Correlative Synchrotron Fourier Transform Infrared
Spectroscopy and Single Molecule Super Resolution Microscopy for the
Detection of Composition and Ultrastructure Alterations in Single
Cells
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Abstract
Single
molecule localization microscopy (SMLM) and synchrotron
Fourier transform infrared (S-FTIR) spectroscopy are two techniques
capable of elucidating unique and valuable biological detail. SMLM
provides images of the structures and distributions of targeted biomolecules
at spatial resolutions up to an order of magnitude better than the
diffraction limit, whereas IR spectroscopy objectively measures the
holistic biochemistry of an entire sample, thereby revealing any variations
in overall composition. Both tools are currently applied extensively
to detect cellular response to disease, chemical treatment, and environmental
change. Here, these two techniques have been applied correlatively
at the single cell level to probe the biochemistry of common fixation
methods and have detected various fixation-induced losses of biomolecular
composition and cellular ultrastructure. Furthermore, by extensive
honing and optimizing of fixation protocols, many fixation artifacts
previously considered pervasive and regularly identified using IR
spectroscopy and fluorescence techniques have been avoided. Both paraformaldehyde
and two-step glutaraldehyde fixation were identified as best preserving
biochemistry for both SMLM and IR studies while other glutaraldehyde
and methanol fixation protocols were demonstrated to cause significant
biochemical changes and higher variability between samples. Moreover,
the potential complementarity of the two techniques was strikingly
demonstrated in the correlated detection of biochemical changes as
well as in the detection of fixation-induced damage that was only
revealed by one of the two techniques