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 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.
Brusnichkin
Anton V
AV
Chemistry Department, M. V. Lomonosov Moscow State University, Vorob'evy Hills 1/3, Moscow 119991, Russia.
Nedosekin
Dmitry A
DA
Galanzha
Ekaterina I
EI
Vladimirov
Yuri A
YA
Shevtsova
Elena F
EF
Proskurnin
Mikhail A
MA
Zharov
Vladimir P
VP
D045304:Cytochromes c / Q000378:metabolism*; D003952:Diagnostic Imaging / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007624:KB Cells / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D010783:Photometry / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*
D045304:Cytochromes c