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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Leica EM GP Features

Environmental chamber

To provide the best conditions for pre-freezing of a suspension,
the environment surrounding the grid and sample has to be pre­cisely controlled. The environmental chamber envelopes the grid
and forceps, providing a temperature and humidity controlled pro­tective environment variable between +4° and +60°C and room
humidity to 99%.

To provide a clear view, an anti-fogging heater keeps the glass
window clear.

Pipette port
(also on right side)

Blotting

Excess fl uid is removed by automatic blotting which
can be initiated in two ways:

1. Autoblotting – after the suspension has been pipetted onto the
grid and the button pressed, the blotter automatically moves to
the pre-set blot position for the user-programmed time before
plunging of the grid.

2. Sensor control – the most automated method of plunging. After
pressing the plunge button the blotter moves towards the grid. A
photosensor detects the moment the droplet touches the grid and
the blotting time countdown begins. The grid is then either held
for the desired length of time (to allow redistribution of fl uid for
example) or immediately plunged into the secondary cryogen.

Blotting and plunging can be activated via the touch screen or
footswitch.

Magnetic holder
for blotting paper

Covered exit port
to ethane

Forceps with
grid in blotting
position

Blotter

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Viewing system

The Leica EM GP has an optional stereomicroscope to aid align­ment and sample preparation. The LED illumination, fi xed both
inside the environmental chamber and beaming down onto the
Dewar, provides excellent high light levels for observation of the
complete preparation and plunging process.

The Dewar

After switching on the Leica EM GP, the 1 liter Dewar can be fi lled
with LN2 before liquefying the secondary cryogen, usually eth­ane. A full Dewar lasts for approximately 1 hour between refi lling.
Liquefying the secondary cryogen is fast, easy and safe with the
liquefying head. The head is connected to the secondary cryogen
regulator of the gas bottle and the gas slowly fed in. It condenses
within seconds, taking about one minute to fi ll the 2.5 ml container.
A cover is provided to prevent LN2 splashing into the ethane on
subsequent refi lling of the LN2. The temperature of the second­ary cryogen can be controlled precisely from the control panel. A
container, fi lled with LN2, sits in the Dewar to hold a grid box for
transfer of prepared, vitrifi ed samples.

Liquefi er in place over ethane container in Dewar

After freezing the grid remains in or above the ethane (de­pending upon user settings) ready for transfer to the grid box

5

Control Panel

Operation is via touch screen control with all adjustable param­eters visible.

Program screen

All parameters can be adjusted and set for up to 10 programs.In
the Setup menu the positioning of the grid relative to the blotter
can be adjusted and also the transfer position after plunging.

Safety

The Leica EM GP operates under strict safety conditions. During
any movement of the environmental chamber a large red STOP

Main screen

Program in use

Current temperature of
environmental chamber

Current humidity of
environmental chamber

Current temperature of
ethane container

LN2 level

button appears on the control panel. Touching this button will im­mediately stop any movement. An alarm signals when either the
secondary cryogen is too warm and may evaporate or the LN2
level is too low.

Bake-out

At the end of a run the bake-out cycle takes 60 minutes to dry the
Dewar and environmental chamber, which allows a second run
within a short time if the user does not wish to maintain the LN2
level in the Dewar.

Sets start position to
accept forceps

Chamber moves downwards
ready for dispensing suspension

Blotting/plunging sequence

Press to raise grid in ethane and
increase Dewar GN2 production
to keep out moisture

Program screen

Program number

Rotate grid after
applying suspension

Rotate grid before
applying suspension

Countdown for blotter exchange

Plunge automatically
after blotting

Blotting with blot
sensor activated

Delay time
before blotting

Blotting
time

Scroll through
programs 1-5 or 6-10

Delay before
freezing after blotting

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Applications

The Leica EM GP is designed for all EM laboratories with a need
to view vitrifi ed fl uid samples or extremely thin samples in the
cryo-TEM, including biological research, virology, protein crys­tallography, pharmaceutical research, cosmetics and industrial
laboratories.

Samples that can be prepared vary for example from suspensions of
viruses, liposomes, microtubules, proteins and other cellular com­ponents to paint or solutions and emulsions in both aqueous and
inorganic solvents. The Leica EM GP can be used to plunge freeze
samples not only on EM grids for the Bare Grid Technique, but also
sapphire discs and samples in freeze fracture planchettes.

Montage overview of plunge frozen grid.
Note the homogeneity of the fi lm thickness.

Micrographs courtesy of Dr. Guenter Resch, IMP/IMBA Electron Microscopy Facility, Vienna, Austria

Liposomes

Micrographs courtesy of Angela Pickl-Herk, MFPL, Vienna, Austria

Microtubules

Rhinovirus particles on holey carbon fi lm

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Leica EM GP - E - 7/09 Order No. 16222002

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