Posted by Geoff Chandler on Tue, Sep 06, 2011 @ 02:59 PM
Now that we have described three methods for attaching muscle to an Aurora Scientific Inc. force transducer and length controller we will now explain how to build the attachment for the tying method.
Motor Attachments
For the motor (315C or 322C) start with a piece of 29 gauge stainless steel tubing that's about 10mm long. "Dress" the ends by inserting the tubing into a "pin vise" (see part numbers and links below) and using a very fine sharpening stone to smooth the ends of the tubing. Aim for a very smooth end with a fairly abrupt taper (a long taper encourages slipping of the prep). An easy method of achieving this smooth end is to hold the pin vise at an angle of about 60 degrees to the sharpening stone and then lightly drag the end of the tube along the stone while spinning the pin vise in your hand. Examine the end of the tubing under a strong magnifying glass or dissection microscope. Make sure there are no "burrs" or other sharp edges that could cut the prep. It is best to smooth both ends of the tube as this gives you maximum flexibility as to which end to use later when you attach the tube to the lever arm.
Photo 1 Dressed 29 gauge Hypodermic Tubing
Next put a 90 degree bend in the tubing by chucking it in the pin vise 1/2 way down its length and then, starting with the pin vise at a very shallow angle with respect to a flat surface, slowly raise the pin vise while keeping the half of the tubing that extends from the vise in full-length-contact with the flat surface. Continue to raise the vise until it is normal to the flat surface (at which time the tubing ought to have a 90 degree bend). Too much bend and the tubing will "crimp" and you will have to start over again. Once you have an L-shaped piece of tubing that you are happy with (the legs of the "L" will be on the order of 4.5mm each), you will epoxy it in such a way that one leg is in line with the long axis of the motor arm and the other leg comes off the end of the motor arm at a 90 degree angle. Use 2 hour epoxy (5 minute epoxy isn't typically waterproof). We suggest that you first "tack" the "L" to the arm using a tiny amount of superglue - this is only to hold the "L" in place while the epoxy is curing. The 315C motor is very sensitive to added mass. You may need to make the “L” about 3mm x 3mm when used with a 315C. Also use the minimum amount of epoxy that will get the job done. Even the added mass of the epoxy can have a detrimental effect on motor tuning.
Force Transducer Attachments
For the 400A series force transducer, the length of tubing will depend in part on bath configuration. As a rule of thumb use the shortest tube that will do the job (since added mass reduces the frequency response). A reasonable length for the tube is approximately 13mm, 5mm of which will be inside the glass capillary output tube of the force transducer with the remaining 8mm outside. Dress the ends of the tubing as described above and then epoxy one end inside the output tube. Don't try to get the stainless tube concentric with the glass output tube, this can be very difficult. Instead let its outside surface rest against the inner surface of the output tube, this ensures that the stainless steel tubing and the glass output tube are lined up. Getting epoxy in the coupling tube is a bit of a trick. The best way to do this is to dip the end of the tubing in the epoxy pool and then insert it. We suggest you rotate the tube as it is inserted to help distribute the glue over the surface. It takes very little glue to make this connection. You will want to keep an eye on the tube as the epoxy cures, making constant minor adjustments to maintain a straight stainless steel extension. We find it quite helpful to mount the force transducer on the apparatus (ASI 801C or 802D) before gluing the stainless tube in place. This way you can use the slot cut into the end of the bath to hold the free end of the tube while the glue dries. The micro-manipulator XYZ stages included on the ASI apparatus allow you to move the transducer with respect to the slot in the bath plate to ensure the tube is directly inline with the transducer's output tube.
Hypodermic Needle Tubing: McMaster-Carr Catalogue #8988K429, or, in a pinch, use a 29 gauge hypodermic needle and cut the needle and Luer fitting off.
Pin Vise: McMaster-Carr Catalogue #8455A16
Sharpening stones: McMaster-Carr Catalogue #4456A17
Posted by Geoff Chandler on Tue, May 10, 2011 @ 03:42 PM
Tip 3: Glue can be used to Attach a Skinned Myocyte to an ASI Force Transducer and Length Controller
Depending on the size of the tissue preparation it is possible to attach muscle tissue to a length controller and force transducer using glue. Typically the glue method is used with very small preps including skinned myocytes.
