Figure 2 |. Tissue-thickness-dependent absorption spectra acquired by conventional mid-IR spectral imaging (MIRSI).

a, Sketch of three tissue sections with different thicknesses on standard IR-transparent substrates characterized in transmission mode. b, Measured absorption spectra from 80 μm, 40 μm, and 20 μm thick tissue sections. The absorption-band amplitudes consistently increase with tissue thickness. c, Second derivative of the absorption spectra for glycogen (1024 cm⁻¹ ▲), deoxyribose (1060 cm⁻¹ ▲), symmetric phosphate (1090 cm⁻¹ ▲), C-OH (1170 cm⁻¹ ▲), asymmetric phosphate (1236 cm⁻¹ ▲), $\text{β}$-sheet (1542 cm⁻¹ ▲), amide II (1574 cm⁻¹ ▲), and amide I (1660 cm⁻¹ ▲). d, Area integral distributions from $n_{\text{pixels}}=12.5\times {10}^3$ different pixels along the second derivative absorption bands plotted for some of the above molecules. The individual pixels’ integral value distributions from three different tissue thicknesses show quantitative and qualitative dependence on the spectral signatures.