Ultrasensitive label‐free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

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. 10 3 lower 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)