High-resolution visualization of Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution transmission electron microscopy

Abstract

High-pressure freeze-substitution and transmission electron microscopy have been used for high-resolution imaging of the natural structure of a gram-negative biofilm. Unlike more conventional embedding techniques, this method confirms many of the observations seen by confocal microscopy but with finer structural detail. It further reveals that there is a structural complexity to biofilms at both the cellular and extracellular matrix levels that has not been seen before. Different domains of healthy and lysed cells exist randomly dispersed within a single biofilm as well as different structural organizations of exopolymers. Particulate matter is suspended within this network of fibers and appears to be an integral part of the exopolymeric substance (EPS). O-side chains extending from the outer membrane are integrated into EPS polymers so as to form a continuum. Together, the results support the concept of physical microenvironments within biofilms and show a complexity that was hitherto unknown.

FIG. 1.

P. aeruginosa PAO1 biofilm prepared by conventional TEM processing. These micrographs clearly show the heterogenous distribution of biomaterials such as membrane vesicles (white arrows), cellular detritus (star), and other extracellular polymers. Unfortunately, little information about biofilm structure can be acquired from such micrographs since conventional processing induces features such as membrane artifacts (white arrowheads) and condensed cytoplasmic material (black arrowheads). Additionally, conventional methods fail to reveal the true nature of extracellular polymers that are known exist between cells. Bar = 2 μm.

FIG. 2.

A) Schematic of the flat specimen holder for freeze-substitution. In short, a biofilm grown on a sapphire disk was briefly dried and transferred to a copper flat specimen carrier. The holder was then filled with a 10% sucrose solution, which served as a cryoprotectant throughout the freezing process. B) The 1.5-mm-diameter specimen carrier (i) was then tightly secured into a loading pod (ii). This entire apparatus was inserted into the high-pressure freezer using a loading rod (iii). Samples were immediately frozen under high pressure, which was applied to the underside of the biofilm substratum as shown in A. An extensive technical description of the high-pressure freezer and the freezing process can be found in Studer et al. (63).

FIG. 3.

Typical temperature and pressure changes of the biofilm specimen throughout high-pressure freezing as established by the EMPACT system (Leica Microsystems). This diagram reveals a pressure of over 2 × 10⁵ kPa (heavy line) was applied, followed by a temperature decrease from 20°C to below −190°C (thin line) at a cooling rate of 1.1 × 10⁴°C per s. These conditions permit excellent vitrification in specimens up to ∼200 μm thick.

FIG. 4.

TEM micrograph of a full biofilm profile in thin section (A), which reveals extensive heterogeneity in terms of cell distribution and physiological status. Prior to sectioning, the substratum was situated at the bottom of the micrograph, while the top represents the biofilm-media interface. Each biofilm shows diffuse (black arrow) and tightly packed cells (arrowhead) of with interstitial regions of “void” space (star). A few lysed cells are also visible (white arrow). These characteristics are represented at higher magnification in B, C, and D. B) Cells towards the biofilm-medium interface were few and spread far apart relative to the rest of the biofilm. C) Cells situated away from the biofilm-medium interface were generally tightly clustered and packed along the same axis. D) Some areas adjacent to healthy cells were characterized by extensive cell lysis. These regions appeared to be randomly distributed, but were not found in all biofilms. Bars: A = 5 μm; B, C, and D = 1 μm.

FIG. 5.

A) TEM micrograph of freeze-substituted PAO1 biofilm cells. In this image and at higher resolution (B), it is evident that conventional embedding artifacts are not present in these cells, as they are characterized by an evenly stained plasma membrane (PM), a semitranslucent periplasmic gel (PG), a taut outer membrane (OM), and evenly distributed cytoplasm with ribosomes spread throughout. The fibrous background material (EPS) emanating from the lipopolysaccharides (LPS) on the outer membrane is absent from planktonic cells (C), which suggests that these features are the extracellular biofilm matrix. Bar = 0.5 μm.

FIG. 6.

A) TEM micrograph of a cluster of biofilm cells surrounded by an extensive EPS network. Most EPS is present near the cell surfaces. B) High-resolution image of the EPS region marked in A (see text for details). Microenvironments of EPS of high (HD), intermediate (ID), and low (LD) density are clearly present. C) Some areas adjacent to the substratum reveal more extensive heterogeneity. At least three polymer types or arrangements are present in this small area of the biofilm (outlined) and are shown at higher magnification in panels D, E, and F. While the exact chemical nature of these polymeric configurations is unknown, these micrographs reveal the extensive complexity of the EPS matrix. Bars = 1 μm.

FIG. 7.

A) High-resolution image of a biofilm PAO1 cell envelope. LPS can be seen extending from the outer membrane as a fibrous polymer brush (arrows), though it appears to be less electron dense than the surface of planktonic cells (see Fig. 5C). The electron density is likely provided by uranyl ions, which suggests that this polymer is B-band LPS. Bar = 100 nm. B and C) Western immunoblots of LPS isolated from planktonic (lane 1) and biofilm (lane 2) PAO1 cells. SDS-PAGE gels were standardized to 25 μg protein per lane and reacted with either (B) anti-A band or (C) anti-B band monoclonal antibodies. These blots confirm the presence of B-band LPS in PAO1 biofilms, though it is evident that, relative to planktonic PAO1 cells, B-band is produced in smaller amounts. D) LPS was not visible on several clusters of biofilm cells, which is suggestive of localized microenvironments in which PAO1 alters the characteristics of its cell envelope. Bar = 100 nm.

FIG. 8.

TEM micrograph of Alcian blue-stained EPS. While this highly positively charged dye provided enhanced contrast of EPS, electrostatic interactions caused condensation of the polymers against each other and against the cells that they surround. Bar = 200 nm.