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Cryo preparation system for in vacuum analysis of biological samples

Schematic of cryosystem

Introduction

The Cryo Preparation System has been designed for the in-vacuo preparation of clean, fresh surfaces of biological materials by cryo-fracturing. The system was created to permit subsequent surface analysis of biological samples.

The system is now in use1-3 for analysis of samples by the mass spectral imaging technique known as ToF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry), but is applicable to any other analytical techniques that require ultra-clean sample preparation. Surface analytical techniques require that the prepared sample surface be ultra-clean and remain so during analysis. In conjunction with Professors Winograd and Vickerman and their research groups at Penn State (U.S.A.) and U.M.I.S.T. (U.K.), Kore has designed the Cryo Preparation System to meet these exacting requirements. Click here for a summary of features and benefits of the design, or see details with each module.

The system comprises a number of different modules:

  1. Fast Entry Probe and Fast Entry Lock
  2. Transfer Rod
  3. Cold Fracture Stage
  4. High Resolution CCTV Camera Plus High Resolution Zoom Optics
  5. Preparation Chamber with Cryo shield
  6. Sample Stub
  7. Cold Stage (in the Analytical Chamber)

Modules (1)-(4) are mounted on the preparation chamber (5). Module (7), the sample stage, is mounted in a separate analytical chamber separated from the preparation chamber by a gate valve.

  1. D.M. Cannon, M.L. Pacholski, T.P. Roddy, N. Winograd & A.G. Ewing, Proc SIMS XII, 1999, p.931, Elsevier
  2. R.M. Braun, P. Blenkinsopp, S.J. Mullock, C.A. Corlett, K.F. Willey, J.C. Vickerman & N. Winograd, Rapid Commun. Mass Spectrom. 12 (1998), 1246
  3. B. Cliff, N.P. Lockyer, C.A. Corlett & J.C. Vickerman, Proc SIMS XIII (to be published)
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(1) Fast Entry Probe and Fast Entry Lock

Features Benefits
Water-free, inert atmosphere during transfer from coolant to fast entry lock No ingress of moist lab air; no frosting during time to transfer to fast entry lock
Small volume around sample (5cc) Short transfer time (
Sorption Pump (5 litre volume) Instant pump-out of sample to ~1mbar by virtue of simple 'volume sharing'
Efficient sorption pumping Sorption pumping more efficient at 1mbar and below.
Fast sorption pumping to transfer pressure 10-3 mbar within 1 minute
'Clean' pumping Oil-free pumping system
Schematic of entry lock

The sample probe assembly allows the analyst to immerse the sample stub into the coolant (nitrogen, propane, ethane etc).

Sample preparation using appropriate materials and techniques results in vitreous quenching of the samples: there is no gross disruption of cell material by water crystal formation during cooling.

After reaching the temperature of the coolant, the probe is removed and the cylindrical cup is lowered over the sample stub. The low volume, combined with evolving coolant gas, ensures that a moisture-free atmosphere is preserved around the sample for the short time it takes to transfer the assembly over to the fast entry lock.

The cylindrical cup mates with a receiving cup on the fast entry lock and a vacuum seal is made with a spring-energised lip seal. A valve to the sorption pump is then opened. Volume sharing (5cc : 5litres) alone produces an instantaneous reduction of pressure to 1mbar, and then sorption pumping in the next minute results in a pressure of ~10-3 mbar.

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(2) Transfer Rod

Features Benefits
Magnetically coupled all-metal probe No seals; high vacuum maintained

Schematic of transfer rodPhoto showing stub in entry probe

The 'Transfer Rod' allows the transfer of the sample stub from the fast entry probe onto the transfer rod.

The fast entry probe is then withdrawn and the entry gate valve is then closed. At this point the sample stub is in the preparation chamber on the end of the transfer rod. The sample is subsequently transferred to the fractured stage parking facility or fracture facility, or onto the cold stage in the analytical chamber. The preparation chamber is separated from the analytical chamber by a gate valve.

There is no active cooling at the end of the transfer rod; a thermal insulating break minimizes the heat flow during the transfer stage.

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(3) Cold Fracture Stage

Features Benefits
In-vacuo cryo-fracturing of biological samples Preparation of ultra-clean fracture surfaces from biological samples
3 independently cooled regions: fracture position, cold parking, fracture 'knife' Can raise local temperature for fracture without affecting parked samples
Active control of sample temperature during fracture to ± 1°C Ability to control the fracture temperature precisely and repeatably
Differential temperature shielding to prevent gas condensation on freshly prepared surfaces Preparation of water-free fracture surfaces from biological samples
Ability to introduce/store two sample stubs while conducting analysis on a sample Higher throughput of samples per day
Active cooling of stored samples to -180°C Safe, clean and cold storage of samples prior to fracturing
Motorised manipulator capable of moving at ~12mm/sec Rapid exchange/processing of samples

Schematic of fracture stage

The cold fracture stage: this unit has two separate functions:

  1. To allow cryo-fracturing of the sample at a precisely controlled temperature
  2. To allow the user to load and store up to 2 samples in a cold 'parking area'

The fracture stage unit is LN2 cooled, and can be maintained at -180°C. The whole unit moves in and out to allow samples to be parked in the 'parking area', picked up for placement in the fracture unit, or transferred (following fracture) into the sample analysis chamber.

