Nanoscale imaging of microbial pathogens using atomic force microscopy

Nanoscale imaging of bacterial surfaces, and their modifications upon treatment with antibiotics. Atomic force microscopy (AFM) deflection images in aqueous solution of M. bovis cells prior (a, b) and after treatment with isoniazid (INH) (c), ethionamide (ETH) (d), ethambutol (EMB) (e), and streptomycin (STR) (f) at the minimum inhibitory concentration. All the drugs induced substantial modifications of the cell surface. (g) Statistical analysis of the images in which the variation of the r.m.s. roughness (Rr.m.s.) is constructed from PSD analysis as a function of the length scale. All drugs give rise to a substantial increase of cell surface roughness, to an extent related to the localization of the target. (Reprinted, with permission, from Ref. 25. Copyright 2007 Springer).

Real-time imaging of single A. fumigatus conidia during germination. Series of height (left, 6 µm × 6 µm) and deflection (right, 4 µm × 4 µm) images in culture medium showing a single spore trapped into a pore, as a function of germination time. During germination, spore swelling and ultrastructural alterations were clearly noted. (Reprinted, with permission, from Ref. 13. Copyright 2007 Biophys Society).

Structural dynamics of single A. fumigatus conidia. Series of high-resolution deflection images recorded on a single spore during germination. Within less than 3 h, the crystalline rodlet layer changed into a layer of amorphous material, presumably reflecting inner cell wall polysaccharides. After 2 h, both rodlet and amorphous regions were found to coexist (separated by dashed line). (Reprinted, with permission, from Ref. 13. Copyright 2007 Biophys Society).

Chemical force microscopy (CFM): principle and application to the probing of hydrophobic forces. (a) Water contact angle (&thetas;) values measured for mixed self-assembled monolayers (SAMs) of CH3- and OH-terminated alkanethiols as a function of the molar fraction of CH3-terminated alkanethiols. (b) Histograms of adhesion forces measured on the mixed SAMs using CFM with hydrophobic CH3-tips. (c) Variation of adhesion forces as a function of the cosine of the water contact angle. (d) Adhesion force as a function of the surface fraction of CH3-terminated alkanethiols computed using Cassie's law. (Reprinted, with permission, from Ref. 32. Copyright 2007 ACS).

Chemical force microscopy (CFM) of A. fumigatus conidia. (a) High-resolution image of a wild type conidial surface in aqueous solution revealing rodlets. (b) Adhesion force map (z-range : 6 nN) and (c) adhesion force histogram (n = 512) recorded with a hydrophobic tip, indicating that the rodlet surface is uniformly hydrophobic. (d–i) High-resolution images, adhesion force maps and histograms (n = 512) obtained on NaOH-treated conidia (d–f) and on the ΔrodA ΔrodB double mutant (g–i). The purely hydrophilic surface is attributed to cell wall polysaccharides. (j–l) High-resolution image, adhesion force map and histogram obtained on detergent-treated conidia, revealing highly correlated structural and hydrophobic heterogeneities. (Reprinted, with permission, from Ref. 12. Copyright 2007 ACS).

Chemical force microscopy (CFM) of germinating A. fumigatus conidia. Series of adhesion force maps (500 nm × 500 nm for 0–120 min, and 750 nm × 750 nm for 180 and 210 min; z range = 5 nN) obtained by recording 32 × 32 force curves with a hydrophobic tip on a single germinating spore. Consistent with the structural dynamics (Figure 3), substantial reduction of adhesion was noted with time, reflecting a dramatic decrease of hydrophobicity. After 2 h, heterogeneous contrast was observed in the form of hydrophobic patches, surrounded by a hydrophilic sea. (Reprinted, with permission, from Ref. 13. Copyright 2007 Biophys Society).

Molecular recognition forces of single adhesins. (a) Schematics of the surface chemistry used to functionalize the atomic force microscopy (AFM) tip and substrate with heparin-binding haemagglutinin (HBHA) and heparin. Recombinant histidine-tagged HBHA were attached onto an AFM tip terminated with Ni⁺⁺-nitrilotriacetate (NTA) groups while biotinylated heparin was bound to a gold surface via streptavidin and biotinylated bovine serum albumin (BBSA) layers. (b) Representative force curves and adhesion force histogram obtained in PBS between a HBHA tip and a heparin surface. The adhesion force histogram revealed a bimodal distribution reflecting the binding strength of one and two adhesins. (c) Same experiment in the presence of free heparin (50 µg/mL) demonstrating a dramatic reduction of adhesion frequency due to the blocking of the HBHA adhesion sites. (Reprinted, with permission, from Ref. 38. Copyright 2005 NPG).

Molecular recognition forces of single antibiotics. (a) Gold tips were functionalized with bis(vancomycin) cystamide while gold supports terminated with propionic acid groups were covalently reacted with D-Ala-D-Ala-D-Ala peptides. (b) Representative force curve and adhesion force histogram obtained in PBS between a vancomycin tip and a D-Ala-D-Ala support. (c) Control experiment showing a dramatic reduction of adhesion frequency when the force measurements are performed in the presence of free D-Ala-D-Ala-D-Ala peptides. (Reprinted, with permission, from Ref. 39. Copyright 2007 ACS).

Molecular recognition imaging of cell surface adhesins. (a) atomic force microscopy (AFM) topographic image recorded in PBS showing two M. bovis Bacillus Calmette-Guérin (BCG) cells on a polymer substrate. (b) High-resolution image of the cell surface revealing a smooth morphology. (c–d) Two representative adhesion force maps (gray scale: 100 pN) recorded in PBS with a heparin-modified AFM tip. Adhesion events (bright pixels) reflect the detection of single adhesins. The adhesin distribution is not homogeneous, but apparently concentrated into nanodomains which may play important biological functions. (Reprinted, with permission, from Ref. 38. Copyright 2005 NPG).

Molecular recognition imaging of antibiotic binding sites. (a) Phase contrast and (b) fluorescence image of L lactis cells during the course of the division process. Fluorescent vancomycin accumulates at the division site, by attaching specifically to D-Ala-D-Ala sites of cell wall peptidoglycan. (c) Atomic force microscopy (AFM) image of a cell showing a well-defined division septum as well as a ring-like structure expected to be rich in nascent peptidoglycan (white box). (d) Adhesion force map (gray scale: 100 pN) recorded with a vancomycin tip on the septum region (highlighted by the white box in c). Adhesion events are essentially located in the septum region (red line), more specifically on the ring-like structure, suggesting that newly formed peptidoglycan is inserted. (Reprinted, with permission, from Ref. 39. Copyright 2007 ACS).

Detection of specific bacterial cell wall constituents using immunogold atomic force microscopy (AFM). Contact mode (deflection) images (a, c, e) and tapping mode (phase) images (b, d, f) of immunogold-labeled M. bovis cells: native cells (a, b), cells treated for 24 h with isoniazid (INH) (c, d) and ethambutol (EMB) (e, f). All cells were incubated with monoclonal anti-LAM antibodies, followed by another incubation with the corresponding gold-conjugated secondary antibodies. (Reprinted, with permission, from Ref. 25. Copyright 2007 Springer).