Quantitative IR microscopy and spectromics open the way to 3D digital pathology

Abstract

Currently, only mass‐spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro‐microscopy is reported. An automated curve‐fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR‐absorption spectrum into a IR‐band spectrum; (3) the reconstruction of an 3D IR‐band matrix (x, y, z for voxel position and a 4th dimension with all IR‐band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

Two-Dimensional Correlation Spectroscopy (2D-COS) for Analysis of Spatially Resolved Vibrational Spectra

“…Hyperspectral imaging from serial sample sections represents the third approach to produce HSV. [47][48][49] Although no application examples of the 2D-COS technique for HSV are known to date, the pre-processing routines described in the next sections would be applicable also for these data types.…”

Extended Multiplicative Signal Correction for Infrared Microspectroscopy of Heterogeneous Samples with Cylindrical Domains

“…Recent applications include examples from biology, [2][3][4][5][6] materials science, [7][8][9] forensics, 10,11 agriculture, 12,13 and medicine. [14][15][16][17][18][19][20][21][22][23][24][25] Although FT-IR microspectroscopy includes the exceptional molecular selectivity of fundamental absorption modes, high spectral accuracy from interferometric measurements, and robust signal-to-noise (S/N) ratios, spectral distortions 26 arise from scattering at interfaces 27 that can complicate the analysis of recorded data. For analytical measurements of samples whose sizes are comparable to the wavelength of impinging electromagnetic radiation, in practice, scattering from the object dominates the total recorded attenuation.…”

3D chemical imaging of the brain using quantitative IR spectro-microscopy

“…Three-dimensional (3D) microscopy is a powerful approach for imaging biological specimens. 1 It offers excellent spatial resolution and facilitates the observation of tissue sub-structures and content 2 under physiological and pathological conditions. However, although it is feasible to achieve a high spatial resolution close to the diffraction limit in small, transparent biological specimens 3 such as cultured cells, it remains difficult to achieve high-resolution images of larger, more optically challenging entities such as tissue blocks, biopsies, or organs.…”

“…Several approaches have already been proposed for ex vivo 3D spectro-microscopy imaging, such as serial sections based electron, 6 X-ray fluorescence, 7 infrared, 2 , 8 mass, 9 and Raman 10 microscopies, with a 3D reconstruction of small tissue volumes at the micro- and nano-scopic scales. Mass spectrometry (MS) imaging was the first spectroscopic technique that was able to create a 3D reconstruction of the chemical information of a tissue sample.…”