Many new techniques were brought to the forefront with the muscle mechanics sessions we have had thus far. Having detailed some in my previous article, I detail some more emergent techniques having a great impact in the field.
Single Myofibril Technique - It was developed in Dr. Poggesis lab in Italy and has been a focus of our lab (Dr. De Tombe) as well (check Ryan Matejas poster on Wednesday). My work heavily employs this technique as well. It enables us to measure kinetics of activation/relaxation in the muscle within milliseconds. The forces recorded are of the order of nano newtons and the muscle preparation itself is approx. 20-80 um long to 2-4 um in diameter. The technique uses phase contrast microscopy. The myofibrils (a single skinned string of sarcomeres ) are bound to the coverslip glass by electrostatic interactions. We pick them up between two glass needles. One needle is stiff and doe snot move while the second needle has an L-shaped tip of about 7um and is compliant. As the solutions are switched from relaxing to activating using a double barreled perfusion pipette, the muscle contracts, pulls on the compliant needle thereby making it move and the movement is then detected by video microscopy. The forces and kinetics are then calculated.
Exchange Mass Spectroscopy - Deuterium water exchange is been exploited to study the structure of various molecules (myosin, troponins, tropomysoin) in the contractile apparatus in various conformations (calcium free, calcium bound, phosphorylated, methylated, binary complexes, ternary complexes). The idea behind the technique is simply to replace the hydrogen ions in the amino acid backbone by the deuterium labeled isoform of hydrogen and then capture the mass difference in form of shifted m/z ratio of peptide/protein peaks by mass spectrometry. A qualitative as well as quantitative analysis ensues.
In Vitro Motility assay - This technique enables us to measure the kinetics of sliding velocity of myosins over the thin filament. The major advantage of this technique is that one can play around with a lot of the contractile proteins individually and study their effects very specifically. The myosin heads are bound on the coverslip. As thin filament molecules (actin, actin with Tropomysoin, actin with tropomysoin and troponin) are injected onto the coverslips they start sliding under unloaded conditions. You could change the isotonic strength of the solutions, the ATP/ADP ratios, phosphomimetic protein modifications and study these changes very easily as the velocity of thin filamnet sliding varies. The movement is captured by video microscopy.
Flourescence Resonance Energy Transfer (FRET) - Intermolecular interactions are studied as two molecules in close proximity to each other are labeled with two different flourophores. The preparation is excited at two different wavelengths and the signal is captured. As the molecules interact based on the experimental conditions, conformational changes might take place which can be recorded using FRET by measuring the distance changes between the two flourescent signals under a confocal microscope. Currently FRET is being used in Dr. Seth Robias lab to take it to the next level wherein intramolecular changes can be recorded by measuring the distances between the signals.
STED - Developed in Dr. Lehnharts lab, STED is an emission microscopy based technique wherein a higher resolution of imaging is obtained in contrast to confocal microscopy. For molecules like t-tubules where sizes are less than 300nm, confocal does not capture all the nuances of the structure as the wavelength it uses is within the same range of 300nm. However with STED you get sharper images and better resolution. A doughnut shape around the area of excitation by laser is exploited in STED. A major advantage of STED is that it can capture structural dimensions in the z-axis as well and reveal in-depth details.