David E. Muirhead
Cellular Pathology Coordinator,
Texas Scottish Rite hospital for Children
2222 Welborn Street,
Dallas TX 75219
USA
214-559-7766 (W)
214 559 7768 (Fax)
E-mail David.Muirhead@tsrh.org

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Item: ULTRAONE
Article #: VC2860240
Type DMK #: 3mm-45_
Serial #: SE00001A
 
A 3 mm diamond knife was received in the Department of Cellular Pathology, Texas Scottish Rite Hospital for Children, Dallas, Texas for ultramicrotomy assessment. The following parameters were assessed and tested;
 
i. Quality of Diamond
ii. Quality of securing cement
iii. Boat/trough
iv. Sharpness Durability
v. Cutting angle
vi. Cutting Speed
vii. Static/wetting of knife edge
viii. Cutting qualities of different tissues
 

REPORT

On opening package and removing the diamond knife from Styrofoam, it was noted that the box seal was not broken. The plastic box was of a good quality. However, after opening and closing of the box the Plexiglas pins used as hinges broke. This could be a problem, as the knife may not be held secure in the box. This is an area where damage to diamond knives can occur.
 
The Diamond Knife was examined under stereo microscope, placed in knife holder and stage examined on the ultramicrotome with diffracted light and the stereo head to assess the cutting edge. Light was varied across the knife-edge with back lighting. No flaws or artifacts could be identified. Knife appeared clean and debris free. The boat was filled with fresh distilled water through a swinnex filter on a 10 ml syringe. It was over filled and water was removed via syringe and filter until a silver shine was obtained on the surface. Water surface was checked for oils and debris, – negative. An epon/alardite block with no tissue was placed in specimen holder. The block was cut at the recommended speed and angle, 1mm/sec and 6 degrees respectively. Ribbons of ultrathin sections, 
15-10 sections per ribbon, of silver diffraction color, approx 60nm thick were easily obtained. The sections showed a minimum compression when compared to the area of the original cut surface. Passing a heated pen over the surface flattened sections. The cutting angle was varied between 3-7 degrees with very little difference, suggested angles setting between 4-6 degrees. 
 
However, on varying the speed a change in thickness and compression was noted. Between 0.8 mm/sec and 2mm sec no change was noted. Speeds above 2mm/sec thicker section were obtained dark gold and below 0.8 some variation was noted. Optimum speed for good ribboning, consistent thickness and minimum compression was between 0.8mm/sec and 1.4 mm/sec.
 
The diamond was assessed in several places, left hand side, middle and right hand side. All areas were found to be flawless and ribbons of 20 sections were obtained with no visible artifacts, scores or chatters identified. They were of a uniform thickness, i.e. silver diffraction colored sections, approximately 60-80nm in thickness. The above tests were repeated with epon/alardite bocks of skeletal muscle and identical results were obtained.
 
It was noted that there was occasional wetting problems with the cutting of the edge diamond, drying back. This was easily remedied by washing diamond knife in a 1:10 dilution of a wetting agent, tween 20 or decon in a diamond knife cleaner from Microsharp. Followed by a good rinse in distilled water and allowed to air dry in dust free atmosphere.
 
The cutting property of the knife was tested with the following tissues under the same criteria and conditions as above; skeletal muscle, nerve, liver, kidney, lung, skin, myotendinous junction, bone – cancellous/trabecular and cortical, plant tissue - leaves, stems roots and root hairs. All tissues produced flat, artifact free ribbons of silver sections with minimum amount of compression. Occasional variation in thickness was seen with the cortical bone i.e. silver sections (approx. 60nmn) with occasional gold section within the ribbon.
 
The new style of boat/trough was excellent it was more practical than previous design allowing better manipulation and retrieval of ultrathin sections onto the copper grids.

 
Comments
The diamond knife has been used every day for the last four months by experienced staff, trainee staff and high school students. To date we still have only utilized the left hand side of the knife. All tissues have been sectioned from human tissue to plant tissues. Ultrasections of a reproducible high quality have been produced. 
 
This diamond knife is of a high quality and durability. In my experience if used properly the life expectancy of this knife could be 1-2 years in the hands of a good microtomist i.e. good user not abuser. If this is the quality we are led to believe will be the standard and cost are kept low this will be an excellent retail item affordable by most EM labs.
 
The following images (below) and electron micrographs were obtained with the above diamond knife.
 
I would like to thank Harris International for giving me the opportunity to test this new product on their behalf. 

 
David E. Muirhead
Cellular Pathology Coordinator/ Diagnostic/Research Electron Microscopist 
Department of Cellular Pathology
 

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Click on a thumbnail for an enlarged view of photo

Cell Culture 1

High power electron micrograph of a human myotube grown in cell culture showing the irregular shaped nuclie with peripheral aggregates of contractile proteins with electron densities characteristic of z-band formation. Several elongated mitochondria can be seen scattered throughout the cytoplasm.
Bar = 2000nm 

 

Cell Culture 2

This is a high power electron micrograph showing the cytoplasm of a 21-day-old myotube. A sarcomere pattern can be clearly identified showing Z-bands, M-bands (myosin) and I-bands (actin) which the basic contractile unit of skeletal muscle.
Bar 2000nm 

