Leica EM GP User Manual

Leica EM GP

Automatic Plunge Freezer for the Bare Grid Technique

The Bare Grid Technique

Many specimens for cryo-TEM can be prepared by immersion
freezing, where a fl uid sample is pipetted onto an EM grid (usually
coated) and the excess removed until a thin fi lm remains, before
plunging into a cryogen such as liquid ethane. The grid can then
be directly transferred under cryo conditions to the cryo electron
microscope (cryo-TEM) for observation. This is the bare grid tech­nique.

The bare grid technique can be used for many types of sample
ranging from biological cell sub units to industrial emulsions.
Imaging macromolecular assemblies, viruses and cells in their
native, hydrated environment in the cryo-TEM is the state-of-the­art technique in electron microscopy, providing maximum resolu­tion with minimal specimen damage.

Although a simple method, it is imperative that the suspension
thickness on the grid can be reproduced and vitreous ice can be
formed, otherwise much time is wasted loading useless samples
into the cryo-TEM. The sample fi lm is only tens to hundreds of
nanometers thick and so can be easily infl uenced by temperature
shifts and humidity prior to freezing. If the humidity is too low then
the fi lm breaks due to it quickly drying. If the sample is adversely
affected by temperature, then the morphology may change before
freezing.

Leica Microsystems has developed a plunge freezer, in conjunc­tion with Dr. Guenter Resch of the IMP/IMBA Electron Microscopy
Facility in Vienna, Austria, to standardize procedures and make
the bare grid technique more reproducible.

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Leica EM GP Form and Function

The Leica EM GP plunge freezes samples into a secondary cryo­gen such as liquid ethane after removing excess fl uid by auto­matic blotting.

After connecting the forceps holding the grid to the Leica EM GP,
an environmental chamber lowers to surround the grid, providing
a protective temperature and humidity controlled environment.

Access ports on both sides of the environmental chamber allow
easy pipetting of solutions and suspensions for both left and right
handed users.

Excess fl uid is then removed by automatic blotting with fi lter pa­per from one side of the grid. Parallel, single sided blotting was
developed to prevent damage to delicate support fi lms. The blotter
touches the complete grid surface in one movement. The grid can
be programmed to automatically turn 180° prior to blotting to allow
fl uid removal from the correct side.

All parameters are displayed on the touch-screen control panel,
where numerous settings can be adjusted and monitored such as
blot time, grid/blotter positioning, temperature, humidity, LN2 level,
and secondary cryogen temperature.

After freezing, the grid is transferred to a pre-cooled grid box in­side a transfer container fi lled with LN
can then be taken to the sample holder of the cryo-TEM to prepare
for loading into the electron microscope.

located in the Dewar. This

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Leica Design by Werner Hölbl

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