VCT100. Vacuum Cryo Transfer

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1 VCT100 Vacuum Cryo Transfer

2 VCT100 Versatile Specimen Transfer Key features include: Contamination free transfer Minimum load on analysis tool Resolution of analysis tool maintained Independent preparation and analysis Repeated preparation of same specimen Full availability of analysis tool during preparation 2

3 Contamination free specimen transfer between preparation and analysis Transferring specimens to the chamber of an analysis system is one of the most critical steps of most preparation methods. It is essential to protect the samples from contamination. The VCT 100 concept was established particularly to cross link preparation units with various analysis systems. Samples are transferred in a well defined environment, e.g. protective gas or high vacuum conditions. In addition the specimen can be kept at low temperature for cryo techniques. The sample holders used for specimen transfer are compatible to an integrated stage. Due to the modular stage concept, the stage can be installed in any (ultra) high vacuum system or glove box and used for both room temperature and cryo applications. With the air lock, the transfer shuttle can be mounted to any SEM, FIB, AFM, SIMS or XPS chambers. The flexible system design enables adjustments to any customer specific application. Setup advantages Contamination free transfer between preparation and analysis unit Versatile transfer environment Minimal interference with the analysis system for highest possible resolution Independent sample preparation and analysis Efficient access to the analysis as well as to the preparation unit Preparation units can be linked to several VCT100 adapted analysis units. Preparation and analysis can be performed at different locations Easy adjustment to configuration changes X - Beam FESEM with VCT100 FESEM stage with installed VCT cryo stage 3

4 ESEM with VCT docking station and shuttle X-ray system with VCT docking station on high vacuum loading chamber and VCT cryo dewar on ultra high vacuum chamber. Cryo SEM Lately Cryo Scanning Electron Microscopy (Cryo SEM) has been established as the surface analysis method of choice for wet (soft condensed and hydrated matter) and beam sensitive specimens. Cryogenic sample preparation is quicker than conventional sample preparation techniques like chemical fixation with subsequent dehydration and critical point drying. With the right experimental setup the entire processing of the specimen from object extraction to immobilization (freezing), surface preparation, coating, and imaging can be done in less than one hour. A typical preparation procedure starts with cryo fixation of the specimen using techniques like: High Pressure Freezing with the HPM010 or HPM100, Jet Freezing with the JFD030 or Plunge Freezing depending on the nature of the specimen. The specimen is subsequently transferred to one of the Leica preparation units: SCD500, MED020 or BAF060 using the VCT100 shuttle. In the preparation unit the specimen can be fractured, etched and coated. In the VCT100 shuttle the specimen is then transferred, protected under high vacuum and by a cold trap, onto the cryo stage in the SEM. For the actual SEM analysis the VCT100 shuttle is detached from the SEM. Cryo SEM advantages Physical preparation (no artefacts deriving from chemical treatment) No handling with toxic reagents Sole preparation technique for many applications Beam damage reduction due to low temperature during imaging X-ray system with VCT cold finger and interface for cryo sample holder 4

5 System components The VCT100 concept is highly flexible and comprised of four main components: the loading box, the preparation unit, the cryo stage and the VCT100 shuttle. Shuttle The VCT100 shuttle is equipped with a cold trap and isolating tip. The shuttle keeps the specimen under high vacuum and cryo conditions for a minimum of 20 minutes. Loading box The specimen is loaded under LN 2 onto the specimen holder and then retracted into the shuttle. Since the shuttle is precooled, the specimen is transferred to the preparation unit contamination free without any exposure to air. 5

6 Preparation units Three preparation units are available for cryo SEM sample preparation: the SCD500, the MED020 and the BAF060. The SCD500 equipped with the freeze fracture/etching unit allows mirror fracturing followed by sputter coating for subsequent cryo SEM analysis. The MED020 configured as freeze fracturing/etching and coating system is the main preparation device for cryo SEM. Cryo sputtering is the main configuration to produce thin and uniform metal layers on the specimen surface. Using the optional E-gun module, films may be produced with shadowing effects as well as replicas for TEM analysis. The BAF060 is a fully automatic freeze fracturing machine that is designed for replica production for high resolution TEM investigation. It is also the perfect tool to produce double layer coated specimens for cryo SEM. 6