Liver 1

Low power electron micrograph of hepatocytes (liver cells) showing the characteristic round nucleus with heterochromatin clumping and margination with a prominent nucleolus, top right hand corner. Cytoplasm contains copious glycogen, mitochondria, cytoplasmic vesicles and a small homogeneous semi-electron dense vacuole is present which is characteristic of lipid droplet.
Bar =10um 

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Liver 2

The electron micrograph consists of four hepatocytes taken at high power. Part of a nucleus showing chromatin clumping and margination is present in the bottom left hand side of the image. The cytoplasm contains small strands of rough endoplasmic reticulum (RER), mitochondria, copious glycogen and a large homogeneous semi-electron dense lipid vacuole. Aggregates of small round electron dense viral particles can be seen surrounding the lipid vacuole and scattered throughout the cytoplasm. The viral particles are suggestive of HIV particles.
Bar = 2000nm

Lung

Low power electron micrograph of a lung biopsy of patient with histocytosis X. The alveolus is patent and lined by type I and II pneumocytes, There is enlargement of the alveolar wall due to infiltration of inflammatory cells, several polymorph leucocytes and scanty large mononuclear cells can be seen within the wall.

Mesothelioma

Low power electron micrograph of a metastatic mesothelioma taken from the base of the pelvis. The image is composed of loosely aggregate of tumor cells, considerable portion of the cell surfaces are covered with microvilli. The microvilli are profuse, long, branching, curved and slender, with an absence of well-defined rootlets. The cytoplasm contains numerous mitochondria, aggregates of cytoplasmic filaments (vimentin filaments) and dilated segments of rough endoplasmic reticulum. The nuclei are somewhat irregular showing chromatin clumping and margination with prominent nucleoli. Adjacent cells are joined by long well developed desmosomes.
Mag 6000x

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Muscle 1

Low power electron micrograph of two muscle fibers showing the characteristic myofibrillary pattern. The fibrils are long and non-branching. Sarcomeres show a normal sarcomeric pattern with no disruption. A small clump of three myonuclie can be is seen in the bottom of the image which appear normal. There is no thickening of the basal lamina. Aggregates of pleomorphic mitochondria are present scattered throughout the fiber and within the para-nuclear space. A prominent small blood vessel can be seen lying between the two fibers. These changes are nonspecific.
Bar = 10um 

Muscle 2

The electron micrograph consists of part of a small atrophic myofiber showing loss of myofilament, taken at a high power. There is total loss and disruption of the normal sarcomeric pattern. Small aggregates of contractile filaments can be seen within the cytoplasm, top right hand corner. The myonucleus is present showing condensation of the chromatin with margination and clumping. This biopsy was taken from a patient with Duchenes Muscular Dystrophy.
Bar 1000 nm

 

Nerve 1

Low power electron micrograph of a nerve biopsy. The image consists of a portion of a transverse section of a nerve fascicle. The perineurium is present, top of the image, with normal flattened polygonal perineurial cells lying in a lamellar formation. The matrix consists of myelinated fibers of varying sizes with clusters of unmyelinated nerves. Several Schwann cells can be identified. Several nerves are enlarged showing thickening of the myelin. In the center of the image a typical ovoid is present showing loss of its axon. Many of the axons are exhibiting shrinkage and vacuolation. There is a definite increase in collagen bundles.
Mag 5,500x

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Nerve 2

The image is a high power electron micrograph taken from the body of nerve fascicle shown in the above image. Several enlarged myelinated nerves showing early Wallerian degeneration can be seen. Many of the axons present exhibit varying degrees of vacuolation.

 

Nerve 3

Low power electron micrograph of a patient with SLE. The image shows Schwann cells devoid of axons and myelin. There are clusters of non-myelinated nerves showing axonal degeneration. These ultrastructural changes are characteristic of a demyelinating process.

Plant 1

The electron micrograph consists of a low power image taken from the alfalfa plant showing normal plant root cells. The plant cells are surrounded by a rigid, amorphous structure known as the cell wall. A plant nucleus can be clearly identified in one of the cells. Several chloroplasts are present showing the stroma with their characteristic thykaloid sacs. The vacuole or tonoplast is a large single membrane structure occupying approximately 70-90% of the cells present leaving a narrow peripheral area of cytoplasm along the cell wall.
Bar 10 nm. 

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Plant 2

Low power electron micrograph of plant tissue showing plant cells with rigid cell wall taken from the alfalfa plant. The plant cell nuclei are present in many of the cells. There is vacuolation of the cytoplasm with some of the cells showing the characteristic tonoplast. Small-elongated ovoid structures can be identified lying in the rims of cytoplasm lying close to the cell wall. These structures are characteristic of rhizobium bacteria, nitrogen fixation bacteria that form nodules on the root hairs and root of plants.
Bar = 10um 

Plant 3

High power of image 2 showing the thin rim of cytoplam lying close to the cell wall. Within the cytoplasm the elongated ovoid structure, rhizobium bacilli bacteria, are seen surrounded by a single membrane. The thick bacteria cell wall can be easily recognized.
Bar = 1000nm 

Kidney Biopsy

IgA glomerulonephritis. An electron micrograph of a kidney biopsy from a 32 year old female patient. The mesangium is markedly enlarged with profiles of four mesangial cell nuclei. The matrix bars are prominent occupying most of the cytoplasm. Large amounts of mesangial and paramesangial of electron dense deposits of IgA immunogloblin are obvious. The endothelial cells are swollen taking up most of the urinary space,
Mag x3200