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Ultrasensitive Label-Free Photothermal Imaging, Spectral Identification, and Quantification of Cytochrome C in Mitochondria, Live Cells, and Solutions

Ultrasensitive Label-Free Photothermal Imaging, Spectral Identification, and Quantification of Cytochrome C in Mitochondria, Live Cells, and Solutions

Anton V Brusnichkin, Dmitry A Nedosekin, Ekaterina I Galanzha, Yuri A Vladimirov, Elena F Shevtsova, Mikhail A Proskurnin, Vladimir P Zharov

J Biophotonics. 2010 Dec;3(12):791-806.

PMID: 20572284

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 x 10(-9) mol/L (80 attomols in the signal-generation zone); that is ca. 10³ lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 x 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42136	pH PT			Price

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