Issue 19, 2013

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 and 1 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.

Graphical abstract: Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells

Article information

Article type
Paper
Submitted
22 Feb 2013
Accepted
26 Jun 2013
First published
03 Jul 2013
This article is Open Access
Creative Commons BY-NC license

Analyst, 2013,138, 5610-5618

Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells

E. C. Mattson, M. Unger, S. Clède, F. Lambert, C. Policar, A. Imtiaz, R. D'Souza and C. J. Hirschmugl, Analyst, 2013, 138, 5610 DOI: 10.1039/C3AN00383C

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