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SEM of Bone , a Summary
HI Biomechanics Interest group,
Thanks a lot for the replies, i recieved for my question on SEM
technique of bones. I am mailing the summary of the mails
I recieved,. Since I am in midst of my research, I am not in
a position to comment authoritively on any of the mails.
Hope this summary helps everyone.
This also is my public acknowledgement for all those who
contributed in the summary.
Sunil Padture
spadture@bobcat.ent.ohiou.edu
>From bfinlay@uwovax.uwo.ca Tue May 25 08:37:10 1993
Date: Tue, 25 May 93 08:28:58 EDT
Subject: Re: Info On SEM technique for Bone
Dear Sunil:
Just one-or-two words of caution on your interpretations of data obtained
from your SEM studies of bone.
Conventional SEM studies will require viewing the sample in a vacuum which
will effectively require a fully dehydrated sample.
Complete dehydration of cortical bone from the diaphysis of the bovine femur
results in anisotropic shrinkage with the following approximate magnitudes:
Axial 0.9% (9,000 microstrain)
Circumferential 2.7% (27,000 microstrain)
Radial 4.0% (40,000 microstrain)
These figures should be viewed in the light of the typical physiological
peak strain of 3,000 microstrain. Also, you will note that these strains
create notable shear strains.
In studying bone in the SEM in the past, I have generally used 10-20 nm of
gold-palladium coating. Removal of glycoproteins is generally not necessary
to view the bone structures - i.e. in the way that it is to view soft tissue
structures.
Hope these comments help...
Best wishes:
Bryan Finlay, PhD 519-663-3063
Director of Orthopaedic Research 519-663-3904 FAX
University Hospital
P.O. Box 5339
London, Ontario, CANADA, N6A 5A5
>From ame_0123@bigdog.engr.arizona.edu Mon May 24 11:46:46 1993
From: ame_0123@bigdog.engr.arizona.edu (Terrance J. Dishongh)
Now this I can answer.. We do bone SEM's all the time. If your
at the end of your experiment with the bone (mechanical testing)
then spudder coat the bone before the SEM. We use a gold mixture
but I have seen it done w/ a carbon compound too. Magnification is purely
up to what your using it for. What are you hoping or wishing to find.
I look a crystalline structure so I use about 150X to 350X. A friend of
mine here uses it to look for begining of tumors so he uses 1500X it depends..
Hope that helps..
Terry Dishongh
>From dingwell@bme.ri.ccf.org Mon May 24 09:09:29 1993
Date: Mon, 24 May 93 09:12:55 EDT
From: Jonathan Dingwell
Whitehouse, Dyson, and Jackson (1971, J. Anatomy, V.108, n. 3, pp. 481-496)
give a pretty good discussion on the preparation of their specimens of
cancellous bone. Their article describes some of the earlier work in this area
and is references quite often in the literature. It would probably be a good
place to start at least. Good luck.
Jon Dingwell
>From MEHTA01@swmed.edu Sun May 23 22:55:16 1993
Hi.
In order to study the structure of bone by SEM, there are a few things
to be wary of before beginning your investigation, that might make it facile to
interpret results later.
1. Bone has various levels of structural organisation and the
magnification you choose depends on what level of structure you wish to
investigate. eg. 30 - 100X should give you some idea of osteonal
organisation, 100-300X should give you some idea of fibrillar bundle
arrangement and so on...
2. Drying and coating: Again, depending on the magnification, the
method of drying may or may not be of importance. Normally, for gross
(low mag) observations, you can air-dry with dessication and coat with
gold or carbon sputter. Coating is important as you will get a lot of
charging otherwise. Some people even infiltrate (wet) specimens with
osmium tetroxide before processing for SEM.
3. Search the papers by A. Boyde for more information. Papers by
Ascenzi et al might also yield some useful info. For BackScattered
Electron Imaging (BEI) of bone, look up H Hagler.
Good luck.
DO post the replies you get in a compiled form. i would like to see them and
i am sure other would too.
