UltraScan™ 4k x 4k Camera Performs and Inspires Under Cryo Conditions

Cick here to download (PDF) the Gatan - National Cancer Institute (NIH) authored paper entitled "Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies."

The superior performance of UltraScan™ CCD cameras for materials science and general bio-medical TEM applications has been widely recognized and accepted by the TEM community. However, there is a lack of performance data available for cryo-electron microscopy applications.

Cryo-TEM has become one of the fast growing market in recent years. The primary objective of cryo-TEM involves imaging frozen hydrated biological macromolecules with high spatial resolution. One of the challenges in cryo-TEM is radiation damage, i.e. the structures of the frozen hydrated molecules are damaged rapidly under the illumination of high-energy electrons in TEM. Although cooling down samples to liquid nitrogen or helium temperature can reduce the damage, structure degradation still remains the number one challenge that electron microscopists have to face. As a result, cryo-TEM must be performed under extremely low electron illumination condition (low dose). Consequently low-dose images are noisy in nature due to statistical noise from the low electron illumination on sample.

Cryo-TEM applications place stringent specifications on the design of suitable CCD cameras. Under low-dose (hence low signal) conditions, the CCD camera must have enough sensitivity to record structures of interest, and yet negligible readout noise to the overall image quality. In other words, the noise in the image must be illumination limited, rather than CCD readout limited. The typical electron dose used in cryo-TEM applications ranges from 10 - 100 electrons per square angstrom. This of course varies with any particular macromolecule samples in the investigation.

In March, 2003, UltraScan™ 4000 (4-port CCD readout) with ultra-high sensitivity phosphor scintillator was tested on FEI Company's Tecnai Polara cryo-TEM. This TEM is a 300kV FEG with a twin lens equipped with a cryo-specimen transfer system. With this TEM, it is possible to load a sample in the microscope at low temperatures (< -155°C or 118K) and carry out TEM imaging at temperatures < 15K by cooling with liquid helium. The objective for the performance test is to explore the suitability of UltraScan cameras with ultra-high sensitivity for cryo-TEM applications.

To demonstrate the performance of the CCD camera for cryo-TEM imaging, TMV (tobacco mosaic virus) samples were used. This is equivalent to the use of graphitized carbon sample in the demonstration of camera resolution for conventional high-resolution imaging.

TMV contains protein building blocks arranged in a helical fashion. As a result, TMV structures contain various specific layer periodicities, which can be seen in electron microscope images. One of the dominant one is the 23A periodicity. Next is 11.5A and so on. The objective in imaging TMV virus is to investigate the minimum electron dose required to resolve the 23A and 11.5A periodicities. This directly shows the performance of the CCD camera that is easily related to imaging biological macromolecules under low-dose condition.

The TMV test showed excellent imaging capability of both the 23A and 11.5A periodicities by the US4000 camera down to electron dose of 5e-/A2. Images below are cryo-TEM images of TMV and their FFT images showing the 23A and 11.5A periodicities.

The amorphous carbon Thon-ring test shows high image quality for electron dose down to 10e-/A2.

TEM magnification: 59,000x
Electron dose: 10e-/A2

TEM magnification: 59,000x
Electron dose: 5e-/A2

Measurement on the CCD readout noise shows that at 59,000x magnification (used to image TMV virus) for electron dose greater than 0.3e-/A2 the readout noise is negligible.

In conclusion, the UltraScan™ 4000 (4k x 4k) CCD with ultra-high sensitivity phosphor scintillator delivers an excellent performance for cryo-TEM applications at 300kV. The fast acquisition speed (~5 sec readout of 16 million pixels) and superior sensitivity and resolution under low dose conditions make this high performance CCD camera indispensable to any modern cryo-TEM systems.

Acknowledgement: Dr. Felix de Haas (FEI Company) and Mr. Gerald van Hoften (FEI Company) for recording the TMV images and assistance with Tecnai Polara cryo-TEM.