Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics

Abstract

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria

“…Previous in vitro photoacoustic approaches (26,27) required a relatively large amount of blood because they used the bulk thermoelastic mechanism of signal generation and thus were unable to detect single iRBCs among many uninfected RBCs. Several other diagnostic methods use the various optical properties of hemozoin (19,(28)(29)(30)(31)(32)(33). All these methods cannot transdermally and rapidly detect low levels of parasitemia in a needle-and reagent-free way, as H-VNBs demonstrated.…”

“…Hemozoin can be found in any parasite type and any blood stage, including gametocytes (8,10,11). High parasite sensitivity and the specificity of H-VNBs result from a much stronger acoustic signal of a vapor nanobubble compared with that from a bulk thermal effect used by traditional photoacoustics (26,27,34) or from the optical effects in hemozoin (28)(29)(30)(31)(32)(33). Vapor bubbles can also be generated via optical absorbance of hemoglobin in uninfected RBCs but at more than 100-fold higher laser fluence (35).…”

Raman spectroscopy: techniques and applications in the life sciences

“…This led to other studies using resonance Raman to create sub-cellular maps of specific molecules including heme-containing enzymes such as cytochrome b 558 [146] and globins such as hemoglobin and neuroglobin [200]. Figure 21 Wood et al used resonance Raman to image trophozoites in malaria-infected cells [201]. Parasite-deposited haemozoin is easily visible in Raman images due to the resonance enhancement see Figure 21(c) .…”

“…(c) Hyperspectral Raman images of malaria-infected cells [201]. (Left) Raman image of the band from 1620-1680 cm −1 assigned to haemozoin, (Right) cluster analysis of Raman spectra.…”

A comparison of differential leucocyte counts measured by conventional automated venous haematology and darkfield microscopic examination of fresh capillary blood

“…identification of parasite-infected cells as seen through DM also 79 permits easy malaria diagnosis [18,19]. infants, the elderly and the infirm [20].…”