Subcellular mapping of living cells via synchrotron microFTIR and ZnS hemispheres

Abstract

FTIR imaging is a label-free, non-destructive method valuably exploited in the study of the biological process in living cells. However, the long wavelength/low spatial resolution and the strong absorbance of water are still key constrains in the application of IR microscopy ex vivo. In this work, a new retrofit approach based on the use of ZnS hemispheres is introduced to significantly improve the spatial resolution on live cell FTIR imaging. By means of two high refractive index domes sandwiching the sample, a lateral resolution close to 2.2 μm at 6 μm wavelength has been achieved, i.e. below the theoretical diffraction limit in air and more than twice the improvement (to ~λ/2.7) from our previous attempt using CaF2 lenses. The ZnS domes also allowed an extended spectral range to 950 cm−1, in contrast to the cut-off at 1050 cm−1 using CaF2. In combination with synchrotron radiation source, microFTIR provides an improved signal-to-noise ratio through the circa 12 μm thin layer of medium, thus allowing detailed distribution of lipids, protein and nucleic acid in the surround of the nucleus of single living cells. Endoplasmic reticula were clearly shown based on the lipid ν(CH) and ν(C=O) bands, while the DNA was imaged based on the ν(PO2−) band highlighting the nucleus region. This work has also included a demonstration of drug (doxorubicin) in cell measurement to highlight the potential of this approach. Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1245-x) contains supplementary material, which is available to authorized users.

Towards identifying the mode of action of drugs using live-cell FTIR spectroscopy

“…Using synchrotron sources, the measurement of infrared spectra of living cells within a reasonable time frame has been successfully demonstrated, 27,28 including the characterisation of normal cells from carcinoma, 29 response of cells to mechanical stress, 30 apoptotic pathways, 31 chemical stress, 12 optical stimulation, 32 protein aggregation, 17,33 drug actions, 10 toxicity in living algae 34 and imaging with subcellular spatial resolution using ZnS hemispheres. 35 However, access to synchrotron facilities are limited and therefore measurements of living cells that can be carried out in an ordinary laboratory remains highly desirable. The use of bench-top (non-synchrotron) FTIR imaging to measure a population of living cells in transmission mode has shown that the SNR can be improved by using principle component reconstruction, allowing the distinguishing of cells before and after the treatment of drugs.…”

Transmission Fourier Transform Infrared Spectroscopic Imaging, Mapping, and Synchrotron Scanning Microscopy with Zinc Sulfide Hemispheres on Living Mammalian Cells at Sub-Cellular Resolution

“…Fourier transform infrared (FT-IR) spectroscopic imaging of live cells has been shown to be a promising tool for cell biology studies. 1–11 However, with the relatively long wavelength of infrared (IR) light especially in the so-called fingerprint region, the spatial resolution obtained is usually limited to approximately 3–30 µm with a standard FT-IR imaging microscope, depending on the numerical aperture (NA) of the objective and the wavelength of light used to generate the FT-IR image. 12,13 This limits the study to the analysis of single cells for most mammalian cells, which have sizes in the 10–50 µm range, with subcellular features of a few micrometers and below, resulting in unresolved subcellular information.…”

“…United Stated Air Force resolution target feature of 2.19 µm could be resolved at wavelength of 6 µm. 1 ZnS has the advantage of having a much lower spectral cut-off value of ∼700 cm −1 than CaF 2 , which has a cut-off value of ∼1000 cm −1 ; therefore, it does not increase the noise level below ∼1100 cm –1 region as in the case when CaF 2 is used. This is particularly beneficial for the linear array and single-point mapping systems where the detectors sensitivity range can reach down to 720 cm −1 and 650 cm −1 , respectively.…”

Characterization of double-deformable-mirror adaptive optics for IR beam shaping in hyperspectral imaging

“…7 Synchrotron IR (SRIR) radiation, with three order of magnitude higher brilliance than thermal source, one order of magnitude larger spectral distribution than laser sources 8 and superior spectral stability to tunable lasers, is an ideal choice for FTIR microspectroscopy and imaging. In addition, the lowétendue of SRIR benefits FTIR such that the diffraction-limited resolution with high spectral quality can be achieved for both single detector 9 and FPA. To cope with the photon flux loss in confocal geometry, a high magnification objective (i.e.74x) is coupled to the FPA such that the oversampling can be implemented to realise the diffraction limited resolution with reasonable SNR level.…”