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Leica VT1000 S Materials Testing

Leica VT1000 S Vibrating blade microtome

Vibrating Blade Microtomes

What is the best way to get my bath cold and keep it cool?

In most cases the use of crushed ice in the ice tray is quite satisfactory. Alternatively, the use of plastic encased water cubes may be used. However, there may be instances, where an exact experimental temperature is required (e.g. some of the newer immunochemical procedures). For that purpose the use of the Vibratome R900 Peltier cooler* or Minichiller with the VT1000 S/VT1200/VT1200 S is recommended. An exact temperature is selected and held +/- 1 °C. *please contact your local Leica representative to clarify whether the Peltier is available in your area.

Do I have to achieve a specific temperature when I chill my bath?

Most users select a temperature of +4 °C. 4 °C is the “natural” temperature and results when storing the buffer on ice prior to sectioning and later when using crushed ice in the ice bath to keep the buffer in the buffer tray cold.

What variables do I have to work with to obtain good sections?

1. Knife forward travel speed, 2. Amplitude, 3. Blade angle, 4. Bath temperature, 5. Section thickness The “Rule of thumb” is that softer tissues require slower forward speed, higher amplitude and frequency, steeper blade angle and cold buffer.  For fresh tissue a knife travel speed below 0.1 mm/s is recommended. The amplitude should be between 1 and 1.5 mm for the VT1200. The frequency is fixed at 85 Hz (+/- 5Hz). For fixed tissue the knife travel speed can be faster (up to 0.5mm/s) and amplitude can be lower (between 0.6 – 0.8 mm amplitude). Sectioning of fresh or fixed tissue is always easier when the specimen is cold by using cooled physiological buffer. The selection of the section thickness depends on the purpose of the study. When sectioning fresh tissue for patch-clamping, the section thickness should not be selected too thin, (must be more than 30 µm) otherwise all cells would be damaged. Most users select a section thickness between 100 and 300 µm. Fixed tissue can be sectioned thinner, the tissue is firmer due to the fixation and living cells are not needed for recording anymore.

Are there Thickness/Thinness limits for sectioning?

The purpose of a Vibratome is to produce sections of fixed or unfixed tissue – without embedding in paraffin or freezing. Due to this fact, the specimen is rather soft and thus the section thickness cannot be as thin as when sectioning harder and stiffer material. One of the main applications of the VT Series is to produce sections for neurophysiology of living tissue. For the patch-clamping technique, undamaged whole cells are needed. The diameter could be 30 µm or more. If the section thickness would be chosen too thin, no cell would be intact. Most users select thicknesses of about 100 – 300 µm for living tissue, fixed tissue, because it is stiffer, can be sectioned down to 10 – 20 µm, this however depends on the type of tissue. Thicknesses as prepared with a rotary microtome or cryostat of 2 – 3 µm cannot be achieved with a vibrating blade microtome.

When is it necessary to embed tissue?

Embedding in Agar: Agar or Agarose is recommended for very small and soft specimens. The embedding has no effect on the specimen itself, it can also be used for fresh tissue. The embedding gives support to the specimen and avoids pushing away the specimen by the vibrating blade. To avoid heat artefacts Agarose with a low melting point of 37 °C is recommended.

Are there recommendations for embedding media available?

2.5 % Agar–Agar or for heat sensitive specimens (fresh tissue) 2.5 % Agarose with a low melting point of 37 °C.

Are there daily maintenance recommendations available?

Remove all removable parts from the microtome – use a spray bottle with destilled water and wash all parts thoroughly. Afterwards use 100 % Ethanol. Dry parts on a dry cloth. Wipe control panel with a moist cloth. Please note: VT1200 does not need lubrication.

I am getting “thick & thin” sections, (i.e. first one really thin or nothing, the next one very thick or double what it should be; the next thin, the next thick, etc.) Why? What can I do?

