Size Limits of Very Small Microorganisms

Introduction

The search for microbial life in terrestrial and extraterrestrial rocks has recently intensified following the announcement of evidence of past Martian life in a meteorite from Mars [1]. Although there is debate about whether a compelling case has been made for evidence of past Martian life in the meteorite, there is no debate that the evidence exists at the nanometer scale [2,3]. Biomarkers include both organic and inorganic species, although inorganic "biominerals" are perhaps more likely to survive geological processing. Microorganisms that precipitate biominerals during their life cycles can exert control over crystal size, crystallographic orientation, degree of crystal perfection, and morphology. In principle, specific biominerals (e.g., magnetite and Fe sulfides) may be used as indicators of past biogenic activity, providing their properties are significantly different from minerals produced by non-biological processes. Evidence of biomineralization may exist only at the nanometer scale [3]. (Biominerals ~10 nm in diameter and containing less than 10,000 atoms have been observed.) One of the biggest challenges in looking for evidence of past (or present) microbial life in geological samples is to develop and refine analytical methods to probe specimens on a scale comparable to that of the biogenic activity.

Electron Microscopy

Electron microscopy is unique among analytical techniques in that it provides the ability to examine the morphologies, internal structures, crystallography, and compositions of materials at close to atomic resolution. The essential elements of an electron microscope are a high-vacuum column, an electron gun (a thermal or field emission electron emitter), a system of magnetic lenses to focus the electrons before (and after) interacting with the specimen, beam-scanning coils for rastering the electron beam across the specimen. A variety of (electron and x-ray) detectors are available for imaging and spectroscopy.

The two major classes of electron beam instruments are the scanning electron microscope (SEM) and the transmission electron microscope (TEM). (Both instruments have proven useful for studying microorganisms and biominerals). Each instrument exploits a specific electron optical configuration and incident beam energy range that targets it toward certain types of microanalysis. The SEM is used for characterizing the surfaces of thick (electron opaque) specimens (Figure 1). Most SEMS operate in the 2-30 keV range and are configured primarily for imaging (using secondary and backscattered electrons) and compositional analysis (using energy-dispersive x-ray spectroscopy (EDS)). Some SEMs are also equipped with one or more crystal spectrometers for compositional analysis using wavelength-dispersive x-ray spectroscopy (WDS). (WDS offers ~10X better detection limits over EDS for some elements.)

Figure 1. Worm-like elongated forms on a fracture surface within the Martian meteorite ALH84001. Since the orientations of many of the elongated forms are parallel to the substrate cleavage direction (vertical ledge at left), it is highly likely that they are mineral lamellae (rather than "nanofossils") with segmented surface structures resulting from deposition of a conductive gold coating [4].

Sample preparation can be of critical importance in SEM. If a specimen is a good conductor, secondary electron images of surfaces with nanometer-scale resolution are possible. If a specimen is a poor conductor or insulator, a conductive coating must first be applied in order to obtain the highest resolution images (see Figure 1). Thin (1-20 nm thick) coatings of carbon, chromium, palladium, or gold are evaporated or sputtered onto specimens to make them conductive. The less conductive the specimen, the more coating must be applied to obtain highest-quality images. However, once a coating has been applied it is primarily the coating rather than the underlying specimen that is being imaged. When imaging nanometer-sized features on a coated surface, great care must be taken to distinguish indigenous surface microstructures from those caused or accentuated by application of the conductive coating. The problem of conductive coating artifacts is particularly problematical with the new generation of field emission scanning electron microscope (FE SEM), because the subnanometer field emission electron beam permits secondary electron imaging with resolution of 1-2 nm. Under these circumstances, coating microstructures that are not resolvable using a lower-resolution SEM are easily resolved using FE SEM.

