FTIR spectroscopic imaging and mapping with correcting lenses for studies of biological cells and tissues.

Histopathology of tissue samples is used to determine the progression of cancer usually by staining and visual analysis. It is recognised that disease progression from healthy tissue to cancerous is accompanied by spectral signature changes in the mid-infrared range. In this work, FTIR spectroscopic imaging in transmission mode using a focal plane array (96 × 96 pixels) has been applied to the characterisation of Barrett's oesophageal adenocarcinoma. To correct optical aberrations, infrared transparent lenses were used of the same material (CaF2) as the slide on which biopsies were fixed. The lenses acted as an immersion objective, reducing scattering and improving spatial resolution. A novel mapping approach using a sliding lens is presented where spectral images obtained with added lenses are stitched together such that the dataset contained a representative section of the oesophageal tissue. Images were also acquired in transmission mode using high-magnification optics for enhanced spatial resolution, as well as with a germanium micro-ATR objective. The reduction of scattering was assessed using k-means clustering. The same tissue section map, which contained a region of high grade dysplasia, was analysed using hierarchical clustering analysis. A reduction of the trough at 1077 cm(-1) in the second derivative spectra was identified as an indicator of high grade dysplasia. In addition, the spatial resolution obtained with the lens using high-magnification optics was assessed by measurements of a sharp interface of polymer laminate, which was also compared with that achieved with micro ATR-FTIR imaging. In transmission mode using the lens, it was determined to be 8.5 µm and using micro-ATR imaging, the resolution was 3 µm for the band at a wavelength of ca. 3 µm. The spatial resolution was also assessed with and without the added lens, in normal and high-magnification modes using a USAF target. Spectroscopic images of cells in transmission mode using two lenses are also presented, which are necessary for correcting chromatic aberration and refraction in both the condenser and objective. The use of lenses is shown to be necessary for obtaining high-quality spectroscopic images of cells in transmission mode and proves the applicability of the pseudo hemisphere approach for this and other microfluidic systems.

Label Free Detection of Sensitive Mid-Infrared Biomarkers of Glomerulonephritis in Urine Using Fourier Transform Infrared Spectroscopy.

More reliable biomarkers using near-patient technologies are needed to improve early diagnosis and intervention for patients with renal disease. Infrared (IR) vibrational spectroscopy/microspectroscopy is an established analytical method that was first used in biomedical research over 20 years ago. With the advances in instrumentation, computational and mathematical techniques, this technology has now been applied to a variety of diseases; however, applications in nephrology are just beginning to emerge. In the present study, we used attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to analyze urine samples collected from rodent models of inflammatory glomerulonephritis (GN) as well as from patients with crescentic GN, with the aim of identifying potential renal biomarkers; several characteristic mid-IR spectral markers were identified in urine samples. Specifically, a 1545 cm-1 band increased in intensity with the progression and severity of GN in rats, mice and humans. Furthermore, its intensity declined significantly in response to corticosteroid treatment in nephritic rats. In conclusion, our results suggest that specific urinary FTIR biomarkers may provide a rapid, sensitive and novel non-invasive means of diagnosing inflammatory forms of GN, and for real-time monitoring of progress, and response to treatment.