By far the most common cause of “thick & thin” is technique. Refer to all the technique comments. The most common technique problem is too shallow a blade angle when cutting “soft” (e.g. fresh brain) tissue. The correct blade angle is a function of the blade bevel. The consequences of incorrect blade angle are worse in soft or inelastic tissue. Increasing the blade angle will usually solve the problem. Then leave it at the new angle, even when going back to harder tissue. Check whether the specimen is properly glued onto the specimen disc with cyanacrylate glue. Check that the specimen is not too high, especially when the specimen is very soft and small. Do not section at a too high knife travel forward speed. A too high cutting speed will push the specimen away and thick & thick sections are the result. To keep the specimen firm cooling of the physiological buffer is recommended while sectioning. Embedding of the specimen in 2.5 % Agarose with a low melting point to avoid heat artefacts might also help. In case all these tips are not helping call service and have the feeding mechanism of your VT checked.

Are there preferred types of blades and why would you select a certain type?

There are 4 different types of blades available: razor blades, injector blades and sapphire blades. In most cases razor blades give a satisfactory sectioning result. They have the advantage of being reasonably priced. In the VT1200 S they can be inserted as a whole. Injector blades are about 3 times as thick as razor blades. Most customers prefer to use the thinner razor blades. Razor and injector blades are made out of metal and some customers might not like the direct contact of metal with the tissue. In case users would like to avoid metal blades in contact with the tissue, a sapphire blade is recommended. Sapphire blades are more expensive, but if handled properly can last very long, as long as no preparation tool hurts the sapphire. Sapphire blades can be re-sharpened.

Are the magnets strong enough to hold specimens in place?

The cutting forces of a vibrating blade are very little. The force of the magnet is strong enough to hold the specimen disc in place.

VT1000 S

Do I have to achieve a specific temperature when I chill my bath?

Most users select a temperature of +4 °C. 4 °C is the “natural” temperature and results when storing the buffer on ice prior to sectioning and later when using crushed ice in the ice bath to keep the buffer in the buffer tray cold.

What variables do I have to work with to obtain good sections?

1. Knife forward travel speed, 2. Amplitude, 3. Blade angle, 4. Bath temperature, 5. Section thickness The “Rule of thumb” is that softer tissues require slower forward speed, higher amplitude and frequency, steeper blade angle and cold buffer.  For fresh tissue a knife travel speed below 0.1 mm/s is recommended. The amplitude should be between 1 and 1.5 mm for the VT1200. The frequency is fixed at 85 Hz (+/- 5Hz). For fixed tissue the knife travel speed can be faster (up to 0.5mm/s) and amplitude can be lower (between 0.6 – 0.8 mm amplitude). Sectioning of fresh or fixed tissue is always easier when the specimen is cold by using cooled physiological buffer. The selection of the section thickness depends on the purpose of the study. When sectioning fresh tissue for patch-clamping, the section thickness should not be selected too thin, (must be more than 30 µm) otherwise all cells would be damaged. Most users select a section thickness between 100 and 300 µm. Fixed tissue can be sectioned thinner, the tissue is firmer due to the fixation and living cells are not needed for recording anymore.

Can the Vibrocheck be used on the VT1000 S ?

No.

Are there recommendations for embedding media available?

2.5 % Agar–Agar or for heat sensitive specimens (fresh tissue) 2.5 % Agarose with a low melting point of 37 °C.

Are there daily maintenance recommendations available?

Remove all removable parts from the microtome – use a spray bottle with destilled water and wash all parts thoroughly. Afterwards use 100 % Ethanol. Dry parts on a dry cloth. Wipe control panel with a moist cloth. Please note: VT1200 does not need lubrication.

Are there preferred types of blades and why would you select a certain type?

There are 4 different types of blades available: razor blades, injector blades and sapphire blades. In most cases razor blades give a satisfactory sectioning result. They have the advantage of being reasonably priced. In the VT1200 S they can be inserted as a whole. Injector blades are about 3 times as thick as razor blades. Most customers prefer to use the thinner razor blades. Razor and injector blades are made out of metal and some customers might not like the direct contact of metal with the tissue. In case users would like to avoid metal blades in contact with the tissue, a sapphire blade is recommended. Sapphire blades are more expensive, but if handled properly can last very long, as long as no preparation tool hurts the sapphire. Sapphire blades can be re-sharpened.

I know something is wrong but I haven’t a clue as to what or why. What is my best approach?

Check the user manual and follow all instructions. In case you are still not satisfied with the results, please call Leica Microsystems Technical Service.

Are the magnets strong enough to hold specimens in place?

The cutting forces of a vibrating blade are very little. The force of the magnet is strong enough to hold the specimen disc in place.