TEM is used primarily for examination of the interiors of thin (electron transparent) specimens. Most TEMs operate in the 100-400 keV range. A TEM without beam-scanning capabilities is referred to as a conventional TEM or CTEM, and a TEM equipped with beam-scanning coils is called a scanning TEM, or STEM. Modern analytical STEMs equipped with secondary and backscattered electron detectors provide most of the capabilities of an analytical SEM plus an additional range of capabilities that are specific to the TEM. These include brightfield and darkfield imaging, high-resolution lattice-fringe imaging (Figure 2), electron diffraction, and electron energy-loss spectroscopy. A STEM with a field emission electron gun (FE STEM) offers high beam currents in extremely small electron "nanoprobes" (0.5 -1 nm diameter). Coupled with high collection efficiency solid state x-ray detectors, this makes quantitative compositional EDS microanalysis and compositional mapping with spatial resolution of a few nanometers possible. Using the newly emerging electron energy-loss (energy-filtered) imaging technology, compositional mapping with resolution ~ 1 nm is possible. Unlike EDS mapping, energy-filtered imaging is only semi-quantitative, but it offers the huge advantage of especially high (collection) efficiency for light element analysis and mapping. Thus, biogenic nanostructures containing organic and inorganic matter could be mapped using energy-filtered imaging. Because thin specimens (ideally <100 nm thick) are required for TEM, specialized sample preparation procedures are required. Ultramicrotomy, ion milling, chemical etching, and precision polishing are the most commonly used methods for producing thin TEM specimens.

Electron Microscopy of Biominerals

Three approaches are potentially useful for detecting evidence of biomineralization in rocks using electron microscopy. They are morphological studies using high-resolution SEM imaging, mineralogical studies using TEM, and compositional studies using TEM.

The morphological approach usually relies on using high-resolution SEM imaging to identify shapes or forms on surfaces (e.g., worms) that are consistent with past biological activity. This approach has been used to search for nanofossils in meteorites and terrestrial rocks [1,5]. However, image interpretation is subject to uncertainties, and it is usually difficult to obtain corroborating compositional and structural data from the same specimen [4]. Conductive coatings produce nanometer-sized morphological forms that have been confused with biological forms [5]. A variety of exotic morphological forms similar to biogenic structures can be produced by strictly non-biological processes [4,6]. Even if the morphology of a particular form is consistent with biogenic activity, it may not be unique to biogenic activity.

The TEM has proven ideal for probing the mineralogy of biominerals [2,3]. Common biominerals include iron oxides (e.g., magnetite) (see Figure 2), iron sulfides (e.g., greigite and pyrrhotite), carbonates, and other minerals. Some biominerals are arranged in distinctive configurations. For example, magnetotactic bacteria are a group of organisms that orient and navigate along geomagnetic field lines, and they do so by precipitating chains of magnetite (or iron sulfide) nanocrystals. Unfortunately, the chains may not survive geological processing, and the individual bacterial magnetosomes that make up the chains can be difficult to distinguish from some inorganically produced magnetites (see Figure 2).

Figure 2. Comparison of twinned biogenic and non-biogenic single-domain magnetite (Fe3O4) nanocrystals. The upper-left and lower-right TEM lattice-fringe images are of synthetic magnetite (Fe3O4), while the other two are bacterial magnetosomes. The morphological, structural, and crystallographic properties of the biogenic magnetites overlap those of the inorganic magnetites. (Images courtesy of M. Pósfai and P.R. Buseck.)

Compositional analyses at the nanometer scale can be useful for investigating biogenic structures. The distribution of heavy elements can be mapped with resolution on the order of ~5 nm using EDS. Electron energy-loss energy-filtered imaging can be used to investigate the distribution and speciation of biogenically important light elements C, N, and O at the nanometer scale. Organic compounds (e.g., PAHs) associated with potential biominerals may be indicators of past biogenic activity [1]. Although molecular species cannot be directly detected using electron microscopy, it is possible to probe the local (atomic and molecular) bonding environment of C, N, and O (and heavier elements), using electron energy-loss spectroscopy.

References

1. D.S. McKay et al. (1996). Science 273, 924-930.

2. M. Pósfai et al. (1998). Science 280, 880-883.

3. A. Iida and J. Akai (1996). Sci. Rep. Niigata Univ., Ser E (Geology) 11, 43-66.

4. J.P. Bradley et al. (1997). Nature 390, 5145-5146.

5. V.A. Pedone and R.L. Folk (1996). Geology 24, 763-765.

6. R. Symonds (1993). Geochem. J. 26, 337-350.

Last update 12/28/00 at 3:59 pm