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Malaria pp 299-305 | Cite as

Atomic Force Microscopy of Plasmodium-Infected Red Blood Cells: Detecting and Localizing Single Molecular Recognition Events

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 923)

Abstract

Atomic Force Microscopy (AFM) is a powerful tool for exploring the interaction between ligands and receptors, as well as their exact locations on the red cell surface. Here we discuss current and future applications for AFM based single-molecule force spectroscopy to study adhesion of Plasmodium-infected red blood cells. A protocol is provided for simultaneous topography and recognition imaging of the surface of Plasmodium falciparum-infected cells using CD36 functionalized tips.

Key words

Atomic force microscopy Single-molecule force spectroscopy Ligand–receptor ­interactions PfEMP1 CD36 

1 Introduction

Atomic force microscopy (AFM) is a surface scanning method that provides nanometer scale positional accuracy and piconewton force sensitivity even under physiological conditions. In contrast to optical microscopy and electron microscopy that rely on detecting deflected or scattered light or electrons, AFM relies on a flexible cantilever with a sharp tip at the end to directly sense the sample surface. By monitoring the tip–sample interaction from cantilever deflection as it scans across the sample surface, AFM can collect accurate three-dimensional (3D) topographical and surface mechanical properties simultaneously. Importantly, AFM enables the investigation of fresh and air-dried erythrocytes without the need for fixation and surface coating/staining that may alter ­delicate nanostructures on the surface of infected red blood cells (iRBC).

The first malaria-related applications of AFM focused simply on the morphological scan of the iRBC. However, even such seemingly straightforward work encountered challenges, with Aikawa et al. publishing AFM images in the 1990s showing artifactual “double headed knobs” (1, 2). More recently, optimized protocols have been developed to allow accurate and repeatable AFM imaging of iRBC, revealing significant nanoterrain variation within and between Plasmodium species (3, 4, 5, 6).

One of the major advantages of AFM for the examination of malaria iRBC versus scanning electron microscopy (SEM) is that the same sample can be examined simultaneously or separately by other imaging methods. For example, an individual iRBC fixed to the glass slide can be located using the coordinates on a micro ­copper grid on the underside of the slide. The cell of interest can be initially scanned using the AFM, subsequently the same cell can be imaged using indirect fluorescent assay and finally the cell can be stained with a standard Giemsa stain to ascertain the developmental stage of the cell under investigation (4). It is important to remember that SEM has two other important disadvantages: first, it is almost impossible to determine the stage of a parasite or even if a cell is really infected; second, accurate measurements of nanostructures (such as excrescences on the RBC infected by P. falciparum and P. malariae) by SEM are subject to significant distortion by electromagnetic interference (7).

Although AFM has, and will continue to provide important quantitative morphological data on the surface nanoterrain of Plasmodium iRBC, single-molecule force spectroscopy is the most exciting application of this technology (8, 9). Force spectroscopy, in the context of this chapter, measures receptor–ligand unbinding under mechanical stretching force. A recent study by Li et al. utilized this method to compare the strength of binding between the PfEMP1 ligand and the endothelial receptors CD36 and thrombospondin (TSP) (9) (Fig. 1). In this study, single-molecule force spectroscopy revealed that CD36-mediated interaction was much more stable than that mediated by TSP at single molecule level. This data supports the hypothesis that TSP acts as a braking (or tethering) system that initiates rolling of RBC on the vascular endothelium (10). The powerful binding strength of the CD36 receptor subsequently provides for the stable adhesion of the P. falciparum-infected RBC to the endothelium.
Fig. 1.

Representative profile of the force curves obtained from a multi-peak rupture of CD36-PfEMP1 receptor ligand interaction. Points 1, 2, 3, and 4 correspond to different states that occur upon retracting the CD36 functionalized cantilever from the PfEMP1 covered knob, including the elongation of the PEG and PfEMP1 and the sequential rupture of the two ligand receptor pairs.

It should also be noted that instead of functionalizing AFM cantilever with a receptor, it is also possible to attach a monoclonal antibody of interest (11). In a potent demonstration of single-molecule force spectroscopy, Zhu et al. used 5-methylcytidine antibody functionalized AFM tips to reveal epigenetic methylation patterns in individual DNA strands with a resolution of 4 Å (12).

