Fig 1.
Raman spectrum of Trypanosoma brucei brucei strain STIB247 and some examples of its associated false colour image obtained with Raman measurements.
Data obtained with a 532 nm laser, 6 s acquisition time and 100x lens. (A) A schematic of a bloodstream trypanosome (Created in BioRender.com), (B) white light image of T. b. brucei mapped where the red square correspond to 25 x 25 μm mapped at 0.5 μm step size, (C) representative Raman spectrum of the parasite, taken from a single point on the parasite, and (D) false coloured image associated of the Raman peaks 1452, 1577, 1666 and 2934 cm-1 with their respective intensity bar, which assign a colour gradient from the lowest (black) to the highest (white) intensity.
Table 1.
Tentative assignment of Raman peaks for the characterisation of the biological composition of T. b. brucei using previously published works from biological samples [31,41,42].
Fig 2.
Raman analysis of T. b. brucei infected (red) and uninfected (blue) murine skin sections (BALB/C) on CaF2 slides showing (A) the resulting PCA scores plot, and their associated averaged normalised Raman spectra in the spectral window (B) 550–1800 cm-1 and (C) 2550–3600 cm-1.
Raman maps measurements were taken using a 532 nm excitation laser and 1 s acquisition time over a 50 x 50 μm square on the skin for each map and 2 μm step across x and y. Spectra were obtained by averaging seven maps from each tissue sample, then normalised to the highest peak intensity. Tentative peak assignment is given in Table 2.
Table 2.
Tentative assignment of Raman peaks specific to the trypanosome infected skin, based on Fig 2B and 2C.
Specific Raman peak were chosen when they showed a clear shift (s), decrease in intensity (d) or increase of intensity (i) compared to the uninfected skin Raman spectrum. The tentative biological assignment was performed using previously published works from biological tissues. [31,40–42,46].
Fig 3.
Spectral comparison between reference spectrum of T. b. brucei and uninfected and T. b. brucei murine infected skin (BALB/C).
Five different T. b. brucei were mapped and averaged into a single Raman spectrum and both skin spectra were shown in Fig 2. Two Raman spectral region are shown: (A) 550–1800 cm-1 and (B) 2550–3600 cm-1.
Fig 4.
In situ Raman analysis of the skin of uninfected and T. brucei infected mice (BALB/C) at 14 days post-infection.
Measurements were obtained using a 785 nm excitation laser with a 60 s acquisition time and ten measurements were taken across each murine skin. The resulting principal component analysis scores plot is shown in (A) where each dot corresponds to the averaged (from the 10 Raman measurements per sample) and normalised spectrum of one murine skin sample, (B) displays the spectral comparison of each Raman spectrum and (C) table regrouping the mean data and its SD from the semi-quantitative measurements of parasites and inflammatory cells in the skin stained by immunochemistry; all data from the immunochemistry staining are grouped in S9 and S10 Figs.
Fig 5.
In Situ Raman analysis of a T. b. brucei infection time course in C57/black6J mice compared to detectable blood parasitaemia and skin parasite burden.
(A) PCA scores plot, (B) spectral comparison of each averaged and normalised Raman spectrum, (C) estimation of the blood parasitaemia and (D) estimated number of parasites/ mg skin by qPCR for PFR2 gene over the same time course as the Raman measurements. Raman measurements were obtained using a 785 nm excitation laser with a 30 s acquisition time and five measurements were taken across each skin sample.
Fig 6.
Graphs showing the evolution of the T. b. brucei infection in C57/black6J mice over the time course using a Raman peak intensity ratio.
Four Raman peaks are displayed: (A) 670 cm-1 (Porphyrin, tryptophan) / 1442 cm-1 (CH2 bending mode), (B) 858 cm-1 (Phosphate group, tyrosine) / 1442 cm-1 (CH2 bending mode), (C) 940 cm-1 (C-C stretch backbone, polysaccharides) / 1442 cm-1 (CH2 bending mode) and (D) 1003 cm-1 (Phenylalanine) / 1442 cm-1 (CH2 bending mode). The grey line corresponds to the Raman peak ratio averaged between both control murine and act as a threshold for the detection of the infection.