7 Cryo stage The cryo stage slides onto the standard SEM stage and is connected via copper bands to the LN 2 dewar. The specimen holder with the specimen is transferred onto the cryo stage using the shuttle manipulator. The cryo stage can be used for any specimen with the advantage of improving the vacuum in the chamber and reducing beam damage. VCT100 control The analysis units as well as the preparation units are equipped with a compact, light weight airlock system through which the specimen is transferred. The air lock is pneumatically controlled without causing a magnetic or electrical field in the SEM. The VCT100 control system enables integrated safety communication for interlocks. 7

8 Applications Cryo SEM is an optimal imaging technique for a wide range of applications in life and material sciences. The following examples comprise technical systems such as cement formulation and liposomes as well as several biological objects. Cryo SEM is not exclusively used to access the ultra-structure of an object. This technique can also be used for the analysis and localization of different components (e.g.elements). High pressure frozen, freeze fractured cement suspension. The sample was fractured at -115 C in a BAF060, etched at -105 C for 4 minutes and coated by electron beam evaporation with 3 nm of Pt/C. P Cement particle, E Ettringites. Electron Microscopy ETH Zurich (EMEZ). Specimen courtesy of L. Holzer, EMPA, Swiss Federal Laboratories for Materials Testing and Research, 3D-Mat group, Duebendorf, Switzerland. High pressure frozen, freeze fractured liposome suspension. The suspension was fractured at -150 C in a BAF060 and double layer coated by electron beam evaporation with 3 nm of Pt/C at 45 and subsequently with 6 nm of carbon. Specimen was imaged using backscattered electrons. Electron Microscopy ETH Zurich (EMEZ). 8

9 High pressure frozen, freeze fractured mouse intestine biopsy. The sample was fractured at -115 C in a BAF060, etched at -105 C for 5 minutes and coated by electron beam evaporation with 3 nm of Pt/C. M Mitochondria, N Nucleus, NP Nucleopores, G Golgi, PM Plasma membrane. Electron Microscopy ETH Zurich (EMEZ). Specimen courtesy of René Fischer, Laboratory of Organic Chemistry, ETH Zurich, Switzerland. High pressure frozen, freeze fractured suspension of Vero cells. The sample was fractured at -115 C in a BAF060 and immediately coated by electron beam evaporation with 3 nm of Pt/C. M Mitochondria, N Nucleus, NP Nucleopores, G Golgi, PM Plasma membrane. Electron Microscopy ETH Zurich (EMEZ). Specimen courtesy of Peter Wild, Institute of Veterinary Anatomy, University of Zurich, Switzerland. 9

10 High pressure frozen, freeze fractured mouse brain biopsy. The sample was fractured at -115 C in a BAF060, etched at -105 C for 5 minutes and coated by electron beam evaporation with 3 nm of Pt/C. MS Myelin sheath, NF Neurofilaments. Electron Microscopy ETH Zurich (EMEZ). Specimen courtesy of René Fischer, Laboratory of Organic Chemistry, ETH Zurich, Switzerland. 10

11 High pressure frozen, freeze fractured suspension of yeast cells (bakers yeast). The sample was fractured at -120 C in a MED020, etched at -100 C for 1 minute and sputter coated with 2 nm of Pt. The high magnification image shows the protoplasmic fracture face of the plasma membrane with invaginations and hexagonal particle arrangements. Electron Microscopy ETH Zurich (EMEZ). Wax surface on tulip leaf. A piece of tulip leaf was plunge frozen in liquid nitrogen, etched at -105 C for 5 minutes in a BAF060 and coated by electron beam evaporation with 3 nm of Pt/C. Small wax tubes cover the whole surface of the leaf. Electron Microscopy ETH Zurich (EMEZ). 11

12 Accessories A variety of specimen holders can be supplied with the system. The selection depends on the cryo fixation technique and the specimen. Blank Specimen Holder The specimen is directly glued to the specimen holder surface. Specimen Holder For Freeze Fracturing For plunge frozen and high pressure frozen specimens. To be used with the various gold and aluminium specimen carriers. Specimen Holder For Mirror Fracturing For four plunge frozen or jet frozen sample sandwiches using copper specimen carriers. Specimen Holder For SEM Stubs With fastening screw thread to fix the SEM stub For plunge frozen specimens using gold specimen carriers. For plunge frozen specimens using gold specimen carrier sandwiches of 4.5 mm in diameter. For plunge frozen and propane jet frozen specimens using copper specimen carriers. 12