Resent advances in AFM technology now allow for the simultaneous collection of topography and molecular recognition imaging (8). Below, we provide a protocol for mapping adhesion sites of CD36 on the surface of iRBC. Future work should extend such a study using other receptors (e.g., ICAM or CSA) or parasites collected from field isolates (especially from severe cases or placental isolates).

2 Materials

  1. 1.

    AFM tips: Model of MSCT/MLCT-E (Bruker AFM probes), cleaned in air plasma for 5 min before functionalization.

     
  2. 2.

    Biotinylated bovine serum albumin (Biotin-BSA) (Thermal Scientific): make up to 2 mg/ml in distilled water, store at 4°C.

     
  3. 3.

    Strepavidin (Thermal Scientific): make up to 1 mg/ml in distilled water, store at −20°C (3).

     
  4. 4.

    NHS-PEG4-Biotin (Thermal Scientific): no-weight format, powder form stored at 4°C, 2 mg dissolved in 170 μl distilled water immediately prior to use.

     
  5. 5.

    Glycine (Sigma): make up to 1 mg/ml in distilled water, store at 4°C.

     
  6. 6.

    Recombinant CD36 (Sino Biological Inc): make up to 0.1 mg/ml in distilled water, store at 4°C.

     
  7. 7.

    Poly-l-lysine (Sigma): solution form of 0.1 mg/ml stored at 4°C.

     
  8. 8.

    Glutaraldehyde (MP Biomedicals): make up 1% glutaraldehyde solution in 1× PBS prior to cell fixation.

     

3 Methods

3.1 Functionalization of AFM Tips

  1. 1.

    Incubate the precleaned tip with Biotin-BSA overnight at 4°C.

     
  2. 2.

    Wash five times in distilled water for 10 min and incubate with streptavidin for 30 min at room temperature (RT).

     
  3. 3.

    Wash three times in distilled water and incubate with NHS-PEG4-Biotin for 20 min and then incubate with CD36 for 2 h.

     
  4. 4.

    Quench the reactions with glycine for 30 min and wash in distilled water three times.

     

3.2 Cell Preparation

  1. 1.

    Enrich the trophozoite and schizont stage-infected RBC using MACS (Miltenyi Biotec) (see Note 1).

     
  2. 2.

    Immerse the cover slips (22  ×  22 mm) in 0.1 mg/ml poly-l-lysine for 20 min and dry in a vacuum desiccator or a dry cabinet.

     
  3. 3.

    Incubate the enriched cells on poly-l-lysine-coated cover slip for 30 min and wash away unbound cells with RPMI (see Note 2).

     
  4. 4.

    Fix the bound cell on the cover slip in 1% glutaraldehyde for 1 h (see Note 3).

     
  5. 5.

    Quench the nonspecific reactions with Glycine for 30 min and wash with 1× PBS three times.

     

3.3 Simultaneous Topography and Adhesion Mapping

  1. 1.

    A Bioscope Catalyst AFM (Bruker) sitting on a Zeiss inverted microscope is used for the peak force QNM test (see Note 4).

     
  2. 2.

    A bare tip is used to locate the cells and to find the proper relatively flat cell surface for subsequent higher resolution functional scanning using functionalized tips (see Note 5).

     
  3. 3.

    The tip is changed to a functionalized one and a force curve is collected in ramp mode on glass surface without cells to calibrate the deflection sensitivity, and then spring constant of the functionalized tip is calibrated using built-in thermal tune module.

     
  4. 4.
    The previously identified cells are relocated and the topography and adhesion images are captured simultaneously in peak force QNM mode. An example of the output is shown below (see Fig. 2).
    Fig. 2.

    The left panel is the adhesion map and the right panel is the topography channel. Yellow circle indicates the correlation between the adhesion site and the knobs. Arrow indicates debris attached on the surface, a good marker to confirm the absence of cross talk from topography on adhesion map. Scale bar: 500 nm.

     
  5. 5.
    The imaging settings are adjusted as shown in Table 1 (see Note 6).
    Table 1

    Initial scan parameter settings for peak force QNM measurement

    Scan size

    Scan rate

    Resolution

    Setpoint

    Peak force amplitude

    Lift height

    2–10 μm

    0.5–1 Hz

    256–512

    300–800 pN

    30–100 nm

    Auto config

     

4 Notes

  1. 1.

    Ex vivo matured clinical isolates of P. vivax and P. falciparum can also be concentrated on Percoll gradients.

     
  2. 2.

