Statement_of_Work_Titan_Halo_v3.doc

Statement of Work

1. Background

In order to advance our cryo-electron tomography program to the state-of-the-art, the NIDCD seeks the procurement of a cryo transmission electron microscope (cryo-TEM) equipped with a robust phase-plate and fast detector. In brief, the system must have a field emission gun, a range of accelerating voltages up to 300 kV (for thick sample tomography), TEM & STEM modes, a long-life phase plate, a fast 4k x 4k scintillated CMOS camera. The following are more detailed specifications.

All specifications will be measured according to the standard site acceptance test (SAT) document available from the vendor, unless otherwise stated:

2. Specifications

Electron Optical Specifications

Accelerating Voltage. The microscope will have an accelerating voltage of 80 to 300 kV, in continuously adjustable steps. At least two kVs should be supplied pre-aligned and accessible as simple recall from stored conditions.

Field Emission Source. The microscope must employ a high-brightness, Schottky field emitter source with a brightness of at least 7.5 x 107 A/m2 sr V. Beam current shall be at least 2 nA in a 1 nm probe at 300kV measured according to the manufactures standard site acceptance test (SAT).

Coherence and Instrumental Resolution. Point-to-point resolution, under SAT conditions will be 0.25nm. STEM resolution under SAT conditions will be <=0.23nm.

Using a sample of thin (50-100 Å) carbon film, Thon rings of significant intensity are to extend to at least 3.0 Å (and in all directions) in Fourier transforms of micrographs taken by a direct detection camera under typical cryo-electron microscopy conditions (at a magnification corresponding to a ~1 Å pixel size, with a half second exposure, and a total micrograph dose of ~15-20 e-/Å2, and a defocus of about 1µm). This resolution must be achieved with and without the phase plate.

Phase Plate. The microscope will come equipped with a Volta phase plate located in the back focal plane of the microscope’s objective lens and produce a tunable phase shift of nominally 90deg for scattered electrons compared to the unscattered beam. The practical application of this under cryo-EM, low dose conditions is to provide high contrast images close to focus (to be demonstrated on site). A Volta phase plate is self-generating and automation should be provided to regenerate phase plates once one is exhausted.

The phase plate design should be a total solution that consists of an aperture holder infrastructure in which a thin film phase plate is mounted in addition to conventional apertures. The phase plate position should be heated to minimize contamination. This combination will allow for a long life time before replacement (at least 4 months in normal usage). The operation of the Phase Plate should be embedded into the main software applications for single particle acquisition and tomography series acquisition with strong linkage to the TEM cameras to ensure an increased information and high quality data output.

Objective Lens. A cryo-Twin objective lens with a large pole-piece gap (>=11mm) should be provided for enhanced contrast.

Parallel Illumination. A full three-condenser electron optical column should be supplied to allow for parallel electron beam illumination over a large range of dose conditions. In addition, all lenses should be of the double-wound variety allowing for constant power dissipation regardless of lens excitation. This ensures maximum stability when changing optical conditions such as those for room temperature standard doses to those of low dose during cryo conditions, as well as major instrument changes such as between TEM and STEM modes and high tension changes. Overall the instrument should allow such changes and be in alignment and operable for sample navigation in <=5 minutes.

Rotation-free Imaging System. The lens system should be configured to prevent image rotation when the magnification is changed, including within the low magnification range.

Free Lens Control. In addition to the stored and automatic electron optics, provision for independent control, including all lenses, stigmators, and alignment coils should also be provided. Once set, these conditions can be saved and recalled.

Apertures. At least 4 apertures should be motor-driven and computer-controlled.

Stage Specifications

Goniometer. The stage must be motorized and computer-controlled, with a eucentric goniometer and have at least ±70° of continuous tilt, unhindered by the pole pieces or anti-contaminator, when using the supplied room temperature or cryo holders. The goniometer must be able to return to within 2 µm of a stored position when recalled by the operator. The stage must have piezoelectric elements which allow an X, Y-motion of 1.25µm with a step size of approximately 20pm that can be used for specimen micro positioning and drift compensation.

Eucentricity. For all holders, eucentricity must be achievable and an in-focus object at X = Y = 0, when tilted across the full tilt range (i.e. +/- 70 degrees) should yield a deviation in the X and Y position of the object of <=1.0 µm. For cryo-holders, this requirement must be met under cryo conditions.

