, is highlighted in blue. b An image sequence showing different orientations of a C. elegans nematode. The specimen is rotated using acoustic streaming. c A stationary C. elegans worm near the microbubbles. Red dots indicate the individual positions of the micro-indenter probe during mechanical characterization. d Schematic illustrating the composition of internal organs which might lead to different stiffness values measured by micro-indentation and which can be used to extract the right orientation via fluorescence imaging. The angle θ denotes the location of the indentation in a cylindrical coordinate system. e 3D projection of the stiffness values onto the geometry of a C. elegans nematode showing the different positions around the characterized specimen. f The unfolded stiffness map with individual indentation positions. Multiple bands with different stiffness ranges can be observed. All presented measurements were performed on a single nematode to avoid noise caused by biological variation between specimens. For multicellular organisms, the stiffness does not necessarily quantify a single biological material, but may include the mechanical properties of several overlapping layers and, hence, should be treated as apparent stiffness. Green bands indicate the possible influence of muscular tissue. Scale bars: a = 100 µm, b = 50 µm, c = 50 µm. Source Data is available as a source data file for f.
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