Transmission Fourier Transform Infrared Spectroscopic Imaging, Mapping, and Synchrotron Scanning Microscopy with Zinc Sulfide Hemispheres on Living Mammalian Cells at Sub-Cellular Resolution.

ABSTRACT

Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by infrared (IR) wavelength making subcellular study challenging. Recently, we have reported, with the use of a prototype zinc sulfide (ZnS) transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron-IR microscopy with a 36× objective with numerical aperture of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron radiation single-point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time, while measurements from the scanning methods produced a smoother image. Synchrotron radiation single-point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.