    For even greater iRBC adhesion to the glass slide, lectin can be used instead of poly-l-lysine (13).

     
  3. 3.

    Unfixed RBC are too soft and deform badly under the tip ­during imaging, which limits the resolution to a few hundreds of nm and thus no detailed features like knobs can be resolved. Unfixed smeared samples might be able to produce similar results of fixed cells.

     
  4. 4.

    Peak force QNM (quantitative nanomechanical mapping) is a patented imaging mode from Bruker that allows simultaneous topographical imaging and quantitative mechanical property mapping of sample surface at the same spatial and temporal resolution (14, 15).

     
  5. 5.

    Normal contact mode or tapping mode can be applied to image the cell surface in this step.

     
  6. 6.

    Peak force QNM comes with automatic imaging parameter adjusting functions. However, the automation is for the purpose of optimizing imaging quality. For quantitative mechanical property mapping, automatic set point adjusting needs to be switched off to achieve constant contact force during property mapping. Besides, since the force curve collection rate for peak force QNM is 1–2 kHz, in order to achieve at least one force curve per imaging pixel to calculate the property, one has to adjust to slower scan rate while capturing at high resolution (0.5 Hz or slower scan rate is recommended for a resolution of 512).

     

References

  1. 1.
    Aikawa M et al (1996) Membrane knobs of unfixed Plasmodium falciparum-infected erythrocytes: new findings as revealed by atomic force microscopy and surface potential spectroscopy. Exp Parasitol 84:339–343PubMedCrossRefGoogle Scholar
  2. 2.
    Nagao E et al (2000) Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy. J Struct Biol 130:34–44PubMedCrossRefGoogle Scholar
  3. 3.
    Arie T et al (2005) Hemoglobin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes. J Struct Biol 150:163–169PubMedCrossRefGoogle Scholar
  4. 4.
    Li A et al (2006) Observations on the internal and surface morphology of malaria infected blood cells using optical and atomic force microscopy. J Microbiol Methods 66:434–439PubMedCrossRefGoogle Scholar
  5. 5.
    Li A et al (2010) High density of ‘spiky’ excrescences covering the surface of an erythrocyte infected with Plasmodium malariae. Br J Haematol 151:1PubMedCrossRefGoogle Scholar
  6. 6.
    Rug M et al (2006) The role of KAHRP domains in knob formation and cytoadherence of P falciparum-infected human erythrocytes. Blood 108:370–378PubMedCrossRefGoogle Scholar
  7. 7.
    Nolze G (2007) Image distortions in SEM and their influences on EBSD measurements. Ultramicroscopy 107:172–183PubMedCrossRefGoogle Scholar
  8. 8.
    Hinterdorfer P, Dufrene YF (2006) Detection and localization of single molecular recognition events using atomic force microscopy. Nat Methods 3:347–355PubMedCrossRefGoogle Scholar
  9. 9.
    Li A et al (2011) Molecular mechanistic insights into the endothelial receptor mediated cytoadherence of Plasmodium falciparum-infected erythrocytes. PLoS One 6:e16929PubMedCrossRefGoogle Scholar
  10. 10.
    Evans EA, Calderwood DA (2007) Forces and bond dynamics in cell adhesion. Science 316:1148–1153PubMedCrossRefGoogle Scholar
  11. 11.
    Ebner A et al (2007) Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers. Ultramicroscopy 107:922–927PubMedCrossRefGoogle Scholar
  12. 12.
    Zhu R et al (2010) Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns. Nat Nanotechnol 5:788–791PubMedCrossRefGoogle Scholar
  13. 13.
    Liu F et al (2003) Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy. Cell Biochem Biophys 38:251–270PubMedCrossRefGoogle Scholar
  14. 14.
    Pittenger B et al (2011) Quantitative mechanical properties mapping at the nanoscale with peakforce QNM. Bruker Application Note #128PubMedCrossRefGoogle Scholar
  15. 15.
    Berquand A (2011) Quantitative imaging of living biological samples by peakforce QNM atomic force microscopy. Bruker Application Note #135PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Singapore-MIT Alliance for Research and TechnologySingaporeSingapore
  2. 2.Singapore Immunology Network, Agency for Science, Technology and Research, BiopolisSingaporeSingapore
  3. 3.Division of Bioengineering, Department of Mechanical EngineeringNational University of SingaporeSingaporeSingapore

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