GEM4labs
GEM4 Summer School 2006 Labs - August 9-11, 2006
Link to GEM4 Summer School website.
Tissue Biomechanics and Mechanobiology
Summary: Mechanical testing of cartilage under static and dynamic compression and shear. Plugs of bovine tissue will be provided for running through a series of tests in the apparatus.
FLTM Lab
A PDF handout of the lab description, background, and protocol.
Magnetic Trap Microrheology
Summary: Using magnetic tweezers to measure viscoelastic parameters of NIH 3T3 fibroblasts.
Recommended Reading
Multiphoton Microscopy
Optical Trap: DNA
Summary: Optical tweezers are an excellent experimental tool to study the biophysics of single molecules including molecular motors (kinesin, myosin, RNA polymerase), mechanical conformations/transitions of molecules (dsDNA, RNA hairpins, filamentous proteins) and receptor-ligand interactions (antigen-antibody). In the most common assays, the mechanical state of the system is monitored by tracking the position of a handle (usually a dielectric microsphere with diameter of 0.5-2um) tethered to the subject of interest (protein, DNA, etc), with nanometer resolution. The handle also serves as probe to apply force to the system to study the energetics of mechanical changes. Single molecule fluorescence allows the direct observation of the mechanical/conformational changes of the system as it is subjected to perturbations, such as force. The combination of these two techniques allows researches to study the biophysical properties of single molecules. In this lab you will learn the basics of operating a high-end optical tweezers to record mechanical transitions of single molecules. The instrument is also equipped with a novel single molecule fluorescence technique to allow simultaneous, coincident optical trapping and single molecule fluorescence. In our demonstration we will measure the force required to unzip a double-stranded DNA molecule, with a resolution of ~5nm and ~0.1pN, while using single molecule fluorescence to confirm the location of the break. Alignment and calibration procedures will also be presented.
See file below for a short description of the experimental set up and what you will see in our demonstration.
"Experimental set up" by J.Ferrer
Recommended Reading
M.J. Lang et al, "Combined Optical Trapping and Single-Molecule Fluorescence," J. Biol. 2 (2003).
(optional) Optical Trapping Review : K.C. Neuman & S.M. Block, "Optical trapping," Rev. Sci. Instrum. 75 (2003).
Optical Trap: cells
Instrumentation Teaching Lab
Atomic force microscopy imaging of cells
Summary: In this laboratory, you will use the atomic force microscope to image the structure and stiffness of living and chemically fixed human microvascular endothelial cells. The pN- to nN-scale mechanical force used to create these images allows you to observe both the micrometer-scale height of these cells, as well as the nanometer-scale cytoskeletal network beneath the cell surface. Because the cells are living and imaged under near in vitro conditions, it is possible to observe cell processes in real time, including migration, response to drugs added to the imaging media, and of course apoptosis. It is also possible to compare the near-surface structure of living and diseased cells. If time allows, you will also observe the near-field optical / fluorescent image of these cell surfaces.
Recommended Reading
AFM: Molecular force spectroscopy on living cells
Summary: In this laboratory, you will use the atomic force microscope to acquire the mechanical interaction forces between the AFM probe and the surface of living human microvascular endothelial cells. By pushing into the cell surface, the stiffness of various points on the cell can be determined qualitatively. By pulling away from the cell surface, the adhesion force between the probe and specific points on the cell membrane can be measured, including the imaging of single cell surface molecules. Both of these loading approaches are used to infer changes in the cell surface / interior as a function of mechanical or chemical environments, and as a function of disease state.
Recommended Reading
K. J. Van Vliet and P. Hinterdorfer, "Probing drug-cell interactions," Nano Today 1
C. Stroh, et al. "Single-molecule recognition imaging microscopy," PNAS 101
bioMEMS Force Sensor
Summary: We will be showing a bioMEMS force sensor and its application in measuring stretch and compression force response of healthy and malaria-infected human red blood cells. The bioMEMS force sensor is made from pure single crystal silicon, and consist of a probe and flexible beams. The probe is used to contact, indent and stretch the cells, and the flexible beams to measure the cell force response. The probe is about 5 µm wide and 5 µm deep. Each of the flexible beams is about 2 mm long, 1 µm wide and 5 µm deep. We will show how to manipulate the sensor and bring it in contact with the cells, and how the cell force response is measured. Every student will have the chance to try out this manipulation process. Two particular cell force response measurements will also be shown: a poly-L-lysine coated sensor probe will be used to measure (1) the stretch force response of a healthy red blood cell and (2) the compression force response of a malaria-infected red blood cell. For more information about this bioMEMS technique, please refer to the following two papers:
Microfluidics
Summary: This laboratory will demonstrate the use of micfrofabricated structures to investigate the mechanical response of cells as they are deformed through narrow microfluidic channels. Specifically, the biorheological behavior of red blood cells at different stages of malaria infection will be studied. Under a known constant pressure differential, it can be seen that the entrance time and velocity through narrow channels (varying from 2 - 8 microns square cross-section) differ between the early and late stage infected and much stiffer red blood cells. It can also be shown that the sufficiently stiff cells cannot pass through the narrowest channels. Analogies can be made between this behavior and that experienced in the body as the cell passes through capillaries of comparable size.
