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The effects of hydration on the DNA conformation in the colon biopsy tissues at different disease stages, hyperplasia, dysplasia, and cancer, and their subsequent classifications were investigated in this study. FTIR spectroscopic imaging was used to study the tissues while controlling the humidity from 16% RH to 88% RH using saturated salt solutions. A nonuniform uptake of water into the tissue at its maximum hydrated state was observed in the chemical images showing the distribution of the absorbance of the ν as OH spectral band. The regions of high absorbance of this band in the tissues overlap with the regions of high absorbance of predominantly the phosphate (1143-1100 cm -1 ) and lipid (2879-2844 cm -1 ) bands. Analysis of the second derivative spectra of the hydrated and dehydrated colon tissues further revealed significant peak shifts and changes in absorbance of the spectral bands that correspond to the vibrations of the phosphate group of DNA. These findings showed some disparities when compared to the effect of hydration on the infrared spectra of live cells and pure isolated DNA, possibly due to the presence of DNA mostly in its A-form in the formalin fixed tissues. Coupled with principal component analysis, the spectral biomarkers that differentiate the healthy colon tissues from the diseased tissues were identified to be in the phosphodiester spectral region (1300-1000 cm -1 ). This differentiation varied under different humidity conditions, with the highest sensitivity of ∼98% found at the dehydrated state of the tissues with random forest supervised classification.

Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by infrared (IR) wavelength making subcellular study challenging. Recently, we have reported, with the use of a prototype zinc sulfide (ZnS) transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron-IR microscopy with a 36× objective with numerical aperture of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron radiation single-point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time, while measurements from the scanning methods produced a smoother image. Synchrotron radiation single-point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.

Fourier transform infrared (FTIR) spectroscopic imaging of colon biopsy tissues in transmission combined with machine learning for the classification of different stages of colon malignancy was carried out in this study. Two different approaches, an optical and a computational one, were applied for the elimination of the scattering background during the measurements and compared with the results of the machine learning model without correction for the scattering. Several different data processing pathways were implemented in order to obtain a high accuracy of the prediction model. This study demonstrates, for the first time, that C-H stretching and amide I bands are of little to no significance in the classification of the colon malignancy, based on the Gini importance values by random forest (RF). The best prediction outcome is found when supervised RF classification was carried out in the fingerprint region of the spectral data between 1500 and 1000 cm -1 (excluding the contribution of amide I and II bands). An overall prediction accuracy higher than 90% is achieved through the RF. The results also show that dysplastic and hyperplastic tissues are well distinguished. This leads to the insight that the important differences between hyperplastic and dysplastic colon tissues lie within the fingerprint region of FTIR spectra. In this study, computational correction performed better than optical correction, but the findings show that the disease states of colon biopsies can be distinguished effectively without elimination of Mie scattering effect. Graphical abstract.

The depth of penetration and effective thickness in ATR-FTIR spectroscopic imaging are dependent on the wavelength and angle of incidence of the incoming light beam. We have demonstrated, for the first time, that variable angle micro ATR-FTIR, which is created via the insertion of circular apertures, is intrinsic at examining embedded components within a prostate tissue specimen. This is done by constructing a 3D model from the stacks of 2D chemical images obtained, each of which represents the spatial distribution of a chosen spectral band assigned to the component of interest at a different probing depth. ATR-FTIR imaging is also shown to have the ability to resolve subcellular components of cells such as organelles. For differentiation of diseased and non-diseased tissues, statistical tests are employed to analyse the spectral datasets obtained. When the second derivative of the spectral datasets was subjected to t-test analysis, the spectral differences between both samples in the fingerprint region are shown to be more significant at a shallow depth of penetration, with the greatest variance at the spectral band of 1235 cm-1 (vasPO2-), depicted by plotting the scores of PCA on its first two PCs. Overall, this paper demonstrates a non-destructive, label-free approach for examining heterogeneous biological samples in the z-direction to construct a 3D model using micro ATR-FTIR imaging, in a qualitative and semi-quantitative manner.