Specimen drift and resolution under cryo conditions Under SAT conditions, the drift rate, without electronic drift compensation, should be <=1.5nm/min at 0deg tilt. Line resolution at alpha-tilt of 0 and +/-45deg will be 0.34nm.

Room Temperature Tomo Eucentricity. After correct holder setup, when tilting +/-70deg, the stage compensations reported in the software should be:

dX=dY<=2um, dZ<=4um When moving through the tilt serious under tomo software control the overall shift in sample position should be <=0.4um.

STEM A fully embedded, native scanning transmission mode (STEM) should be provided within the standard TEM software and hardware, without need for external components such as scan generators and software from third-parties. STEM mode should allow for up to 4k x 4k pixel scans, simultaneously detecting signals from a variety of scattering angles. Four detectors should be provided allowing for BF/DF1/DF2/HAADF signals to be detected simultaneously.

Change in operation from TEM to STEM (and vice versa) should require only a simple push button in the standard TEM user interface software.

Vacuum System Specifications

Vacuum System. The vacuum system should be entirely oil-free, differentially pumped and fully automated and using turbo-molecular, ion getter and dry roughing pumps only.

Anticontaminator. An automatic retractable cryo box anticontaminator must be an integral part of the objective polepiece. It must be easily inserted and retracted by push button computer control. This allows the user to easily switch between cryogenic temperature and room temperature operation. The cryo-box prevents contamination of vitrified cryo-TEM specimens, allowing for long sample life time within the TEM column.

The anticontaminator will be designed to effectively trap extraneous ice contamination. It must not diminish tilt range or resolution in any way. Transmission loss measured according to the SAT under cryo conditions should be <= 10% per 24 hr. The ice contamination rate of a cryo specimen must be less than 5 Å/hour.

3. Additional Hardware and Compatibility

Specimen Holders. At least three holders should be provided for room temperature operation, tomography and cryo-temperatures.

Cameras. The microscope must come equipped with a high dose compatible, CMOS camera for that will allow for alignments, calibrations and high resolution TEM image capture up to 4k x 4k. Frame readout should allow for 4k x 4k images to readout at speeds of at least 25fps.

The microscope shall come equipped an embedded, 4k x 4k direct electron detector (DED) compatible with the included TEM camera and operable by the standard TEM software without the need for additional third party hardware or software. The DED should be upgraded (when available) to the next generation detector (to include, but not limited to, speed improvements and electron counting mode with noise discrimination). Camera performance at 300 kV and a typical dose rate of 10 - 20 e-/pixel/second where Nyquist is the outermost (highest resolution) spatial frequency sampled in the Fourier transform of the image: Detective Quantum Efficiency (DQE) at 0.5 Nyquist ≥ 0.4. A DQE measurement is to be made after installation in the field to show that the measured curve meets or surpasses the DQE curve in the vendor’s literature.

4. Software

Microscope operating software running under Windows 7 will be supplied for the general operation, control, and use of the microscope.

Embedded software from the supplier of the TEM should be supplied to allow automatic acquisition of tomographic tilt series in both TEM and STEM modes. Offline software for processing tilt series data should also be supplied.

Low-dose mode software should be supplied, which employs electromagnetic beam/image shifting to provide search, focus/tracking, and exposure modes along with appropriate beam blanking.

As they become available, the vendor will supply free revisions* and upgrades to their software products required to operate the instrument. This will include existing and future software related to the phase plate and tomography.

*Free upgrades occur for customers under warranty or service contract and are limited to those that do not require concomitant hardware. Further, major integer software revisions will be available at a reduced fee.

5. Measurement of Electron Dose

The conversion efficiency of both embedded cameras will be used along with the provided software to calculate the actual electron dose, which will be verified on site using a faraday cup current measurement and calibrated area measured at the specimen.

6. Future enhancements The TEM should allow for the retrofit of post column image filter or spectrometer for EFTEM or EELS.

7. Installation Items

All items required to operate the TEM will be included, specifically a 15kVA UPS, SF6 leader detector, SF6 and N2 regulators, chiller and compressor.

8. Remote Control

Provided a LAN connection via another computer is supplied, remote operation/diagnostics should be possible. Health monitoring software should also be provided, which will utilize this LAN connection for service diagnostics.