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Duncan, Neil
04-02-1995, 05:10 PM
Hi,

I would like to thank all who replied to my original posting:

"We are attempting to make very fine noninvasive strain measurements on
intervertebral disc tissue. For these measurements we have being using India
ink markings on the tissue (the fine lines need to be less than 0.1mm; if
possible on the order of 0.05mm). However, the India ink has a tendency to
'bleed' (even if the tissue is blotted dry), thereby distorting the very fine
lines. We have also used non-water soluble inks but these do not adhere as well
to the tissue.
We are searching for a possible alternative to the India ink or ways to reduce
the tendency of the India ink to "bleed" into the tissue."

I think the replies can be divided into three groups:
1) Alternative inks and stains
2) Alternative techniques for appling the ink
3) Alternative techniques

The suggestions brought a fresh perspective to our project and we again thank-
you all.

Neil Duncan
Orthopaedic Bioengineering Lab.
Department of Orthopaedic Surgery
University of California, San Francisco



1) Alternative inks and stains

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Neil,

Savio Woo uses a Verhoff stain (check with you pathology department) for
ligament strain. There are other histo stains with should not run as much,
which the path dept can tell you about. Check also Kai-Nan An work in J.
Biomechanics and J Ortho Res (I forget which one) where he uses a florescent
paint.

Another possible method for the disc might be gluing metal beads to the disc
using crazy glue.

Whatever methods, put on a grid of markers for 2D strain distributions.

Best regards,
Peter Torzilli
Lab Soft Tissue Res
Hospital for Special Surgery
torzillip@hss.edu

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What do radiation oncologists mark the skin with to aim their radiation beams?
I see patients walking around with skin markings, but don't know what they use.
Good luck.
Robert McAnelly

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Neil,

I've worked with pericardium, and I am sceptical that any dye-based marker will
serve your application: One needs a minimum quantity of dye for the sensors to
pick up the marker. Have you considered some sort of chemical marking/
staining?

The stain could be painted on with a mask.

Santosh Zachariah

Postdoctoral Senior Fellow
Centre for Bioengineering, FL-20 internet: zach@limbs.bioeng.washington.edu
Univ. of Washington, ph: usa (206) 685 3488
Seattle WA 98195 fax: 543 6124
USA



2) Alternative techniques for appling the ink

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I know that this might sound crazy,but have you tried using a fine tatoo needle?

Brian

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"You are not dressed for work until you put on a smile."
B.J. Plamer, D.C., PhC.

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Neil,

Can you use a selection of points, i.e. dots of ink, to make the
measurements, rather than lines? This may reduce the problem by reducing
the amount of ink applied.

Pete

************************************************** *********
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* Associate Professor of Physical Therapy *
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3) Alternative techniques

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I have never tried this, but could you heat-up a fine wire
(with electric current) and burn a line into the surface?
Perhaps one of those cheap plastic dental floss holders would help
hold the wire. A brief, light exposure of the tissue surface
with the hot wire may mark it without much damage to the tissue.

John Hipp
Beth Israel Hospital
Orthopedic Biomechanics Lab
Boston

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Neil,

I use small circular markers that I manufacture by slicing small-diameter
plastic rods on an isomet histology saw. These markers are ~1 mm in dia.
and ~200 microns thick (although this dimension is easily changed).

I adhere them using cyanoacrylate (crazy clue). A *very* small amount
is needed, and I applly it to the back of the marker using the tip of
a pin.

Jeff

Jeff Weiss
Utah

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Dear Neil,
There may be a variety techniques, but first a question: Are you
measuring strain in 2D or 3D? Certainly disc bulging is a 3D phenomena.


Trey Crisco, Ph.D.
joseph_crisco_iii@brown.edu

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Dr. Duncan--
I found your inquiry on biomch-l regarding very fine noninvasive strain
measurements on intervertebral disc tissue very interesting.
I, too, do noninvasive strain measurements--but on the ligaments. I
tried the India ink technique and had the same 'bleeding' problem. Then I
used small markers (500-800 microns in diameter) attached to the tissue.
[We attached them with skin glue or by dipping them in hairspray and then
placing them on the tissue. (The latter method worked just as well as the
former.)] The problem with this method was that there was not much
contrast between the marker and the tissue, so it was difficult to digitize
the target positions.
When I came here, I had to start over (without much money). I did a lot
of reading and a lot of browsing through other people's labs and my
students and I developed a new--and we think better--technique, based on a
technique used in fluid flow visualization. We call this technique
LIPPM--Laser Induced Photochemical Position Measurement. The targets are a
small amount of either a photochromic or photoluminescent chemical which
are mixed with a water-insoluble binder and painted or dipped into the
tissue. These chemicals (which are available commercially) have the
property that when excited by ultraviolet light (laser) they either change
color (photochromic) or emit light (photoluminescent) for some short time
(5--50 ms) after the laser light has been removed. We record target motion
synchronously using two gated charge collecting device (CCD) cameras, each
of which views the structures/tissues of interest from a different angle.
We use NIH's Image 1.44 software package to digitize the two sets of
two-dimensional data, then we use Walton's procedure to convert the two
sets of two-dimensional data to three-dimensional data.
The advantages to this technique are: (1) the contrast between the
target and the tissue is tremendous and (2) the resolution is great. The
disadvantage is that the photochemical is a very fine powder and, no matter
how careful you are, it gets places you don't really want it to be.
There are some equipment requirements also. First of all you need a
laser. The laser has to be pulsed while the specimen is loaded, flexed,
etc. If you are taking multiple views, the cameras have to be
synchronized. You need gated CCD cameras because of the low light level
and to get the necessary shutter opening delay between the laser pulse and
when you film. The pulse generator driving the laser also has to be used to
drive the cameras or the pulse generator driving the laser has to be sync'd
to the pulse generator driving the cameras.
I hope you find this helpful. If you have any questions, please contact me.

