Leica Microsystems EM ACE600 Application Note

Brand: Leica Microsystems

Model: EM ACE600

Type: Application Note

Leica Microsystems EM ACE600 is a versatile, easy-to-use carbon coater for a wide range of applications in the life sciences and materials science. The EM ACE600 enables high-quality, reproducible carbon coating for improved imaging and analysis in transmission electron microscopy (TEM). With its unique combination of flexibility, accuracy, and reproducibility, the EM ACE600 is the ideal solution for preparing ultra-thin carbon films for TEM specimen preparation.

Key features of the Leica Microsystems EM ACE600:

Adaptive carbon thread evaporation: Ensures precise and flexible carbon coating, enabling the preparation of ultra-thin carbon films with high accuracy and reproducibility.

Key features of the Leica Microsystems EM ACE600:

Adaptive carbon thread evaporation: Ensures precise and flexible carbon coating, enabling the preparation of ultra-thin carbon films with high accuracy and reproducibility.

Application Note

Ultra-Thin Carbon Films

related instruments: Leica EM ACE600

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Ways to Reveal More form your Samples:

Ultra-Thin Carbon Films

Much of the battle involved in obtaining good transmission electron

microscopy data is in the specimen preparation itself. Even though

some nanomaterials are already electron transparent (e.g.

nanoparticles and proteins) and often do not require further thinning

procedures, they need to be dispersed onto thin support ﬁlms.

Carbon ﬁlms provide a continuous electron transparent support

enabling the analysis of specimens without excessive interference

from underlying support material. The quality and characteristics of

such carbon support ﬁlms have a major inﬂuence on the analysis,

whether studying nanomaterials or biological nanostructures. This

application note describes the process of preparing ultra-thin carbon

ﬁlms in a reproducible manner. These support ﬁlms can be used for

a wide variety of nanostructures and allow to obtain a complete

insight into the specimens being studied.

CARBON FILMS: HOW TO DEFINE 'QUALITY'?

Carbon ﬁlms are preferred because of their mechanical strength,

conductivity and thermostability. The preparation of ultra-thin

carbon ﬁlms in a straightforward manner can be a laborious task

owing to the non-reproducible characteristics of conventional

carbon deposition methods. The structure and quality of the carbon

ﬁlms are governed by the evaporation characteristics (evaporation

mode, vacuum level, evaporation rate, work distance and

temperature). Before going into detail on these parameters or on the

evaporation of high quality amorphous carbon ﬁlms, it is essential

to deﬁne the term ‘quality’ and hence to discuss which criteria the

carbon support ﬁlms should fulﬁll in order to be suitable for a broad

range of electron microscopy applications.

• High transparency to electrons: the thickness and density of the

support ﬁlm has an important effect on the contrast and

resolution of the image. When mass thickness is comparable to

that of the specimen the ﬁlm can attenuate the intensity of

structural details in the image.

• Adequate strength to withstand electron bombardment

• Uniform thickness. Film thickness is crucial for analytical

investigations, quantitative imaging, or electron tomography.

• Free of any intrinsic structures, surface irregularities and

contaminants

• Conductive, in order to prevent the accumulation of charges

• Easy to prepare and reproducible

Nevertheless, obtaining such carbon ﬁlms is only the ﬁrst step in the

process of making a suitable TEM specimen. In a second step, the

sample needs to be applied on the carbon support ﬁlm. Samples are

usually dispersed in water or a solvent. Unfortunately, dispersions

in solvents often contain remaining reaction products (e.g.

surfactants, capping agents …) which are hard to eliminate and are

a source of contamination. Even though the carbon ﬁlms were clean

prior to the application of the sample, the sample itself introduces

contaminants which can diffuse across the carbon surface to the

area of interest where they locally decompose and polymerize under

the electron beam. This carbon build-up results in poor signal

strength. Obviously, the carbon support ﬁlms should fulﬁll some

additional criteria when used for TEM specimen preparation:

• If the sample is dispersed in water, the support ﬁlm should be

rendered hydrophilic using a glow discharge or mild plasma

treatment. Such treatments are essential in order to disperse

nanomaterials or biological structures more evenly and may

not damage the carbon ﬁlm.

