Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer

Abstract

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 μm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Fig. 1. Experimental protocol used to analyse the breast tissue sections using FTIR, O-PTIR and Raman spectroscopy and multiphoton imaging.

Fig. 2. IR and Raman spectra extracted from a microcalcification. (a) White light image of the H&E stained breast tissue section with a microcalcification in the middle (purple staining) of the stroma (×5 objective), scale bar: 500 μm. (b) IR image and (c) Raman map at the phosphate peak intensity (1030 and 961 cm⁻¹, respectively). (d) Mean of five IR and (e) Raman spectra were extracted within the MC, truncated onto 1800–900 cm⁻¹ and 350–1800 cm⁻¹, respectively.

Fig. 3. O-PTIR and Raman spectra acquired in a middle of the microcalcification, at the same time and same spot. (a) White light image of the H&E stained breast tissue section (×5 objective), scale bar: 500 μm. (b) O-PTIR and (c) Raman spectra collected within the BMC (purple staining) between 1796–786 cm⁻¹ and 197–1796 cm⁻¹, respectively.

Fig. 4. Example of a breast tissue section with BMCs analysed at different single IR wavelengths using the photothermal system. (a) White light image (×10 objective) showing a microcalcification in dark (red box). Single IR frequencies collected at (b) 1656 cm⁻¹, amide I band, at (c) 1044 cm⁻¹, phosphate band and (d) 872 cm⁻¹, carbonate band. Illustration of different intensity ratios (e) phosphate to amide I ratio (1044 : 1656 cm⁻¹) and (f) carbonate to amide I ratio (872 : 1656 cm⁻¹). (g) Example of O-PTIR spectra collected from the tissue (orange dot, orange arrow) and (h) and within the BMC (black dot, black arrow).

Fig. 5. Example of breast tissue analysis combining different vibrational spectroscopic techniques. (a) White light image (×15 objective) of a BMC defining the area of the FTIR analysis. The red arrows show the BMCs. (b) Single wavelength image extracted from FTIR image of sample at the phosphate intensity at 1030 cm⁻¹. The red arrows show the BMCs. (c) Raman maps based on the phosphate peak intensity at 962 cm⁻¹ taken from the red square in (a). (d) O-PTIR Image at a single IR wavelength corresponding to the phosphate band (1044 cm⁻¹) and taken from the red square in (b).

Fig. 6. (a) Raman map of breast tissue section at the phosphate peak intensity (961 cm⁻¹), the red arrows shows the BMCs in the soft tissue. (b) Single Raman spectrum of a pure mineral TCP (tricalcium phosphate) used as a reference. (c) K-Means cluster analysis with five clusters performed on the Raman map in (a). (d) Two centroids corresponding to the BMC clusters (orange and yellow in c) obtained from the K-means cluster analysis. (e) IR hyperspectral map and (f) Raman hyperspectral map from the red square in (c) at the phosphate peak intensity at 1044 and 961 cm⁻¹, respectively. (g) Mean of five O-PTIR spectra and (h) mean of five Raman spectra extracted from the hyperspectral maps at the same position.

Fig. 7. Multiphoton imaging of a breast tissue section containing a BMC. (a) Composite SRS (in green) at 2930 cm⁻¹ (CH₃ band), SHG (in red) and TPF (in blue) images of a breast tissue section containing a microcalcification (red arrow) (b) zoomed image of the region of interest delimited by the red square in (a), showing a BMC (in blue, red arrow). (c) Composite of SRS images at 2930 cm⁻¹ (CH₃ band) and 2845 cm⁻¹ (CH₂ band) (d) stimulated Raman histology image (SRH). Histology-like section reconstructed by false colour. (e) Adjacent breast tissue section was stained with H&E (×5 objective) used as reference and for comparison with the SRH.