BackgroundCell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, C PFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of C PFA is optimized.MethodsHere, we investigated the mechanical properties of cell fixation as a function of C PFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of C PFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells.ResultsWe found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, a m, were almost constant in a lower C PFA (<10−4%). Interestingly, in an intermediate C PFA between 10−1 and 4%, E dramatically increased whereas a m abruptly decreased, indicating that entire cells begin to fix at C PFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher C PFA (>4%), indicating that the cell fixation is stabilized at C PFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols.ConclusionsTaken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols.
This paper reports a microfabrication-free approach to make hollow channel mass sensors by pulling a glass capillary and suspending it on top of a machined jig. A part of the pulled section makes simple contact with an actuation node and a quartz tuning fork (QTF) which acts as a sensing node. The two nodes define a pulled micro capillary tube resonator (PμTR) simply supported at two contacts. While a piezo actuator beneath the actuation node excites the PμTR, the QTF senses the resonance frequency of the PμTR. The proposed concept was validated by electrical and optical measurements of resonant spectra of PμTR. Then, different liquid samples including water, ethanol, glycerol, and their binary mixtures were introduced into the PμTR and the resonance frequency of the PμTR was measured as a function of liquid density. Density responsivity of −3,088 Hz-g−1 cm3 obtained is comparable to those of microfabricated hollow resonators. With a micro droplet generation chip configured in series with the PμTR, size distribution of oil droplets suspended in water was successfully measured with the radius resolution of 31 nm at the average droplet radius, 28.47 μm. Overall, typical off-the-shelf parts simply constitute a resonant mass sensing system along with a convenient electrical readout.
Scanning Ion Conductance Microscopy (SICM) is an emerging nanotechnology tool to investigate the morphology and charge transport properties of nanomaterials, including soft matter. SICM uses an electrolyte filled nanopipette as a scanning probe and detects current changes based on the distance between the nanopipette apex and the target sample in an electrolyte solution. In conventional SICM, the pipette sensor is excited by applying voltage as it raster scans near the surface. There have been attempts to improve upon raster scanning because it can induce collisions between the pipette sidewalls and target sample, especially for soft, dynamic materials (e.g., biological cells). Recently, Novak et al. demonstrated that hopping probe ion conductance microscopy (HPICM) with an adaptive scan method can improve the image quality obtained by SICM for such materials. However, HPICM is inherently slower than conventional raster scanning. In order to optimize both image quality and scanning speed, we report the development of an alternative configuration scheme for SICM signal amplification that is based on applying current to the nanopipette. This scheme overcomes traditional challenges associated with low bandwidth requirements of conventional SICM. Using our alternative scheme, we demonstrate successful imaging of L929 fibroblast cells and discuss the capabilities of this instrument configuration for future applications.
The range of scanning probe microscopy (SPM) applications for atomic force microscopy (AFM) is expanding in the biological sciences field, reflecting an increasing demand for tools that can improve our fundamental understanding of the physics behind biological systems. However, the complexity associated with applying SPM techniques in biomedical research hampers the full exploitation of its capabilities. Recently, the development of scanning ion conductance microscopy (SICM) has overcome these limitations and enabled contact-free, high resolution imaging of live biological specimens. In this work, we demonstrate the limitation of AFM for imaging biological samples in liquid due to artifacts arising from AFM tip–sample interaction, and how SICM imaging is able to overcome those limitations with contact-free scanning. We also demonstrate that SICM measurements, when compared to AFM, show better fit to the actual dimensions of the biological samples. Our results highlight the superiority of SICM imaging, enabling it to be widely adopted as a general and versatile research tool for biological studies in the nanoscale.
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