High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron beams

Abstract

Conventional Fourier-transform infrared (FTIR) microspectroscopic systems are limited by an inevitable trade-off between spatial resolution, acquisition time, signal-to-noise ratio (SNR) and sample coverage. We present an FTIR imaging approach that substantially extends current capabilities by combining multiple synchrotron beams with wide-field detection. This advance allows truly diffraction-limited high-resolution imaging over the entire mid-infrared spectrum with high chemical sensitivity and fast acquisition speed while maintaining high-quality SNR.

Figure 1

FTIR imaging with a multibeam synchrotron source. (a) Schematic of the experimental setup. M1–M4 are mirror sets. (b) A full 128 × 128 pixel FPA image with 12 overlapping beams illuminating an area of ~50 μm × 50 μm. Scale bar, 40 μm. (c) A visible-light photograph of the 12 beams projected on a screen in the beam path (dashed box in a). Scale bar, ~1.5 cm. We display the beams as one beam from then on in the schematics. Each beam exhibits a shadow cast by a cooling tube upstream, which is not shown in a. (d) Long-exposure photograph showing the combination of the 12 individual beams into the beam bundle by mirrors M3 and M4. Scale bar, ~20 cm.

Figure 2

Chemical images from various FTIR systems. (a–d) The same cancerous prostate tissue section (area, ~280 μm × 310 μm) measured with different instruments, using the integrated absorbance of the CH-stretching region (2,800–3,000 cm⁻¹), without dyes or stains. We processed all images identically (baseline correction only) and used the same color scale (color bar in a; AU, absorbance units). Scale bars, 100 μm and in insets, 10 μm. Images acquired with a conventional table-top system (PerkinElmer Spotlight) equipped with a thermal source in raster-scanning mode (10 μm × 10 μm; a) and linear array mode (6.25 μm × 6.25 μm; b), with an FTIR imaging system (Varian Stingray) equipped with a 64 pixel × 64 pixel FPA (5.5 × 5.5 μm per pixel at the sample plane; c) and with our multibeam synchrotron-based imaging system (pixel size, 0.54 μm × 0.54 μm; d). (e) Hematoxylin and eosin (H&E)-stained prostate tissue (diameter, 0.75 mm). Scale bar, 100 μm. Dashed box specifies the corresponding area of a serial, unstained section from which we generated images in a–d. (f) Typical unprocessed spectra from a single pixel acquired with each instrument (crosshairs in a–d indicate corresponding pixel positions in the infrared images).

Figure 3

High-resolution multibeam synchrotron FTIR imaging. (a) Hematoxylin and eosin (H&E)-stained image of cancerous prostate tissue with chronic inflammation obtained using visible light microscopy. (b,c) Multibeam synchrotron absorbance images obtained from an unstained serial section of the sample shown in a. Spatial detail in images from the new system is highlighted for lymphocytes (blue arrow) and red blood cells (red arrow). (d) Image of the same unstained section imaged with a conventional table-top system (PerkinElmer Spotlight, linear array mode). (e) Expanded views of the boxed area in b showing the typical appearance of lymphocytes in H&E stained samples (top), the new system (bottom left) and a conventional table-top instrument (bottom right). (f) H&E-stained visible light image (top), asymmetric CH-stretching (2,840 cm⁻¹, center) and collagen-specific (1,245 cm⁻¹, bottom) infrared images of an unstained section of normal breast tissue (terminal ductal lobular unit region). Epithelial (green arrow) and intralobular stromal regions (magenta arrow) are highlighted. (g) Spectra of epithelial and stromal cells recorded with a multibeam synchrotron versus a thermal source. (h) Absorbance image (2,840 cm⁻¹; top) of an unstained cancerous prostate tissue showing two benign prostate glands. Inset, potential presence of basement membrane at the interface between stroma and epithelium is marked (arrows). Image (bottom) showing epithelial (green) and stromal (magenta) cells classified using previous algorithms. (i) Average spectra from epithelial, stromal (two each: one closer to the interface, one farther away), and interface pixels identified manually from data obtained using two different instruments. AU, absorbance units. Scale bars, 50 μm.