Ciao
Shreefal Mehta
>From stjohn@fiona.umsmed.edu Mon May 24 10:14:13 1993
From: Kenneth St John
You don't say why you are attempting SEM on bone nor what types of equipment
you have available to you so I will give you some general information.
We have performed SEM on fractured bones from dogs and found that we got
good results from dehydration in acetone but you need to be aware that
a more gentle dehydration through graduated alcohol and then perhaps
into acetone might be a better idea. You will need to get the bone
very dry and then dry it some more in vacuum after you think it is dry.
The bone will need to be coated for observation in the microscope and
if it is not as dry as possible before it is coated, the further drying
which will occur in the microscope will disrupt the coating and give
you charging problems.
Another method we have used for 2D analysis of bone is drying of the bone
through graduated alcohols and embeddding in methyl methacylate polymer.
We then saw the block and polish a face on it which can be coated and
evaluated by backscattered electron imaging.
Another possibility, if you have the equipment, is to use an environmental
scanning microscope. This allows you to image wet samples without drying
or coating. We have used the microscope at the University of Southern
Mississippi with reasonably good results and were able to maintain hydrated
specimens. This eliminates or reduces drying artifacts but introduces
other artifacts like biofilms and the artifacts due to the effects of the
variations in hydration which can occur during viewing.
I hope this has been of some help. Please summarize all responses you
receive and post them on BIOMCH-L.
Kenneth R. St. John
Assistant Professor of Orthopaedic Surgery
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-6199
FAX (601) 984-6014
Internet stjohn@fiona.umsmed.edu
END OF SUMMARY, SEM OF BONE
************************************************** *****************************
__ __
Sunil P Padture /~~\_/~ \ ,,, Graduate Student
spadture@bobcat.ent.ohiou.edu | #=#==========# | Dept.of Mech.Engineering
Tel No: 614-593-3917 \__/~\__/ ``` 251 Stocker Center
38,N. McKINLEY AVE. #406 Ohio University
ATHENS OH-45701 ATHENS OH-45701
Imitation Is Suicide| |The Best way to
Only way to get rid of| |Get rid of Temptation
Imitators, is to commit one| |Is to yield to it.
************************************************** *****************************
HI Biomechanics Interest group,
Thanks a lot for the replies, i recieved for my question on SEM
technique of bones. I am mailing the summary of the mails
I recieved,. Since I am in midst of my research, I am not in
a position to comment authoritively on any of the mails.
Hope this summary helps everyone.
This also is my public acknowledgement for all those who
contributed in the summary.
Sunil Padture
spadture@bobcat.ent.ohiou.edu
>From bfinlay@uwovax.uwo.ca Tue May 25 08:37:10 1993
Date: Tue, 25 May 93 08:28:58 EDT
Subject: Re: Info On SEM technique for Bone
Dear Sunil:
Just one-or-two words of caution on your interpretations of data obtained
from your SEM studies of bone.
Conventional SEM studies will require viewing the sample in a vacuum which
will effectively require a fully dehydrated sample.
Complete dehydration of cortical bone from the diaphysis of the bovine femur
results in anisotropic shrinkage with the following approximate magnitudes:
Axial 0.9% (9,000 microstrain)
Circumferential 2.7% (27,000 microstrain)
Radial 4.0% (40,000 microstrain)
These figures should be viewed in the light of the typical physiological
peak strain of 3,000 microstrain. Also, you will note that these strains
create notable shear strains.
In studying bone in the SEM in the past, I have generally used 10-20 nm of
gold-palladium coating. Removal of glycoproteins is generally not necessary
to view the bone structures - i.e. in the way that it is to view soft tissue
structures.
Hope these comments help...
Best wishes:
Bryan Finlay, PhD 519-663-3063
Director of Orthopaedic Research 519-663-3904 FAX
University Hospital
P.O. Box 5339
London, Ontario, CANADA, N6A 5A5
>From ame_0123@bigdog.engr.arizona.edu Mon May 24 11:46:46 1993
From: ame_0123@bigdog.engr.arizona.edu (Terrance J. Dishongh)
Now this I can answer.. We do bone SEM's all the time. If your
at the end of your experiment with the bone (mechanical testing)
then spudder coat the bone before the SEM. We use a gold mixture
but I have seen it done w/ a carbon compound too. Magnification is purely
up to what your using it for. What are you hoping or wishing to find.