ToF-SIMS researchers have found empirically that water-containing samples should be prepared by fracturing at between -105°C to -110°C. If the temperature is too cold then subsequent surface spectra are dominated by water molecules. Too high a temperature appears to allow excessive mobility of surface species leading to smearing of surface features. Thus, accurate control of the sample temperature during fracture is essential.

During fracturing a large pressure burst is observed. Condensation of this water back onto the sample will tend to obscure the freshly fractured surface. To minimise this, both the 'knife' and the surrounding material of the fracture stage are held at -180°C during fracture. Also, the inside of the preparation chamber is lined with a cryo shield.

The fracture tool itself is specifically for the preparation of fresh surfaces on biological materials. It too is actively cooled with LN2. The sample usually comprises a layer of biological/soft sample material sandwiched between two flat plates (Si wafers, copper plates, etc), and it is this 'sandwich' that is cleaved apart by the fracture tool. The fracture tool is not strictly a knife. The tool is really a 'finger' that can be hooked under the top plate in order to pull it free, thereby causing fracture.

Once the sample has been fractured, the sample stub is picked up again on the transfer rod and transferred through the analytical chamber gate valve and onto the cold stage.

Photo of fracture stage

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(4) High Resolution CCTV Camera and High Precision Optics for Sample Observation During Fracturing

Features Benefits
Variable Zoom Optics >3mm to 450µm field of view
High lateral resolution 2-3 µm at high magnification
Colour Greater contrast

Schematic of viewing system

To allow the analyst to position the fracturing tool precisely, a high resolution CCTV camera is mounted on an optically flat glass window on the preparation chamber. The zoom camera covers a field of view from >3mm to ~450µm. At high magnification, the lateral resolution is ~2-3µm.

Sample illumination is achieved using a halogen lamp with an integral dichroic reflector assembly mounted externally on the preparation chamber (not shown).

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(5) Preparation Chamber with Cryo Shield Assembly

Features Benefits
Custom-designed preparation chamber Allows integration of the various cold fracture modules
Turbo molecular vacuum pumping High capacity pumping: ideal for biological/high surface area samples
LN2 cooled cryo-shield assembly Active trapping of water and other evolved gases produced during fracturing

Modules (1)-(4) are mounted on a sample chamber which features a turbo molecular pump and an inner cryo shield assembly (shown in blue below). The cryo shield assembly is LN2 cooled and has various openings into which the cold fracture modules are inserted. By maintaining the cryo shield at liquid nitrogen temperature, researchers have observed a significant reduction in the magnitude of the pressure bursts during fracturing of biological material. This results in cleaner, fractured surfaces.

Schematic of chamber arrangement
Photo of shield, optics and fracture stage

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(6) Sample Stub

Features Benefits
On-stub thermometer Precise measurement of sample temperature
On-stub heater cartridge option Additional fine control of sample temperature and also above ambient heating to ~100°C
OHFC copper construction Allows for excellent mechanical and hence thermal contact

Schematic of stubPhoto of stub

The sample stub designed by Kore has powerful features such as on-stub thermometry and on-stub heating in an OHFC copper block. It has a sample mounting surface area of 22mm x 22mm. In order to accommodate heating and thermometry the stub is 16.5mm deep. Naturally, the stub has been designed to fit both the fracture stage and the analytical cold stage designed by Kore. For fitting a cryo preparation system onto an instrument with an existing stage design, or for other questions concerning stub and/or stage modification please contact sales.

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(7) Cold Stage

Features Benefits
Active LN2-cooled sample block Permits analysis of otherwise volatile materials in a vacuum
2 separate cooling circuits Permits a cold plate near the sample to be held at lower temperature than sample
On-stub heater cartridge option Additional fine control of sample temperature and also above ambient heating to ~100°C
On-stub thermometry Precise temperature measurement
Very stiff mechanical design Permits high resolution imaging (minimise drift during data acquisition)
X, Y, Z motion stage: ±12mm on all axes Full analytical coverage of the 22mm x 22mm sample stub
Pneumatic sample clamp Ensures good thermal contact (<80 K on stub). Released for easy sample exchange
Option for digital micrometer readouts Precision positioning
Photo of cold stage

Kore has designed a number of high-precision sample stages for operation in UHV environments. For matching the cryo preparation system, we have designed an X, Y, Z stage with ±12 mm of motion in all directions. The stage fits on to a 6" flange and is designed to maintain samples cold for surface analysis of otherwise volatile materials. Using on-stub thermometry and control of the LN2 flow, the sample can be held at any temperature from −180°C to room temperature to within ±1°C. The X, Y and Z motions are achieved through manual, rotary motion, and an option exists to equip the drives with digital micrometer readouts for greater positioning precision.

If you would like to discuss custom requirements for stages please contact sales who will be happy to discuss your specific needs.


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Last updated: Tuesday, September 06, 2005, 17:53

© Kore Technology Limited 2005