• The support ﬁlm should be mechanically stable because it can

undergo excessive handling during a speciﬁc protocol.

• The support ﬁlm should withstand other post-treatments e.g.

high vacuum heating in order to remove contaminants

Taking the above mentioned criteria into account, it is clear that a

trade-off exists between thickness and stability. Recently graphene

is used as a support ﬁlm, however coating a complete holey carbon

ﬁlm with graphene can be challenging and in terms of stability (e.g.

300 kV measurement, increased acquisition times, excessive grid

handling…), ultra-thin carbon ﬁlms are preferred. To obtain a good

stability and charge dissipation, the ultra-thin carbon ﬁlm should be

supported onto a holey carbon ﬁlm, Quantifoil or a mesh grid (> 600

mesh grid). Unsupported ultra-thin ﬁlms will show local deviations

from planarity and can break during electron beam irradiation or

handling.

Brieﬂy, two steps in the preparation are crucial:

(1) obtaining a ‘high quality’ thin carbon ﬁlm on a substrate and

(2) subsequently transferring this ﬁlm onto a suitable support

structure.

EVAPORATING ULTRA-THIN FILMS ON A SUBSTRATE

Several approaches are used in order to prepare ultra-thin carbon

ﬁlms. Typically, a 5 nm layer of amorphous carbon is evaporated

onto a polymeric support ﬁlm (Formvar, Butvar, Collodion, ...) and

subsequently only the polymeric ﬁlm is dissolved. This method,

however, introduces wrinkling of the remaining carbon ﬁlm.

Additionally, not all of the polymer is dissolved causing the ﬁlm

to be not uniform or clean. The most suitable and thinnest ﬁlms

can be prepared by evaporating a carbon ﬁlm directly onto freshly

cleaved mica. This ﬁlm is released in water and transferred onto

a Quantifoil or holey carbon ﬁlm. The supporting structure is

necessary in order to improve the stability and charge dissipation

and limit vibrations during imaging.

Amorphous carbon ﬁlms can be obtained through different carbon

evaporation modes. Arc-evaporation of pre-shaped graphitic rods

is the most widespread technique to obtain amorphous carbon

ﬁlms. Macroparticles associated with the arc process are often

seen in the deposited ﬁlms and result in increased thickness

irregularity. Another disadvantage is the high temperature during

evaporation (radiant heat), which can damage sensitive samples,

result in wrinkled carbon ﬁlms but more important in larger average

sizes of the carbon clusters in the ﬁlm. The large pressure rise

during evaporation even further contributes to the formation of

these clusters (increase in granularity). As a result a severe

surface roughness is observed in ﬁlms with a thickness of 3 to 5

nm. The large heat also affects the frequency of the oscillating

quartz crystal, making accurate and reproducible layer deposition

difﬁcult. Moreover, the high energetic evaporated carbon species

impact the sample, causing a strong adherence which makes

releasing the carbon ﬁlm from the mica substrate difﬁcult.

Obviously carbon rod evaporation is not the most optimal method

to produce smooth ultrathin 3 nm carbon ﬁlms in a reproducible

manner.

ADAPTIVE CARBON THREAD EVAPORATION:FLEXIBLE,

ACCURATE AND REPRODUCIBLE

Adaptive carbon thread evaporation enables a precise and ﬂexible

carbon coating of which its applicability far exceeds that of the

ultrathin carbon ﬁlms shown in this application note. Its

unparalleled ﬂexibility originates from its unique adaptive process

and multi-segment conﬁguration. However, not only the evaporation

mode but also the underlying electronics are crucial, especially

when evaporating the thinnest ﬁlms where uniformity and accuracy

are of the outmost importance. The ultra-thin carbon ﬁlms are

obtained through multiple evaporation. The multi-segment

conﬁguration allows to evaporate carbon from 4 carbon thread

segments. This enables the use of thin carbon threads and results

in the highest accuracy as all layers are build up sequentially

through adaptive pulsing.

