Tip-Enhanced Raman Scattering (TERS) from Hemozoin Crystals within a Sectioned Erythrocyte

Abstract

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Raman spectroscopy: techniques and applications in the life sciences

“…Other bacteria-derived samples, such as isolated membrane patches from Halobacterium salinarum have also been measured with TERS, with different patches able to be distinguished [373]. Wood et al demonstrated measurement of hemozoin crystals in sectioned erythrocytes which had been infected with malaria parasites [374], and were also able to identify nano-oxidation sites with TERS [375]. [353]).…”

“…(e) TERS of a hemozoin crystal in a section red blood cell infected with a malaria parasite with AFM topographs indicating the localization of the measurement [374] (Reprinted (adapted) with permission. Copyright 2011 American Chemical Society.…”

Tip enhanced Raman scattering: plasmonic enhancements for nanoscale chemical analysis

“…The use of a metal surface to enhance TERS signals has become known as gap-mode TERS and it has seen use with both STM [39][40][41][42][43][44] and AFM [45,46] based TERS instruments. In the absence of a gap-mode, most TERS measurements require acquisitions on the order of a few seconds to generate appreciable signals from collections of molecules [47,48].…”

“…Examples of cells and organisms studied by TERS include: Staphylococcus epidermidis [140,141], HaCaT cells [142], tobacco mosaic virus [143], pox and adeno viruses [144], colon cancer cells [145,146], rod photoreceptor cells [48], erythrocytes [47], and Halobacterium salinarum [147]. Because TERS images are typically collected using raster scanning that requires each nanometer pixel to be acquired in series, the acquisition time for each image is long.…”

“…The 10 5 -10 8 cm 2 /s diffusion constants associated with the motion of membrane biomolecules means that molecules can easily be oversampled or missed entirely during the course of a scan, complicating analysis in live cells [140]. In fixed cellular systems [47,48,142,146] One approach that offers potential for the investigation of intact membranes is the use of functionalized nanoparticles that bind to membrane receptors. In situ work with streptavidin discussed above suggests that the TERS signals, which originate from the interaction of a TERS tip with a nanoparticle bound to a protein, provide information about the bound protein.…”