--sal

----------------------------------------------------
Susan A. Lantz, Ph.D.
Associate Professor
Dept. of Mechanical Engineering
University of Utah
Salt Lake City, UT 84112
(801)581-7104
lantz@me.mech.utah.edu
----------------------------------------------------

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Neil,
Have you thought about using a Hall Effect transducer?

Nat Ordway
Department of Orthopedic Surgery
SUNY Health Science Center (315) 464-6462 -->voice
750 E. Adams St (315) 464-6470 -->fax
Syracuse, New York 13210 ordwayn@vax.cs.hscsyr.edu -->email

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Hi Neil,

We are working on some related issues (strain measurement in soft tissue)at
the Biomechanics Lab at UC berkeley.

Are you familier with the soft tissue extensometer that is made by MTS? If
not, give me a call and I can give you some more info. Frank Ashford has my
number.

Good Luck,

Ed Wachtel

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Neil,

I had the same difficulty when using a video dimensional analyzer to
measure strains in a thin fiber of collagen 50 mm long with a diameter of
0.1 mm in a bath of 37C water. I tried several methods for applying
strain markers that had enough contrast to generate a clean video signal
for the VDA to latch onto, including sputtered wax, various stains and
paints applyed with brushes, sutures, etc.

Two methods worked very well. The first was having a vascular
microsurgeon lay in two or three stitches of 10-0 suture across the
specimen at 1 mm intervals (amazes me to this day...) and the second
involved aerated water in the bath. I discovered that very small bubbles
would form on the tissue and grip quite nicely to the surface. If they
were lit from above in a darkened lab, an extremely sharp high contrast
image could be clearly seen at the top edge of the bubble were the light
reflected near the meniscus.

The second method worked well but was discarded due to uncertainties
about whether or not you could prove that the bubble was anchored firmly
to the surface of the tissue or not while the surface was straining. I
think that a mathematical proof could be worked out involving buoyancy
forces balanced by surface tension varying with surface strain, or
something along those lines.

Any comments on this second method? I look forward to any discussion on
this idea...


- Ed -


+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= +=+=+=+=+

Ed Morra
Orthopaedic Research Laboratories
Mt. Sinai Medical Center
One Mt. Sinai Drive
Cleveland, Ohio 44106
(216) 421-4697 (vox)
(216) 421-4843 (fax)
r2eam@dax.cc.uakron.edu

+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= +=+=+=+=+

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It seems that you are wanting do do something similar to what we
described in:
Stokes IAF, Greenapple DM: Measurement of surface deformation of soft
tissue. J Biomech 18:1-7, 1985.

Stokes IAF: Surface strain on human intervertebral discs. J Orthop Res
5:348-355, 1987.

Stokes IAF: Bulging of intervertebral discs: non-contacting measurements
of anatomic specimens. J Spinal Disorders 1:189-193,1988.

This work was done with an optical (photogrammetric) method. Two
key things we found were:
1. Don't use india ink, use little target markers
2. Attach the marker by smearing the soft tissue with vaseline, don't use
glue.

Let me know if you could use some target markers - I have a few left.
Hope this is helpful.

Ian Stokes
University of Vermont, Department of Orthopaedics and Rehabil.
Burlington, VT 05405-0084, USA
Phone: (+1) 802 656 2250 fax: (+1) 802 656 4247

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Neil,

As a suggestion, how about applying some of that spray which is used to seal
large but superficial open wounds, and then putting ink lines on top of this? I
am not sure how flexible the stuff is :-(

Edsko Hekman
Twente University
Fac. Bio-Mechanical Engineering (WB-BW)
Postbus 217
7500AE Enschede
The Netherlands

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Dear Neil,

How about glewing thin threads on the tissue surface?

Goodluck,

Jos

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Have you considered very fine pins or thread. These can be inserted
into soft tissue and work well for uniaxial tests but may need more
thoughtful placement if you are looking at biaxial strain measurement



************************************************** ***********************
James Smeathers
Rheumatology and Rehabilitation Research Unit, Leeds University, England
Internet: RRR6JES@LEEDS.AC.UK Phone: +44-113-233-4955 Fax: +44-113-244-5533

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I am interested on your work on disc strain measurement. It is a dificult
problem particularly on intact discs. As to your problem, have you tried
gluing hairs onto the disc using cyanoacryate adhesive?

Regarding the relevance of your measurements. The largest gradient in the
compressive stress within the matrix of the disc, which corresponds (in a
hand wavey sort of way) to the region or greatest stress in the fibres (and
therefore perhaps strain) occurs about 2mm below the surface of the disc in
healthy young discs - more in degenerate ones. This region would be even
more interesting.

Donal McNally
Bristol

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