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

Abstract

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 infrared range. In this work, FTIR spectroscopic imaging in transmission using a focal plane array (96 x 96 pixels) has been applied to the characterisation of Barrett's esophageal adenocarcinoma. Spectral images were acquired in transmission mode. To correct optical aberrations, infrared transparent lenses were used of the same material 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 is presented where spectral images obtained with added lens are stitched together such that the dataset contained a representative section of the esophageal tissue. Images were also acquired in transmission mode using high-magnification optics for enhanced spatial resolution, and were also acquired using a germanium micro-ATR objective for comparison. 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 added lens using high-magnification optics was assessed by measurements of a sharp interface of polymer laminate, which was also compared with the spatial resolution achieved in micro ATR-FTIR imaging. The resolution was determined from the change in absorbance of spectral bands from one side of the polymer interface to the other. In transmission mode using the lens, it was determined to be 12 µm and using micro-ATR, the resolution was 3 µm for the band at ca. 6 µm The spatial resolution was also assessed with and without the added lens, in normal and high-magnification mode using a USAF target.Spectroscopic images of cells in transmission 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 in 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.2

Clinical applications of infrared and Raman spectroscopy: state of play and future challenges

“…The long standing problem of optical aberrations in FTIR microspectroscopy have been systematically addressed [103][104][105][106][107][108], by introducing a novel, practical, yet elegant, approach to remove scattering artefacts, whereby the tissue biopsy is inverted such that the CaF2 window sits between the biopsy and objective. Then, an added lens, of the same material as the window, is placed on top of the window, centred to the optical axis, to form a pseudo hemisphere.…”

“…Both these advantages make this added lens approach ideal for measuring biopsies. [106,108] The originality and simplicity of this approach has surprised many working in the field, as it was thought that optical aberrations are unavoidable when infrared windows were used as a substrate. [109] The other important advantages to this added lens approach are the increase in magnification and the removal of chromatic aberration, the latter of which would otherwise occur when measuring samples through a window, for example imaging of live cells in microfluidic devices.…”

“…[109] The other important advantages to this added lens approach are the increase in magnification and the removal of chromatic aberration, the latter of which would otherwise occur when measuring samples through a window, for example imaging of live cells in microfluidic devices. [108,110] As such, this method is a state-of-the-art method to acquire high-quality spectra without chromatic aberration and scattering.…”

“…In particular, a stringent test for the assessment of the spatial resolution achieved in FTIR spectroscopic imaging in both ATR and transmission modes has been presented and it has been demonstrated that the true spatial resolution is much lower than what is expected from the projected pixel size, which is often used, albeit incorrectly, as a value for spatial resolution. [103,105,108] Recently micro ATR-FTIR spectroscopic images of living cells have been analysed with disrelation mapping which allowed differentiating molecular states of water as well as studying hydration of proteins within living cells (Figure 4) [112]. he most recent developments in the area of FTIR spectroscopic imaging have been reviewed, where the current state of the art is summarised in detail.…”

Infrared imaging in histopathology: Is a unified approach possible?

“…Furthermore, some of the algorithms available require intensive computation that is not easily available if billions of spectra are to be processed. Correcting lenses could bring help in dealing with some chromatic aberration and scattering effects [18,28,63].…”