Single cell analysis

Although there are numerous drivers for the development of single cell IR spectroscopy measuring an infrared absorbance spectrum from a single biological cell, is an inherently flawed process. Given that the diameter of typical human cells are in the region ~8–30 µm and the size of nuclei and other organelles range from 1 – 10 µm, it is clear that mid-infrared radiation with wavelengths of 3-10 µm will scatter strongly from such samples. This scattering, dominated by Mie scattering, will significantly distort the measured spectrum such that it appears significantly different from the pure absorbance spectrum. This distortion often termed the dispersion artefact has hampered progress in this field since both intensity and position of absorption bands can alter significantly as a result. In this project we have identified the underlying causes of the most prominent spectral distortions namely resonant Mie scattering (RMieS). In collaboration with Achim Kohler at Nofima in Norway, we have now developed a correction algorithm (RMieS-EMSC) that removes the distortions to give a corrected spectrum that resembles a pure absorbance spectrum.

Single cell analysis

Work is continuing on this project to improve the correction method further however a preliminary version of the algorithm is available under License and is free to use for all academic users. (A final version will shortly be available on this website but for those interested in trying the preliminary version please e-mail Paul Bassan: paul.bassan @ manchester.ac.uk)

Publications associated with this project

RMieS-EMSC correction for infrared spectra of biological cells: Extension using full Mie theory and GPU computing
P. Bassan, A. Kohler, H. Martens, J. Lee, E. Jackson, N. Lockyer, P. Dumas, M. Brown, N. Clarke and P. Gardner
Journal of Biophotonics (accepted 05/05/2010)

Resonant Mie Scattering (RMieS) Correction of Infrared Spectra from Highly Scattering Biological Samples
Paul Bassan, Achim Kohler, Harald Martens, Joe Lee, Hugh J. Byrne, Paul Dumas, Ehsan Gazi, Michael Brown, Noel Clarke, Peter Gardner
Analyst, DOI:10.1039/B921056C.

Resonant Mie scattering in Infrared spectroscopy of biological materials – understanding the “dispersion artefact”
Paul Bassan, Hugh J. Byrne, Frank Bonnier, Joe Lee, Paul Dumas, Peter Gardner
Analyst, 134 (2009), 1586—1593

Reflection contributions to dispersion artefact in FTIR spectra of cellular samples
P Bassan, H J Byrne, J Lee, F Bonnier, C. Clarke, P Dumas, E Gazi, M D Brown, N W Clarke, P Gardner
Analyst, 134, (2009), 1171-1175

Optical artefacts in transflection mode FTIR microspectroscopic images of single cells on a biological support:the effect of back-scattering into collection optics
J. Lee, E Gazi, J Dwyer, M. D. Brown, N. W. Clarke, P. Gardner
Analyst 132 (2007) 750 – 755

Funding

EPSRC/RSC Analytical Studentship programme

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