Transmission Electron Microscopy (TEM) is an imaging technique that easily yields atomic resolution on typical Materials Science specimens. When applied to Life Sciences, however, one is confronted with several technologic difficulties. Two main issues are a) the low intrinsic contrast of the organic specimens, and b) the radiation damage caused by the very electrons that are being used to create the images. Newly developed direct electron detectors are now revolutionizing the application of TEM in the field of Structural Biology research. They have led to a resolution revolution and will broaden the use of cryo TEM amongst structural biologists in the years to come.
For many years, Structural Biologists have been fighting the limitations of TEM applied to their specific specimens. Besides the fundamental difficulty of specimen preparation (TEM only accepts very thin, solid specimens) the intrinsic problems of contrast and radiation sensitivity had to be overcome. Low contrast calls for the use of an intense electron beam in the microscope, in order to increase the signal to noise ratio. However, the radiation sensitivity of the organic compounds imposes an absolute limit on the number of electrons that can safely be used before destroying the specimen too much. As a consequence, high resolution imaging has to be done under extremely “low dose” conditions, of typically 10 to 20 electrons per square Ångström. The resulting images are extremely noisy and, at first glance, show little or no information. In practice, the images of tens or even hundreds of thousands of individual molecules are required to end up with a high-resolution three-dimensional reconstruction (image courtesy of James Conway, University of Pittsburgh). Improvement of the signal to noise ratio in the images can only be obtained by maximizing the sensitivity of the detector, while keeping its contribution to the noise to an absolute minimum.
Well into the 1990s, the only way to record a TEM image was by means of photographic film. After being illuminated, these film plates had to be removed from the microscope, developed in a dark room, and then optically scanned to yield a quantitative image. A breakthrough was achieved when CCD (Charge Coupled Device) technology became available and was applied to electron detection. In CCD based cameras (shown in the image, top), the electrons hit a scintillator, generating light, which is partially captured by fiber optics, and directed onto the cooled CCD chip. This generation of electron detectors dominated the TEM market for more than a decade, with detector sizes eventually reaching even 10 k x 10 k pixels in some cases. One drawback of these cameras was the so-called point spread function: a 300 kV electron has quite a large interaction volume in the scintillator, thus generating light in a region far exceeding an individual pixel on the CCD chip. The best 300 kV CCD cameras had typical pixel sizes of 15 to 30 micron. Also, the two-step conversion from electrons to light to charge build up in the pixels by definition implied a limitation on the achievable signal to noise. To eliminate the light conversion step, several academic groups and companies have developed active pixel sensors, mostly based on CMOS technology, which are capable of capturing and detecting the impinging electrons directly (see image, bottom). The application of these detectors are now revolutionizing TEM in Structural Biology, due to their significantly better performance than CCD cameras. See for example
“The Resolution Revolution”, Science Vol. 343, 28 March 2014.
As part of the Cyttron II project,
FEI Company has developed the Falcon family of direct electron detectors. The first version, Falcon I, already resulted in a huge improvement in performance compared to CCD cameras. The performance of a detector can be expressed by the so-called DQE (Detective Quantum Efficiency), which is a measure of the signal to noise contribution of the detector as a function of spatial frequency. As shown in the image, the blue Falcon I DQE curve is about a factor of 3 better than a typical CCD curve, shown in green. More recently, the Falcon II detector was developed, which was based on so-called backthinning technology. Backthinning means that the backside of the CMOS chip, which is not taking part in the detection of the electrons, is being thinned as much as possible. The DQE curve of the Falcon II detector is shown by the red curve, and presents yet another significant improvement in performance. For more information, please visit the FEI website on Falcon II. Meanwhile, developments are continuing to include the next big improvement: electron counting. With this technology, individual electrons can be detected and assigned to a specific pixel in the sensor. Thus, we can look forward to more exciting TEM results in Structural Biology, moving closer and closer towards the ultimate goal of atomic resolution.