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Keywords:

  • photothermal spectroscopy;
  • photothermal microscopy;
  • imaging;
  • trace analysis;
  • molecular targeting;
  • cytochrome c;
  • mitochondria;
  • live cells

Abstract

Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single-channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 × 10–9 mol/L (80 attomols in the signal-generation zone); that is ca. 103lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 × 10–21 mol) in the ultrasmall irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)