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. 2011 Sep 1;2(9):2470-83.
doi: 10.1364/BOE.2.002470. Epub 2011 Jul 29.

CARS Microscopy for the Visualization of Micrometer-Sized Iron Oxide MRI Contrast Agents in Living Cells

Free PMC article

CARS Microscopy for the Visualization of Micrometer-Sized Iron Oxide MRI Contrast Agents in Living Cells

Gianluca Rago et al. Biomed Opt Express. .
Free PMC article

Abstract

Micrometer-sized iron oxide particles (MPIOs) attract increasing interest as contrast agents for cellular tracking by clinical Magnetic Resonance Imaging (MRI). Despite the great potential of MPIOs for in vivo imaging of labeled cells, little is known on the intracellular localization of these particles following uptake due to the lack of techniques with the ability to monitor the particle uptake in vivo at single-cell level. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy enables non-invasive, label-free imaging of MPIOs in living cells with sub-micron resolution in three dimensions. CARS allows simultaneous visualization of the cell framework and the MPIOs, where the particles can be readily distinguished from other cellular components of comparable dimensions, and localized inside the cell.

Keywords: (020.4180) Multiphoton processes; (160.4236) Nanomaterials; (170.3880) Medical and biological imaging; (180.6900) Three-dimensional microscopy; (190.4380) Nonlinear optics, four-wave mixing; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering.

Figures

Fig. 1
Fig. 1
Energy schemes of the nonresonant (a and b) and resonant CARS (c) process. (d) Normalized CARS spectrum of tripalmitin; the arrows at 2845 cm−1 and 3000 cm−1 indicate the typical response of biological matter at the frequencies used for on- and off-resonance CARS measurements.
Fig. 2
Fig. 2
Brightfield microscopy (a), on-resonance (b) and off-resonance (c) CARS images of a 10 × 30 μm2 –sized region of dried solution of MPIOs. Scale bar 5 μm. The normalized intensity of the signal measured from MPIOs on- and off resonance is identical within the variations that result from frequency tuning.
Fig. 3
Fig. 3
(a) Brightfield microscopy, (b) on-resonance CARS and(c) off-resonance CARS images of a HuH7 cell in absence of MPIOs labels. Grayscale values vary from 3 to 60 in (b) and from 6 to 20 in (c)
Fig. 4
Fig. 4
(a) On-resonance and (b) off-resonance CARS images of a HuH7 cell incubated with MPIO solution. (c) is the overlay of the on- and off-resonance images where the former appears in red, and the background corrected off-resonance response appears in green. Grayscale values vary from 3 to 40 in (a) and from 3 to 25 in (b). The overlay image obtained from on and off-resonance measurements allows identification of a single iron oxide particle in the lower part of the cell (green spot).
Fig. 5
Fig. 5
(a) Brightfield microscopy image of a HuH7 cell incubated with MPIOs. (b-f) Overlay of on-resonance (red) and off-resonance (green) CARS images of the same cell with descending axial position (separated by 1 μm). Two internalized iron oxide particles (green) can be identified from the overlay images.

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