Energy filtered STEM imaging of thick biological sections

Abstract

Oak Ridge, TN 37830, U.S.A. KEY WORDS. STEM, energy filtered images, biological thick sections, EELS. S U M M A R YEnergy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection setup. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in predicting the quality of thick specimen images formed with energy filtered electrons.It is known that increasing specimen thickness leads to a decrease of the zero energy loss intensity and an increase in higher (multiply scattered) energy loss electrons. We show that contrast decreases gradually with increased energy loss but, most important, the signal to noise ratio is maximal at an energy loss position slightly below the intensity maximum. This is the optimal position for imaging thick specimens. Moreover our studies confirm that the following parameters have similar effects on the energy loss spectra: (1) increased thickness (t); ( 2 ) higher average Z number elements (or lower mean free path Ai); and (3) lower primary voltage (VJ. I N T R O D U C T I O NCell biological organelles are usually asymmetric in three dimensions and traverse distances of several microns. Such structures must be studied by both light and electron microscopy in order to comprehend adequately their three-dimensional organization at various levels of resolution. Thick (i.e. 30.25 pm) embedded and stained sections present special problems to electron microscope imaging, The origin of the difficulties lies in the nature of the interaction process of the high energy primary electrons with the specimen. Both elastic and inelastic scattering cross-sections are relatively high (of the order of =lo-'* cmz) and consequently the mean free paths are short (typically = 100 nm for A, and A, in organic substances). As soon as the section thickness is of the order of a few hundred nanometres to the micron range, multiple scattering occurs so that the outgoing beam at the specimen exit surface is composed of a broad electron distribution both in energy (energy losses ranging to a few hundred electron volts) and in angle (scattering angles of a few tens to one hundred milliradians). The result is a degradation in intensity, contrast and resolution in an conventional mode where all these effects are combined as a net limitation in penetration power.In order to overcome this inherent penetration limit of electrons through matter, several solutions have been put foward and investigated over the last twenty years. The first is the use of higher primary voltages advocated by Dupouy (Dupouy, 1968;Dupouy et al., 1970;Favard et 0 1989 The Royal Microscopical Society 1 2 C. Colliex et al. al., 1971), with the Toulouse 1.2 and 3 MeV microscopes. They clearly demonstrated an increase in penetration power and an improvement in resolution for thick sections, due to the reduction in chromatic error. Since that time...

Three-Dimensional Aberration-Corrected Scanning Transmission Electron Microscopy for Biology

“…In tilt-series transmission electron microscopy (TEM) the best obtainable resolution is 3 nm at a dose of 20-80 e À =Å 2 ; often the resolution is worse (5-20 nm) and the resolution determination itself is not trivial [26,[36][37][38][39][40]. For samples thicker than 100-200 nm other limiting factors are beam blurring and defocusing effects, which can be partly solved by energy filtering [41][42][43] and through the use of high voltages. Examples of 3D reconstructions obtained with tilt-series TEM are those of muscle actinin [44], the work on the Golgi complex (see Figure 13.3) [45], the structure of the nuclear pore complex [46], and the visualization of the architecture of a eukaryotic cell [41].…”

“…Fourth, phase contrast is very sensitive to inelastic scattering, which is problematic especially for thick samples. Highquality images of biological samples are, therefore, sometimes recorded using an image energy filter, such that only elastically scattered electrons are used to form an image [41][42][43].…”

“…More rigorous calculations of scattering cross sections [78], led to quantitative means for determining molecular weights [79][80][81], and to an optimized combination of the different detector signals to eliminate the effect of variation of the sample thickness in the field of view of an image [82]. Several STEMs are equipped with an EELS [60] that are used to investigate the inelastic scattering at low angles, for example, to reduce effects of sample thickness variations [43,83]. EELS has been widely used in materials science to provide chemical information of the sample with atomic resolution by recording simultaneous signals for all detectors [84,85].…”

Energy Filtered STEM Imaging at 30kV and Below - A New Window into the Nano-World?

“…This is surprising because BF-STEM by itself has an advantage over TEM: inelastically scattered electrons have less of an effect on the final image quality when compared to TEM. Furthermore, EF (energy-filtered) BF-STEM imaging has been reported as a promising approach [3]. However, EF BF-STEM imaging is handicapped by the requirement of a fast EELS detector: for acquiring a 512 × 512 pixel image in under 1s, an EELS detector capable of 512 × 512 = 262,144 EELS spectra /s is required.…”

Energy-filtering transmission electron microscopy in materials science

“…-When the zero-loss transmission Tfil falls below 10-2 the EELS consists of a broad Landau maximum formed by multiple plasmon losses and convolved ionisation edges. Up to mass-thicknesses x ~ 300 Mg/cm 2the intensity in a bE = 5-10 eV window at the most-probable energy loss is larger than 10-3 of the incident intensity and can be used for most-probable-loss imaging, [26,59,60]. This avoids the very strong chromatic aberration.…”