|
|
 |
|
|
Ultramicrotomy
|
|
Ultramicrotomy is a technique to produce thin sections of a sample material for examination with an electron microscope. Many materials in their natural state are too thick to be examined directly with an electron microscope. Ultrathin sections can be created in the range of 10-100nm thick with the use of a diamond knife. Thin sections of materials such as plant and animal tissue, can then be penetrated by an electron beam to an appropriate depth to reveal intricate structures. Others materials such as metals, plastics or synthetic fibers can be processed in a similar fashion reveling their underling structure.
|
|
|
|
|
The ability to cut thin
sections from a material dictates that it must be in a
fairly hard state. Most animal and plant tissues are
relatively soft and must first be embedded in a hard
resin. Some additional information on this process can be
found in our
User Information
area.
A diamond histo knife is typically used to create sections
on the order of 0.5 – 2.0 micron when a light microscope
is used to view the material understudy.
An alternative method to embedding the material for
hardening is to freeze and section at low temperature
using a diamond cryo-knife. This so called
cryo-ultramicrotomy can be applied to both biological and
non-biological material.
|
|
|
Return
to top >
|
|
|
|
|
Application
notes
|
|
The following micrographs and descriptions are a sampling of various materials cut with the classic
Drukker diamond knife. Additional information on applications can also be found in the User Information page.
|
|
|
|
|
|
Ultrathin sections for Transmission Electron Microscopy (TEM)
|
|
| |
|
Ultrathin section of GA perfusion fixed rat liver, showing hepatocyte with cell oganelles, such as mitochondria (mi), nucleus (nu), RER, peroxisomes (pe) and unstained glycogen(gl). The hydrophilic K4M has been embedded with CS-Auto freeze substitution method. Bar = 1µm.
The specialty of this image is the water sensitivity of K4M, a lowicryl mixture, which is very water sensitive. Due to the hydrophobic properties of most diamond knives this is very difficult and almost impossible to cut with a standard diamond knife. Due to the hydrophilic surface of the
Drukker diamond knife, these sections can be done on a routinely basis using a low water level in the trough, resulting in a very detailed clear section.
|
|
|
| Click
image for larger view |
Ultrathin section of GA perfusion fixed rat liver |
|
| Courtesy of University Groningen, Lab. For Cellbiology and Electronmicrsocopy, Oostersingel 69/2, 9713 EZ Groningen, The Netherlands. |
|
|
Return
to top >
|
|
|
|
|
|
|
Titanium clip used for suturing rat aorta
It has been always stated that sectioning of titanium is impossible due to the carbide formation during the process.
This is a series of micrographs showing sections of a titanium clip and its surrounding tissue.
|
|
|
|
|
|
| a.
Elastic aorta wall bordering the clip covered by connective tissue (Ct) generated by mussel cells and fibroblasts.To make the soft tissue visible the micrograph is taken at a low voltage, which lets appear the thin Ti section very dark.
(right)
b. Detailed view on the Ti clip structure, taken at a higher voltage. The image
shows the irregular distribution of the
titanium particles in the clip. (below)
|
|
|
|
| Click
image for larger view |
a. Epon embedded titanium clip
(ti).
Bar = 2µm |
|
|
|
|
|
|
| Click
image for larger view |
b. Epon embedded titanium clip, detailed view
of figure image a.
Bar = 2µm.
|
|
|
|
|
| Click
image for larger view |
|
c. FE-SEM image of titanium clip.
Bar = 100µm |
|
Courtesy of University Groningen, Lab.For Cellbiology and Electronmicroscopy,
Oostersingel 69/2, 9713 EZ Gronigen,
The Netherlands. |
|
Return
to top >
|
|
|
|
|
|
|
Un-decalcified bone tissue
sectioned with a 45° Drukker diamond knife. Pre-fixation was performed in Karnovski fixative and post-fixation in OsO4. The sections, 40nm thick were made on a LKB Ultramtome IV at a clearance angle of 5° and a cutting speed of 1mm/sec. The sections were stained during 10min with lead citrate only. It clearly can be seen that the interface between the hard mineral matrix and the soft cellular part remained perfectly intact. Sectioning artifacts like scratches or material displacement due to variation in hardness, are absent.
|
|
|
|
| Click
image for larger view |
|
Un-decalcified bone tissue |
|
| Courtesy of Laboratory for Electron Microscopy Medical Faculty, Leiden University Rijnsburgerweg 10, 2333 AA
Leiden, The Netherlands |
|
Return
to top >
|
|
|
|
|
|
|
|
|
|
| |
|
Epon embedded outer layer of the Zebrafish embryo, showing the three layered constructions (a,b,c,) of the chorion with different density and pores (po) at the inner side bordering the peri-0vital-line space. A special embedding and cutting technique made the visibility of the glycocalyx (*) possible. Bar = 0.5µm |
|
Epon embedded internal layer of the same Zebra fish embryo (12-h stage). In this syncitical layer zip-like desmosomes (de) surrounded by filamentous material (fi) are found. In this stage the cellular structure is not fully developed yet. Bar = 200nm |
| |
|
|
Courtesy of University Groningen, Lab. For Cellbiology and Electronmicrsocopy,
Oostersingel 69/2, 9713 EZ Groningen,
The Netherlands. |
|
Return
to top >
|
|
|
|
|
|
|
Cryo-sections
|
|
|
Human blood derived dendrite cell after endocytosis of biotinylated BSA
The ultrastructural components (Golgi G, Iysosomes L) are well preserved, the sections are constant in thickness, smooth and free of section artifacts.
The cells were fixed in 0.1% glutar aldehyde in 0.14 M cacodylate buffer and embedded in 10% gelatine. Thereafter cells were impregnated in 2.3M sucrose, cooled in liquid nitrogen and sectioned on a Reichert FCS cyro-microtome. The BSA was visualized on ultrathin sections by a rabbit polyclonal anti biotin antibody, followed by 10 nm protein-A-go |
|
|
|
| Click
image for larger view |
Human
blood derived
dendrite cell |
Courtesy of Laboratory for Electron Microscopy Medical Faculty, Leiden University
Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands |
|
|
Return
to top >
|
|
|
|
|
|
|
Rat jejunum
Low magnification of an ultrathin cryosection of rat jejunum. The section is stretched over a large area of the grid without any extra preparation. No compression can be recognized and the section is straight and flat.
Courtesy of Utrecht University, Med. Fac., Dept for Cell Biology, Heiberglann 100, 3584 CX Utrecht, The Netherlands. |
|
|
|
| Click
image for larger view |
Cryo
section of a
rat jejunum
|
|
|
|
|
|
|
|
|
|
|
Semithin sections for Light Microscopy
Inner hair cells of non-decalcified guinea pig organ of Corti embedded in Epon-Araldite
It shows the inner hair cell (IHC) and three outer rows of hair cells (OHC I, II, III) with stereocilia with optimal morphology.
Courtesy of University Groningen, Lab. For Cellbiology and Electronmicrsocopy, Oostersingel 69/2, 9713 EZ Gronigne, The Netherlands. |
|
|
|
| Click
image for larger view |
Cross-sectioned non-decalcified guinea pig organ of Corti.
|
|
|
|
Return
to top >
|
|
|
|
|
|
|
User
Information
|
|
|
|
|
|
|
|
|
|
|
|
Return
to top >
|
|
|
|
|
|
|
|
;)
;)
;)
;)
;)
;)
)
