Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions
✍ Scribed by Anton V. Brusnichkin; Dmitry A. Nedosekin; Ekaterina I. Galanzha; Yuri A. Vladimirov; Elena F. Shevtsova; Mikhail A. Proskurnin; Vladimir P. Zharov
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 457 KB
- Volume
- 3
- Category
- Article
- ISSN
- 1864-063X
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✦ Synopsis
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. 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)