Between each pulse, the thickness of the carbon ﬁlm and properties

of the remaining carbon thread segment are measured in order to

adapt the parameters for the next pulse. The high vacuum (5x10-6

mbar) and stable vacuum during evaporation allow the deposition

of uniform ultra-thin carbon layers. The deposited ﬁlms are free of

any intrinsic structures, surface irregularities and contaminants.

Moreover, radiant heat to the sample is decreased considerably. In

the image shown above, consecutive layers were deposited on a

ﬁlter paper (from left to right: 1, 2, 3, 4 and 5 nm). Carbon thread

evaporation is also uniform over a large area (see images below).

A round ﬁlter paper of approximately 10 cm is shown before (A)

and after (B) carbon coating.

Even more important is the combination of ﬂexibility and

reproducibility. Properties of amorphous carbon ﬁlms are strongly

dependent on the evaporation characteristics as e.g. evaporation

rate, vacuum, mode used etc. Once an evaporation protocol is

optimized and stored, reproducible results can be expected coating

after coating, without surprises. This reproducibility is crucial,

considering the fact that carbon coating is always a part of an

elaborate specimen preparation workﬂow.

LNT Application Note - ULTRA THIN CARBON FILMS

ULTRA-THIN CARBON FILMS: PROCEDURE

A A clean glass petri dish is ﬁlled with ultra-pure water.

B A ﬁlter paper is placed in the bottom of the petri dish and

Quantifoils or holey carbon ﬁlms are carefully placed on the ﬁlter

paper.

C Using the adaptive carbon thread evaporation mode, a 3 nm

layer of carbon is deposited onto freshly cleaved mica at a vacuum

level of 5x10-6 mbar. Next, the carbon coated mica foil is lowered

slowly into the water at an angle of 30 degrees. Water will

penetrate the space between the carbon ﬁlm and mica surface

through capillary forces and the ﬁlm is released.

D The ﬁlter paper is slowly withdrawn out of the water assuring

that the square carbon ﬁlm is deposited onto the grids.

E The ﬁlter paper is transferred onto a new ﬁlter paper in order

to drain most of the water before drying it on a hotplate of 40

degrees for 10 minutes.

F The ﬁlms are ready to be used.

In contrast to the supporting Quantifoil ﬁlm (see image F) the

ultrathin ﬁlms covering the holes are impeccably clean and

uniform. The best procedure to prepare a TEM specimen by

applying a dispersion of any sample is to hold the grid with a

tweezer and apply a small drop of approximately 5 µl. Next, the

excess is blotted away by touching the edge of the grid on a ﬁlter

paper. If required the ultra-thin carbon ﬁlm can be glow discharged

or plasma cleaned (low power and only 5% oxygen for

approximately 30 seconds). Below the difference can be seen

between a conventional carbon ﬁlm (left, 15 nm carbon) and an

ultra-thin carbon ﬁlm (right, 3 nm). The lattice of the CdSe quantum

plates can be easily observed in the right image.

Images acquired by Eva Bladt and Sara Bals (EMAT, University of

Antwerp)

Courtesy of: Frederic Leroux and Jan de Weert

Sample courtesy of: Daniel Vanmaekelbergh,

Debye Institute for Nanomaterials Science, University of Utrecht

Leica Microsystems EM ACE600 Application Note

Brand: Leica Microsystems

Model: EM ACE600

Type: Application Note

Key features of the Leica Microsystems EM ACE600:

Adaptive carbon thread evaporation: Ensures precise and flexible carbon coating, enabling the preparation of ultra-thin carbon films with high accuracy and reproducibility.

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Leica Microsystems EM ACE600 Application Note

Leica Microsystems EM ACE600 Application Note

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