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(current research)
Tissue
Engineering FCS,
FPR, FIDA Publications
We study mechanical characteristics of individual cells by means of the cell poker, which has gone through several cycles of re-invention since we made the first one at Cornell University in the 70's. It is similar to an atomic force microscope, in that both use a flexible probe to measure the force needed to produce some amount of deformation in the sample. The cell poker has a wider probe and produces much greater deformations than the AFM, and is therefore sensitive to deeper structures rather than mainly feeling the membrane and underlying actin networks. This cartoon illustrates the principles of operation. The force-sensing part is the horizontal glass beam, about 2 cm. long, which connectes the piezoelectric motor to the stylus. The stylus is made from stretched glass about 25 microns in diameter, and is sharpened to about 2 microns by dipping in hydrofluoric acid, making a tip with a gentle taper and flat end. That is brought into contact with cells adhering to a coverslip on the bottom of the sample chamber. The upper end of the stylus carries a metallized mylar reflector. The top edge of the reflector passes through the focal point of an integrated LED and photodiode sensor, giving a vertical position measurement with about 0.01 micron sensitivity and bandwidth about 3 KHz. The resonant frequency of the probe structure is about 30 Hz. The lower part of the figure represents the probe lowered to be just barely in contact with the cell, and then lowered by the additional amount y. The cell is compressed by the stylus, and the reaction force bends the glass beam, so that the stylus moves down by the amount x. That is the deformation of the cell, and the static force is G*(y - x) where G is the independently calibrated bending force constant of the beam. Actually, the experix experiment control system solves the linear harmonic oscillator differential equation to get a better approximation to the force. |
The next picture shows the experix cell poker analysis screen for a fairly typical experiment, in which the probe is moved down and then up at constant rate over a range of 16 microns in a measurement taking 1 second total. On the left, the motor motion is red, the stylus is blue, the work rate is green and the force is black. The right side shows force vs. stylus height. The green "arrow" marks indicate the time range that is selected for data fitting to determine the point of contact with the cell, and the "target" mark is the contact point determined by a 5-parameter fit to that data. This is from a study that compared cells with different types of myosin suppressed.
This is a partial list of research projects involving the cell poker, with
links to our publication list for those that have been published.
...... Roles of different myosin types, using knockdown mutants
pub Coating Drosophila cells
with the spam gene product increases their stiffness up to 10-fold.
pub A myosin-dependent
viscoelastic force resists deformation in spreading cells.
pub Bending stiffness and
axial compression stiffness of cochlear outer hair cells may originate from
the same cytoskeletal structures.
pub Increased myosin
light chain phosphorylation correlates with increased cytoskeletal
stiffness.
pub Lack of distrophin in
muscle cells results in substantially lower cortical stiffness.
pubCyclic AMP modulates
properties and microfilament assembly in neutrophils.
pub
Mechanical response of leukocytes to indentation is not consistent with simple
models developed on the basis of micropipette-aspiration experiments.
pub
Lymphocytes stiffen in response to crosslinking cell surface antigens.
pub Viscosity values from
three polydimethylsiloxane samples are in agreement with results of
measurements on macroscopic samples.
pub
Conventional myosin is essential both for capping and for the concomitant
increase in cell stiffness in Dictyostelium.
pub Cross-linking
cell-surface IgE- receptor complexes on RBL cells with multivalent ligands not
only triggered secretion but also caused the cells to stiffen.
pubEffect of Salivary
Amylase on the Viscosity Behavior of Gelatinised Starch Suspension and the
Mechanical Properties of Gelatinised Starch Granules.
pubResistance of
lymphocytes to deformation strongly increased when sIg or Con A acceptors on
the cell surface were cross-linked.
pub Cross-linking surface
immunoglobulin on B-lymphocytes causes a substantial increase in the
mechanical stiffness.
pubElastic area
compressibility modulus of osmotically swollen human erythrocytes agrees with
results from micropipette aspiration.
pub
Modulation of lymphocyte deformability by concanavalin A
pub Measured cellular
deformability depends on: location on the cell; temperature; cytochalasin
B.
pub
Correlation of myosin light chain phosphorylation with isometric contraction
of fibroblasts
pub
Studies of mechanical aspects of amoeboid locomotion
pub
Capping of surface receptors and concomitant cortical tension are
generated by conventional myosin.