High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass

Abstract

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

Infrared spectral histopathology using haematoxylin and eosin (H&E) stained glass slides: a major step forward towards clinical translation

“…Despite this, there have only been a limited number of studies utilising alternative substrates. 27,36 Glass slides have the clear advantage that they are readily available, robust, low cost and are already used ubiquitously by the pathologist in the clinic. Although promising, using unstained tissue serial sections on glass adjacent to the H&E stained section introduces challenging image registration issues, and there is no guarantee that both sections will contain any abnormalities present.…”

Spectroscopic imaging of biomaterials and biological systems with FTIR microscopy or with quantum cascade lasers

“…FTIR imaging at high spatial resolution using infrared microscopes has been used to study cryosectioned brain tissues for research into Alzheimer’s disease [ 18 ], intracellular imaging [ 19 ] and discrimination between malaria-infected single cells [ 20 ]. The push for high spatial resolutions can come at the expense of the SNR, and for statistical classification, high resolution may not always be necessary.…”

“…However, the theoretically achievable spatial resolution is fundamentally limited by the NA of the optics rather than the projected pixel size. For a discrete detector array, the sampling frequency should also be at least double the desired resolution, so the use of ‘high-magnification’ mode is still beneficial when imaging is performed at the diffraction limit of infrared light [ 18 , 20 ]. A germanium ATR (Ge-ATR) crystal combined with high-magnification mode allows higher spatial resolutions to be achieved because n Ge = 4, producing a measured resolution for optical contrast of 780 nm with a chrome/glass 1951 USAF target [ 18 ].…”

High and ultra‐high definition of infrared spectral histopathology gives an insight into chemical environment of lung metastases in breast cancer

“…This setup coupled with 15× (NA: 0.62) and 25× (NA: 0.81) Cassegrain condenser/objective couple allows for spectroscopic imaging with a pixel size of 1.1 × 1.1 (HD) and 0.66 × 0.66 (UHD) μm 2 , respectively. For such optics, the total IR system magnification is 36× and 60× and results in a spatial resolution of 2.4 and 1.4 μm at 3750 cm −1 , respectively . The staining with hematoxylin and eosin (H&E) of lung cross sections indicates that morphology of lung tissue in the third and fourth week after cancer cells transplantation is already characterized by clear‐cut metastatic sites in lung parenchyma and pleural cavity surrounded by the lung tissue exhibiting features of local inflammation and healthy tissues in some sites.…”