The Effects of Cell Sizes, Environmental Conditions, and Growth Phases on the Strength of Individual W303 Yeast Cells Inside ESEM

Abstract

We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM)-nanomanipulator system. Compression experiments to penetrate the cell walls of single cells of different cell sizes (about 3-6 micro m diameter), environmental conditions (600 Pa and 3 mPa), and growth phases (early log, mid log, late log and saturation) were conducted. The compression experiments were performed inside ESEM, embedded with a 7 DOF nanomanipulator with a sharp pyramidal end effector and a cooling stage, i.e., a temperature controller. ESEM itself can control the chamber pressure. Data clearly show an increment in penetration force, i.e., 96 +/- 2, 124 +/- 10, 163 +/- 1, and 234 +/- 14 nN at 3, 4, 5, and 6 micro m cell diameters, respectively. Whereas, 20-fold increase in penetration forces was recorded at different environmental conditions for 5 micro m cell diameter, i.e., 163 +/- 1 nN and 2.95 +/- 0.23 mu N at 600 Pa (ESEM mode) and 3 mPa (HV mode), respectively. This was further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e., ESEM mode (3.31 +/- 0.11 MPa) and HV mode (26.02 +/- 3.66 MPa) for 5 micro m cell diameter. Finally, the penetration forces at different cell growth phases also show the increment pattern from log (early, mid, and late) to saturation phases, i.e., 161 +/- 25, 216 +/- 15, 255 +/- 21, and 408 +/- 41 nN, respectively.

Recent advances in nanorobotic manipulation inside scanning electron microscopes

“…By using piezoresistive force sensors, mechanical indentation for stiffness determination of 2D materials and scanning for surface topography were achieved 45 . When AFM probes are used for force sensing, an applied force can be determined from Hooke's law 85,86 . The stiffness of an AFM probe can be calibrated using a reference cantilever of a known stiffness 87 .…”

“…The AFM cantilever structure can be modified by FIB etching and deposition to create various types of application-specific tools. For instance, AFM probes were modified to have a flat tip 86,89 , stiff needle tip 87,[90][91][92][93] , knife blade tip 94 and fork tips 95,96 for cellular and intracellular characterization and cell manipulation. Tungsten probes are fabricated and sharpened by electrochemical etching to obtain a high-aspect ratio.…”

“…Therefore, water-containing samples cannot be directly observed inside an SEM or TEM 152,153 . Environmental scanning electron microscopes (ESEMs) overcome this limitation and permit the observation of liquid-phase materials, such as biological cells without metal coating and other electrically insulating materials that use a special secondary electron detector 86,154 . The first hybrid AFM/ESEM system was developed for accurate topography measurements and tip-sample interaction observations 52,155 .…”

“…Standard AFM cantilevers were modified using FIB etching and deposition to produce different types of functional tools, such as a soft buckling nanoneedle 86,90,91 , a nano-fork and a nano putter 95,168 , as shown in Figures 6a, b and c, respectively. These tools were mounted on a nanomanipulation system in ESEM to perform indentation to determine cell stiffness and viscoelastic properties 87,90 .…”

Acoustic cardiac signals analysis: a Kalman filter–based approach

“…Peptide-coated nanoparticles can bind to a cancer tumor; once the nanoparticles are attached to the tumor, the magnetic property of the iron oxide enhances the images from a magnetic resonance imaging scan. In related work, 3,4 characterization of cellular mechanics of cast cells was carried out using nanoneedles inside an environmental scanning electron microscope. The nanoneedles are used to measure local stiffness characteristics of single cells.…”