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Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions
| Content Provider | PubMed Central |
|---|---|
| Author | Brusnichkin, Anton V. Nedosekin, Dmitry A. Galanzha, Ekaterina I. Vladimirov, Yuri A. Shevtsova, Elena F. Proskurnin, Mikhail A. Zharov, Vladimir P. |
| 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. 103 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 ultra-small 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. |
| Related Links | http://dx.doi.org/10.1002/jbio.201000012 |
| Starting Page | 791 |
| File Format | |
| ISSN | 18640648 |
| e-ISSN | 18640648 |
| Journal | Journal of Biophotonics |
| Issue Number | 12 |
| Volume Number | 3 |
| Language | English |
| Publisher Date | 2010-12-01 |
| Access Restriction | Open |
| Subject Keyword | Research in Higher Education |
| Content Type | Text |
| Resource Type | Article |
