Heavy Atoms

2. Detection of Heavy Atom Labels STEM is one of the few electron microscopes in the world that can image single heavy atoms. The microscope is suitable to visualize tiny gold clusters in biological macro molecules.

Gallery of antibodies labeled with 1, 2,... undecagold clusters. From: J.F. Hainfeld, Microscopy: The Key Research Tool, Vol. 22(1), 91-93 (1992).

11-gold-atom clusters attached to antibodies. Single 1.4nm gold clusters on antbodies

3. Additional strengths of the STEM include:

Intuitive Imaging

The contrast transfer function (CTF) of the STEM annular detector signal is very intuitive: signal intensity increases monotonically with specimen thickness and does not oscillate. Defocused images are blurry with none of the fringes or contrast reversals encountered with normal phase contrast TEM imaging. Therefore any feature seen reproducibly in STEM images must be generated by some feature in the specimen itself.

Quantitation

The STEM image, besides being intuitive, is also quantitative. Image intensity is directly proportional to the local mass per unit area in the corresponding region of the specimen. Given absolute scattering cross sections or a calibration specimen of known mass, intensity in the STEM can be integrated over an isolated particle, a known length of filament, or any arbitrary area and converted to a molecular weight. This provides a direct link to biochemistry if total or subunit molecular weights are known. The homogeneity or variation in mass from one object toanother may be interesting in itself or can provide a further check on the quality of the specimen. Internal details are faithfully rendered in the projected mass distribution. For example the tobacco mosaic virus (TMV) radial mass profile of control particles (included in most STEM specimens) provides a sensitive measure of the quality of specimen preparation, as well as the extent of radiation damage. Fall-off of the signal due to Beer's Law (multiple scattering) in thick specimens is easily corrected point by point in a look-up table, making linear measurements possible for viruses and other objects 0.25µm thick or larger.

Low Dose

The STEM optics, illustrated at left, provide both high resolution and efficient collection of scattered electrons. The dark field annular detector signal typically comprises more than 80% of the elastically scattered electrons compared to <5% for tilted beam dark field in a conventional transmission electron microscope. Furthermore, the STEM scintillator detectors are directly coupled to photomultipliers and provide high quantum efficiency and linearity. The STEM scan is computer controlled so that the operator viewing a low magnification and low dose image can select sub-areas of interest for focusing and data recording. This guarantees minimum pre-irradiation of the sample before recording data. We strive to keep the total specimen dose below 1000 electrons/nm².

High Contrast

The mass thickness of the specimen plus substrate relative to the 2nm thick substrate alone defines the image contrast. With the STEM it is easy to visualize unstained DNA (double stranded), protein, carbohydrate, single heavy atoms or heavy atom clusters. From simple geometry, it is straightforward to calculate the expected signal to noise ratio (S/N) for any expected specimen feature and dose. In fact, we obtain excellent agreement between such simulations and experimental data for specimens such as TMV where the structure is known.

High Resolution

Diffraction-limited resolution of 0.25nm is routinely obtained with a cold field emission electron gun operating at 40 keV. Focusing at 2 million direct magnification and high contrast on grain structure in the carbon substrate (in an area adjacent to the area of interest) guarantees that every image will be precisely focused and stigmated.

Clean Background

Freeze drying of purified unstained objects deposited on a 2nm thick carbon film gives a very clean background and eliminates contrast variations due to uneven staining. Any problems with sample breakdown or extraneous material are immediately obvious as amorphous objects on what should be a clean dark background. Deposition and wash buffers can be selected from a wide range of composition, pH and ionic strength, making conformational studies possible without compromising on background quality. One is seldom tempted to over-interpret STEM images of poor preparations and is directed instead to improve specimen purification.

Image Analysis

The STEM image is stored in digital form on the computer disk eliminating the tedium, non-linearities and noise associated with photographic processing and subsequent densitometry. In fact our STEM images are available on separate analysis terminals immediately after scanning and saved to disk. Standard image analysis packages are available (eg. SPIDER) as well as simple programs for mass analysis and automatic particle analysis (compute background, find TMV and calibrate, find specified objects, align, compare to reference particle, measure mass, compute statistics and image-average selected particles).

Heavy Atom Labeling

Since no stain is needed to "see" the molecule or complex, one is free to add heavy atoms in a highly selective manner. The resulting added mass (at low dose) or bright spots (at higher dose) can be compared with the unlabeled control. J. Hainfeld (BNL) and others have developed a battery of 11-gold and 11-tungsten clusters with various specificities or linkers such as Fab antibody fragments. One can label selected sites in an assembly or complex, one part of a multi-component system prior to reassembly, or an active or functional site.

EELS and Signal Combinations

The STEM is ideally suited for electron energy loss spectroscopy (EELS) since the probe is only a few atoms wide. Inelastic (energy loss) as well as elastic scattering events occur as a result of irradiation of a given pixel. Since the elastic signal has a strong "Z" (atomic number) dependence and the inelastic signal has a weak "Z" dependence, one can estimate the proportion of low versus high "Z" atoms in a given pixel. Furthermore, the elastic and inelastic signals can be separated in space using an annular detector and an energy loss spectrometer to give simultaneous signals from the same pixel. Thus, signal combinations can be made point by point with exact registration ("Z" contrast).

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