I look a crystalline structure so I use about 150X to 350X. A friend of
mine here uses it to look for begining of tumors so he uses 1500X it depends..
Hope that helps..
Terry Dishongh
>From dingwell@bme.ri.ccf.org Mon May 24 09:09:29 1993
Date: Mon, 24 May 93 09:12:55 EDT
From: Jonathan Dingwell
Whitehouse, Dyson, and Jackson (1971, J. Anatomy, V.108, n. 3, pp. 481-496)
give a pretty good discussion on the preparation of their specimens of
cancellous bone. Their article describes some of the earlier work in this area
and is references quite often in the literature. It would probably be a good
place to start at least. Good luck.
Jon Dingwell
>From MEHTA01@swmed.edu Sun May 23 22:55:16 1993
Hi.
In order to study the structure of bone by SEM, there are a few things
to be wary of before beginning your investigation, that might make it facile to
interpret results later.
1. Bone has various levels of structural organisation and the
magnification you choose depends on what level of structure you wish to
investigate. eg. 30 - 100X should give you some idea of osteonal
organisation, 100-300X should give you some idea of fibrillar bundle
arrangement and so on...
2. Drying and coating: Again, depending on the magnification, the
method of drying may or may not be of importance. Normally, for gross
(low mag) observations, you can air-dry with dessication and coat with
gold or carbon sputter. Coating is important as you will get a lot of
charging otherwise. Some people even infiltrate (wet) specimens with
osmium tetroxide before processing for SEM.
3. Search the papers by A. Boyde for more information. Papers by
Ascenzi et al might also yield some useful info. For BackScattered
Electron Imaging (BEI) of bone, look up H Hagler.
Good luck.
DO post the replies you get in a compiled form. i would like to see them and
i am sure other would too.
Ciao
Shreefal Mehta
>From stjohn@fiona.umsmed.edu Mon May 24 10:14:13 1993
From: Kenneth St John
You don't say why you are attempting SEM on bone nor what types of equipment
you have available to you so I will give you some general information.
We have performed SEM on fractured bones from dogs and found that we got
good results from dehydration in acetone but you need to be aware that
a more gentle dehydration through graduated alcohol and then perhaps
into acetone might be a better idea. You will need to get the bone
very dry and then dry it some more in vacuum after you think it is dry.
The bone will need to be coated for observation in the microscope and
if it is not as dry as possible before it is coated, the further drying
which will occur in the microscope will disrupt the coating and give
you charging problems.
Another method we have used for 2D analysis of bone is drying of the bone
through graduated alcohols and embeddding in methyl methacylate polymer.
We then saw the block and polish a face on it which can be coated and
evaluated by backscattered electron imaging.
Another possibility, if you have the equipment, is to use an environmental
scanning microscope. This allows you to image wet samples without drying
or coating. We have used the microscope at the University of Southern
Mississippi with reasonably good results and were able to maintain hydrated
specimens. This eliminates or reduces drying artifacts but introduces
other artifacts like biofilms and the artifacts due to the effects of the
variations in hydration which can occur during viewing.
I hope this has been of some help. Please summarize all responses you
receive and post them on BIOMCH-L.
Kenneth R. St. John
Assistant Professor of Orthopaedic Surgery
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-6199
FAX (601) 984-6014
Internet stjohn@fiona.umsmed.edu
END OF SUMMARY, SEM OF BONE
************************************************** *****************************
__ __
Sunil P Padture /~~\_/~ \ ,,, Graduate Student
spadture@bobcat.ent.ohiou.edu | #=#==========# | Dept.of Mech.Engineering
Tel No: 614-593-3917 \__/~\__/ ``` 251 Stocker Center
38,N. McKINLEY AVE. #406 Ohio University
ATHENS OH-45701 ATHENS OH-45701
Imitation Is Suicide| |The Best way to
Only way to get rid of| |Get rid of Temptation
Imitators, is to commit one| |Is to yield to it.
************************************************** *****************************