Specimen preparation techniques

for soft-matter materials at LCI TEM Lab

Plunge Freezing

ContactContact Dr. Min Gao (Lab Supervisor) for lab tour or using the TEM for research and teaching purposes at mgao@kent.edu or 330-672-7999.

Lab website: http://www.lcinet.kent.edu/organization/facility/characterization/tem/

fac_TEM_main.html or Google “ LCI TEM” and follow the “lcinet” links.

90°C 80°C 70°C 65°C

Version: 10/2013

Excellence in Action

• Basic idea: preserve the native structure of asoft matter sample by throwing it rapidlyinto cryogen (e.g., liquid nitrogen or liquidethane) manually or using a machine.

• For aqueous samples (for example, manybiological materials and lyotropic liquidcrystals), >104 degree/second cooling rate isrequired for vitrification.

• For very thin specimens, above cooling ratecan be achieved routinely using liquid ethaneas cryogen.

• For non-aqueous samples, slower coolingrate may still lead to good preservation ofthe native structure. It is possible to freezethick samples to make TEM specimens usingcryo-sectioning or freeze fracture.

Introduction• A series of specimen preparation techniques for soft materials

have been established at the LCI TEM Lab.

• Freezing: plunge freezing, and high pressure freezing

• Processing: cryo-ultramicrotomy, and freeze fracture

• Materials we have studied: thermotropic and lyotropic liquidcrystals, biological materials, polymers, and oils.

Microvesicles produced during blood macrophageDifferentiation. Ismail, et al., Blood 121 (2013) 984.

FEI Mark IV: an automatic plunge

freezing machine with controlled

temperature and humidity

High pressure freezing

• Basic idea: To slow down the crystallization ofwater in aqueous samples by applying a highpressure (~2000 times the atmospherepressure). So a much slower cooling rate can beused for vitrification of thick aqueous samples.

• The frozen samples can then be cryo-sectionedor freeze-fractured to obtained TEM specimens.

High pressure freezer (Leica EM Pact2)

Freeze fracture

• Basic idea: A frozen sample is fracturedinside a vacuum chamber at lowtemperature . Supposedly, the tomographyof the fractured surface may represent thenative structure. The fractured surface isshadowed and replicated by depositing Pt/C(or other heavy metals) at an angle (forexample, 45°), and carbon from the top.The real material is then dissolved/removed.The replica is studied by room temperatureTEM.

• Can work for a variety of materials.

Cryo-ultramicrotome(Leica UC7/FC7)

Freeze fracture (BalTec/Leica BAF060)

Thermal

treatment

Plunge freezing

CryogenFreeze

Fracture

Pt/CC

Replication of fractured surface

Copper planchette

Sample

Cryo-ultramicrotomy

• Basic idea: Use a diamond knife to sectionfrozen samples into electron transparentslices which are then collected on carboncoated TEM grids.

• The cryo-ultramicrotome at LCI TEM lab isequipped with a discharge device and amicro-manipulator for challenging materials.

Aqueous sample

Copper tube

P

LN2

Diamond knife edge

Ribbon of thin sections

High pressure freezing

Cryo

ultramicrotomy

Freeze fracture TEM(FFTEM) imagesshowing the 1Dperiodic structure(left) and Bouligandarches (right) in thetwist-bend nematicphase of CB7BC.

Borshch, et al., Nature Communications 2013

Nanometer scale smectic domains in a three-ring bent-core nematic LC material as a function of the quenching temperature. Zhang, et al., Physical Review Letters 109 (2012) 107802.

A scanning TEM (STEM) Z-contrast image showing the distribution of Au nanoparticles in a thermotropic liquid crystal.

Distribution of Au nanoparticles in PDMS. Specimen prepared by plunge freezing and cryo-sectioning. Sample from Dr. Quan Li’s group.

Column structure in a chromonic lyotropicliquid crystal with 70% water. Specimen prepared by high-pressure freezing and vitreous sectioning. Sample from Dr. Oleg Lavrentovich’s group.