13 Technical specifications Loading Box LN 2 reservoir 2.5 l for ½ day operation Preparation Units SCD500 Dimensions Metrics 410 mm (W) x 390 mm (D) x 280 mm (H) Weight 11.5 kg Shuttle Temperature Cold trap -170ºC for 20 min Isolating tip Temperature of specimen stage maintained (+0.5ºC per minute in high vacuum) Vacuum High vacuum 2 5 x 10-7 mbar for ~20 min Dimensions Length 580 mm Manipulator transfer length 390 mm Weight 3.6 kg Airlock Weight: 2 kg SEM Cryo Stage Cooling time < -140ºC within 45 min Cooling Capacity ~3 h Cold trap < -170ºC Stage temperature: -140ºC 60ºC Specimen movement Free translation and ± 90º rotation Drift < 2 nm/min Vacuum Pumping System Pumping time Ultimate vacuum Cooling Cryo stage LN 2 filling system LN 2 capacity Sample Preparation Specimen handling Fracturing / Etching Coating Sputter Coating Sputter Control Sputter material Automatically controlled Oil free Membrane pump Turbo molecular pump to < 1 x 10-6 mbar 20 min < 5 x 10-7 mbar Constant temperature control of the specimen between -160Cº +60ºC Specimen protected by Meissner trap Automatic bake out system Manual ½ day operation Via shuttle Mirror fracturing Simultaneous fracturing and sputter coating possible Etching by exact controlled specimen temperature Planar Magnetron Sputtering Permanent sputter coating control by quartz crystal film thickness monitoring (auto termination) Cr, W, Pt Dimensions Metrics Weight 490 mm (W ) x 585 mm (D) x 553 mm (H) Opened 792 mm (H) 87 kg 13

14 Technical specifications MED020 BAF060 Vacuum Pumping System Pumping time < 1 x 10-6 mbar Ultimate vacuum Cooling Cryo stage LN 2 filling system LN 2 capacity Sample Preparation Specimen handling Microtome Fracturing/Etching Coating Sputter coating (standard) Sputter control Sputter material E-beam evaporation (optional) Evaporation sources Angle setting Evaporation techniques Evaporation control Coating material Dimensions Metrics Weight Automatically controlled Oil free Scroll pump Turbo molecular pump 20 min < 1 x 10-7 mbar Electronically controlled by stepper motor Temperature control of the specimen between -160ºC ºC Specimen protected by Meissner trap Automatic bake out system Manual ½ day operation Via shuttle Manually driven for fracturing Automatic knife advance, precision ± 5 µm Etching by exact controlled specimen temperature Planar Magnetron Sputtering Permanent sputter coating control by quartz crystal film thickness monitor (auto termination) Cr, W, Pt Two individual digital programmable electron beam guns Shuttle manipulator Unidirectional, bidirectional Permanent evaporation control by quartz crystal film thickness monitoring (auto termination) Pt/C, Ta/W, Cr 850 mm (W) x 670 mm (D) x 615 mm (H) 60 kg (without accessories) Vacuum Pumping system Pumping time to < 1 x 10-6 mbar 20 min Ultimate vacuum: < 5 x 10-8 mbar Cooling Cryo stage LN 2 filling system LN 2 capacity Sample Preparation Specimen handling Fracturing/Etching Coating Electron beam evaporation Evaporation source Angle settings Evaporation techniques Evaporation control Coating material Dimensions Metrics Weight Automatically controlled Oil free Status displayed by logic mimic diagram Membrane pump Molecular drag turbo pump Rotary cryo stage electronically controlled by stepper motor with variable speed Constant temperature control of the specimen between -180ºC ºC Specimen protected by Meissner trap Automatic bake out system for the entire cooling system Automatic 60 l for one day operation Microtome Motor or manually driven Precision advance ± 1µm Simultaneous fracturing and evaporation possible Etching by exact controlled specimen temperature Two individual programmable electron beam guns In-situ motorised angle adjustment 0º..90º Unidirectional, bidirectional, rotar shadowing, Double Axis Rotary Shadowing (DARS) Permanent evaporation control by quartz crystal film thickness monitoring (auto termination) Pt/C, Ta/W, Cr, W 1170 mm (W) x 885 mm (D) x 1590 mm (H) 300 kg

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