Towards Translation of Discrete Frequency Infrared Spectroscopic Imaging for Digital Histopathology of Clinical Biopsy Samples.

Fourier transform infrared (FT-IR) spectroscopic imaging has been widely tested as a tool for stainless digital histology of biomedical specimens, including for the identification of infiltration and fibrosis in endomyocardial biopsy samples to assess transplant rejection. A major barrier in clinical translation has been the slow speed of imaging. To address this need, we tested and report here the viability of using high speed discrete frequency infrared (DFIR) imaging to obtain stain-free biochemical imaging in cardiovascular samples collected from patients. Images obtained by this method were classified with high accuracy by a Bayesian classification algorithm trained on FT-IR imaging data as well as on DFIR data. A single spectral feature correlated with instances of fibrosis, as identified by the pathologist, highlights the advantage of the DFIR imaging approach for rapid detection. The speed of digital pathologic recognition was at least 16 times faster than the fastest FT-IR imaging instrument. These results indicate that a fast, on-site identification of fibrosis using IR imaging has potential for real time assistance during surgeries. Further, the work describes development and applications of supervised classifiers on DFIR imaging data, comparing classifiers developed on FT-IR and DFIR imaging modalities and identifying specific spectral features for accurate identification of fibrosis. This addresses a topic of much debate on the use of training data and cross-modality validity of IR measurements. Together, the work is a step toward addressing a clinical diagnostic need at acquisition time scales that make IR imaging technology practical for medical use.

Differentiation of group I and group II strains of Clostridium botulinum by focal plane array Fourier transform infrared spectroscopy.

A method was developed for whole-organism fingerprinting of Clostridium botulinum isolates by focal plane array Fourier transform infrared (FPA-FTIR) spectroscopy. A database of 150,000 infrared spectra of 44 strains of C. botulinum was acquired using a FPA-FTIR imaging spectrometer equipped with a 16 x 16 array detector to evaluate the ability of FTIR spectroscopy to differentiate the 44 strains. The database contained strains from C. botulinum groups I and II producing botulinum neurotoxin of serotypes A, B, E, and F. All strains were grown on each of three agar media (brain heart infusion, McClung Toabe agar base, and universal) prior to spectral acquisition. Given the dependence of the infrared spectra of microorganisms on the composition of the growth medium, the spectra were initially separated into three subsets corresponding to the three growth media employed. However, the replicate spectra of all strains, regardless of growth medium, were properly clustered by hierarchical cluster analysis based on differences in their infrared spectral profiles in three narrow spectral regions (1,428 to 1,412, 1,296 to 1,284, and 1,112 to 1,100 cm(-1)). The dendrogram generated from the FTIR data revealed complete separation between group I and group II strains. The spectral differences between group I and group II strains allowed accurate classification of C. botulinum strains at the group level in two blind validation studies (n = 40). These results demonstrate that FPA-FTIR spectroscopy has the potential for rapid discrimination of group I and group II C. botulinum strains in less than 3 min per sample.