Fig 1.
Comparison of H&E-stained optical microscopy and IR images of lymph node tissue.
(a) H&E stained image from a healthy submandibular lymph node biopsy alongside (b) an HD IR image of a serial section of the lymphoid tissue and (c) the same region imaged with a lower resolution FT-IR instrument. The IR images show the absorbance at 3075 cm-1 after baseline correction. Sample spectra, (d) and (e), are plotted from the pixel marked with a red ‘x’ in (b) and (c), respectively. The black scale bar below the zoomed-in image in (c) corresponds to 50 μm, and above the H&E stained image to 300 μm.
Fig 2.
HD training and classification using three demonstrative examples.
For each sample (a-c), H&E (i) and IHC stained serial sections CD3+ (ii), CD10+ (iii), and CD20+ (v) were used to draw ROIs on the HD IR images, (vi), to train the classifier. The resulting classified images, (iv), are shown. The absorbance for the IR images is at 3300 cm-1, and the solid bar in each classified image is 500 μm.
Fig 3.
Classification of an HD IR image of a normal submandibular lymph node.
A qualitative comparison of the classified HD IR image, (b), can be made with the stained serial sections (a) H&E, (c) CD3+, (d) CD10+, and (e) CD20+, showing excellent agreement. Zooming in on the secondary follicle in the HD classified image, (g), shows that the classifier is capturing the naïve B cell mantle (blue), T cells (green), and the light and dark zone regions of activated B cells (cyan and dark blue, respectively) of the germinal center. The dashed boxes correspond to the images shown in Fig 1. The solid bars in (a) and (f) are 1 mm and 175 μm, respectively.
Fig 4.
Classification of HD IR images from a reactive iliac lymph node.
(a) H&E stained serial section presented for comparison to HD classification images overlaid on a low-resolution 4cm x 2cm IR image displaying the intensity of the 1654 cm-1 band, (b). The zoom-in, (d), of the white box in (b) shows the classification of RBCs, T cells, naïve and memory B cells, along with the light and dark zone activated B cells in the sinusoid. The black bars in (a) and (c) are 1.5 mm and 125 μm, respectively.
Fig 5.
Classification of HD IR images from a malignant mesenteric lymph node.
Five individually classified HD IR images overlaid on a low-resolution IR image of an abnormal lymph node biopsy with the band intensity at 1654 cm-1. The zoomed-in regions clearly show consistent activated B cell (more specifically, light zone) classification, in agreement with the diagnosis of diffuse, large B-cell lymphoma made by flow cytometry. The white bar is 2 mm.
Fig 6.
Comparison of malignant lymphoid tissue.
H&E stained serial section (a), HD IR image at 3300 cm-1 intensity (b), and classified image, (c), from Fig 5 (bottom left) confirm the resolving power of HD spectroscopic imaging and the consistent light zone B-cell classification of the abnormal lymph node. The solid bar in (a) and below the zoom are 140 and 35 μm, respectively.