title: Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells.
abstract: Advancements in widefield infrared spectromicroscopy have recently been demonstrated following the commissioning of IRENI (InfraRed ENvironmental Imaging), a Fourier Transform infrared (FTIR) chemical imaging beamline at the Synchrotron Radiation Center. The present study demonstrates the effects of magnification, spatial oversampling, spectral pre-processing and deconvolution, focusing on the intracellular detection and distribution of an exogenous metal tris-carbonyl derivative 1 in a single MDA-MB-231 breast cancer cell. We demonstrate here that spatial oversampling for synchrotron-based infrared imaging is critical to obtain accurate diffraction-limited images at all wavelengths simultaneously. Resolution criteria and results from raw and deconvoluted images for two Schwarzschild objectives (36×, NA 0.5 and 74×, NA 0.65) are compared to each other and to prior reports for raster-scanned, confocal microscopes. The resolution of the imaging data can be improved by deconvolving the instrumental broadening that is determined with the measured PSFs, which is implemented with GPU programming architecture for fast hyperspectral processing. High definition, rapidly acquired, FTIR chemical images of respective spectral signatures of the cell 1 and shows that 1 is localized next to the phosphate- and Amide-rich regions, in agreement with previous infrared and luminescence studies. The infrared image contrast, localization and definition are improved after applying proven spectral pre-processing (principal component analysis based noise reduction and RMie scattering correction algorithms) to individual pixel spectra in the hyperspectral cube.
authors.affiliation: Physics Dept., University of Wisconsin-Milwaukee, Milwaukee, WI, USA. cjhirsch@uwm.edu.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Mattson
authors.forename: Miriam
authors.initials: M
authors.lastname: Unger
authors.forename: Sylvain
authors.initials: S
authors.lastname: Clède
authors.forename: François
authors.initials: F
authors.lastname: Lambert
authors.forename: Clotilde
authors.initials: C
authors.lastname: Policar
authors.forename: Asher
authors.initials: A
authors.lastname: Imtiaz
authors.forename: Roshan
authors.initials: R
authors.lastname: D'Souza
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Hirschmugl
mesh_terms: D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*