Near-field infrared nanospectroscopy and super-resolution fluorescence microscopy enable complementary nanoscale analyses of lymphocyte nuclei
G. C. Ajaezi, M. Eisele, F. Contu, S. Lal, A. Rangel-Pozzo, S. Mai and K. M. Gough
Analyst, Advance Article (2018)
Recent super-resolution fluorescence microscopy (3D-Structured Illumination Microscopy, 3D-SIM)
studies have revealed significantly altered nuclear organization between normal lymphocyte nuclei and
those of classical Hodgkin’s Lymphoma. Similar changes have been found in Multiple Myeloma (MM)
nuclei, as well as in a premalignant condition, Monoclonal Gammopathy of Unknown Significance
(MGUS). Using 3D-SIM, an increase in DNA-poor and DNA-free voids was evident in reconstructed
3D-SIM images of diseased nuclei at 40 nm pixel resolution (x,y: 40 nm, z: 80 nm). At best, far-field FTIR
imaging yields spatially resolved images at ∼500 nm spatial resolution; however, near-field infrared
imaging breaks the diffraction limit at a scale comparable to that of 3D-SIM, providing details on the order of
30 nm spatial resolution. We report here the first near-field IR imaging of lymphocyte nuclei, and far-field IR
imaging results of whole lymphocytes and nuclei from normal human blood. Cells and nuclei were mounted
on infrared-compatible substrates, including CaF2, undoped silicon wafers, and gold-coated silicon wafers.
Thermal source far-field FTIR images were obtained with an Agilent-Cary 620 microscope, 15× objective,
0.62 NA and 64 × 64 array Focal Plane Array detector (University of Manitoba), or with a similar microscope
equipped with both 15× and 25× (0.81 NA) objectives, 128 × 128 FPA and either thermal source or synchrotron
source (single beam) infrared light at the Advanced Light Source (ALS), LBNL, Berkeley CA. Near-field IR
spectra were acquired at the ALS, on the in-house SINS equipment, as well as with a Neaspec system, both
illuminated with synchrotron light. Finally, some near-field IR spectra and images were acquired at Neaspec
GmbH, Germany. Far-field IR spectra of normal cells and nuclei showed the characteristic bands of DNA and
proteins. Near-field IR spectra of nuclei showed variations in bands assigned to protein and nucleic acids
including single and double-stranded DNA. Near-field IR images of nuclei enabled visualization of protein and
DNA distribution in spatially-resolved chromosome territories and nuclear pores.