In this method a small amount of glue is placed on the tip of micropipettes attached to a force transducer and to a length controller. The tissue is then brought into contact with the glue and the myocyte is attached.
Two main types of glue are commonly used. The first is an expanding foam called GREAT STUFF manufactured by The Dow Chemical Company. It is readily available in the USA in stores such as Home Depot. The other is a silicone based glue manufactured by Dow Corning Corp.
Figure 1 Myocyte Glued to Micropipettes
The glue method of attaching a skinned myocyte to a force transducer and high speed length controller is explained well in the following paper by Sweitzer and Moss.
The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation. N K Sweitzer and R L Moss J Gen Physiol 1990 96:1221-1245. Published December 1, 1990, doi:10.1085/jgp.96.6.1221
The method consists of forming micropipettes with a tip diameter of 4 microns and attaching these micropipettes to the force transducer and lever arm using paraffin wax, shellac or dental impression compound. A drop of glue is placed on a microscope slide or cover slip along with a drop of cell suspension. An appropriate myocyte is chosen by viewing the suspension droplet with a microscope. The two micropipette tips are then dipped into the glue and lowered onto each end of the myocyte. The glue is left to cure for about 45 minutes and then the pipettes are translated along with the suspended myocyte to the test chamber. Aurora Scientific's 803B Skinned Myocyte Test Apparatus was designed for exactly this experiment and it provides a large cell mounting and gluing area along with 8 test wells for different calcium concentrations. Force-pCa data can be obtained with this system.
P. de Tombe, Loyola University, Chicago and G. Stienen, VU University Medical Centre, Amsterdam are two researchers using glue to attach skinned myocytes to an Aurora Scientific high speed length controller and force transducer.
Posted by Geoff Chandler on Wed, Apr 20, 2011 @ 02:05 PM
Tip 2: T-Clips can be used to Attach a Muscle Fiber to an ASI Force Transducer and Length Controller
T-clips are small clips formed in a "T" shape and typically cut from aluminum foil. The bottom of the T has a small hole in it.
Figure 1 Drawing Showing T-Clip Dimensions
The top of the T is wrapped around the fiber and squashed down onto the fiber. This leaves the vertical part of the T sticking out the end of the fiber. A small hook is created and attached to the lever arm of the high speed length controller and a separate hook is attached to the force transducer. In operation the hole in the T-clip is slipped over the hook to attach the fiber.
Figure 2 T-Clip Attachment to Fiber
T-clips work well for low force applications such as single permeabilized fibers. If you intend to test higher force fibers, for example frog fibers or bundles or strips, you may find that the tissue slips inside the T-clip and a more positive method of attachment such as tying should be looked at.
Refer to Y.E. Goldman, R.M. Simmons, Control of Sarcomere Length in Skinned Muscle Fibres of Rana Temporaria During Mechanical Transients, J. Physiology (1984), 350, pp. 497-518 for a good description of T-Clip fabrication and use. Research groups routinely using T-Clips include Pieter de Tombe's lab at Loyola in Chicago and Frank Brozovich at the Mayo.
Posted by Geoff Chandler on Sun, Apr 03, 2011 @ 12:46 PM
Tip 1: Tying a Muscle Fiber/Small Muscle to an ASI Force Transducer and Length Controller
Tying requires adding a short length of fine gauge hypodermic needle tubing to the force transducer and length controller. The fiber is then tied to these two needles using fine suture (10-0 monofilament nylon suture). See the attached photo of a small intact muscle tied to a force transducer and length controller. Neither the force transducer output tube nor the lever arm are visible in this photo but the researcher was using an Aurora Scientific model 400A force transducer and a model 318B length controller. In the photo the wire at the bottom is a platinum stimulation electrode and the needle at the top is a thermocouple.
The expected force generated by the fiber will determine the number of suture loops to use. This photo shows four on each end which was required to prevent slippage of this mouse lumbrical muscle. Note: whenever you tye an intact muscle you must ensure that no sutures are tied to the muscle tissue itself, only attach the muscle using the tendons.
Photo 1 Lumbrical Muscle Tied to 29 AWG Tubes
When tying permeabilized fibers, or even fiber bundles, it is more likely that you will only require 2 or 3 sutures per end.
Photo 2 Permeabilized Fiber Tied to 29 AWG Tubes
(photos courtesy of Muscle Mechanics Lab - University of Michigan)