The spectral-domain OCT system consists of a Ti:Al2
femtosecond laser (KMLabs, Inc.) producing 800 nm light with a bandwidth of 120 nm (providing lc
~3 μm axial resolution). This is pumped by 4.5 W of 532 nm light from a frequency-doubled Nd:YVO4
laser (Coherent, Inc.) The broadband light is launched into a single-mode fiber interferometer which is divided into the sample arm and a stationary reference arm. The sample beam is steered using galvanometer mirrors placed one focal length above a 30 mm achromatic imaging lens (providing Δx
~12 μm transverse resolution). A water-jacketed electromagnet described previously [18
] is placed between the lens and sample allowing the beam to pass through the central bore. A 250 W power supply is used to achieve a magnetic field of ~0.08 T and gradient of ~15 T/m within the sample imaging volume. The interference of the reference and sample beams is measured with a spectrometer described previously [24
], composed of a grating, imaging lens, and line camera (Pirahna 2, Dalsa Inc.) with capability of 33 kHz line rates. The spectrometer resolution was designed to provide an optical imaging depth of 2 mm.
The magnetic modulation frequency fB
was chosen to be 55.6 Hz for tumor tissues and 100 Hz for tissue phantoms, based on the best response (highest A
) achieved from these samples. A lower axial scan rate of 1 kHz was chosen to avoid excessive oversampling. The camera exposure time was 250 μs. The root-mean-square phase noise measured from a stationary tissue specimen at 1 kHz without transverse scanning was 0.2 rad. B-mode scans over 2.5 mm were performed with a scan velocity v
of 0.625 mm/s, corresponding to a right-hand-term in Eq. (10)
of 104 Hz and thus satisfying the criterion for fB
> 52 Hz. Each frame consisted of 4000 pixels width by 1024 pixels depth, taking 4 seconds to acquire. Each image was acquired twice, once with the magnetic field modulated and once with the field off, resulting in a total acquisition time of 8 s per MMOCT image. 3-D sampling was performed on each sample by acquiring 6 B-mode images with 0.5 mm spacing in y
, resulting in a total imaging area of 2.5 × 2.5 mm. (The large spacing in y
was chosen as a tradeoff between larger sample areas and shorter imaging times.)
MMOCT images were generated according to Eqs. (8)
, and (13)
. Initially, the data collected from the line camera is resampled to provide S
) evenly sampled in frequency ω
. Median filtering of Smm
was performed over 23 × 23 μm. The bandpass filter width was chosen to pass transverse features of Smm
up to a spatial frequency of 1/(32 μm). All images were downsampled by a factor of fz
for portability and cropped to 800 pixels in z
to avoid edge effects near the bottom and top of the image. The mean Smm
with and without the mechanical phase lag filter
were computed for each image. MMOCT images were rendered for display by applying
pointwise to Smm
at each pixel. Then, for each set of 6 images, the mean and standard deviation of the image-averaged Smm
and image-averaged ϕ
were computed. This was all performed in post-processing using a Matlab® script which requires 15 s per image.
Two types of MNPs with similar properties were used (see ). The first type, Sigma-Aldrich #637106, are approximately 20–30nm in diameter, composed of pure magnetite (Fe3O4) and are without any surface coating. We will refer to these as bare MNPs. These were used for preparation of tissue phantoms because of their miscibility in silicone oils. The second type, Ocean NanoTech #SHP-20, are significantly more monodisperse in size at ~20nm, composed of a combination magnetite/maghemite core (exact ratio unknown) and a polymer coating with a hydrophilic, COOH-terminated outer surface. We will refer to these as COOH-MNPs. These are stable in aqueous solutions (including saline solutions), and were used for the tissue imaging study.
Transmission electron micrographs of MNPs. (a) 20–30nm bare MNPs. (b) 20nm COOH-terminated MNPs.
Transmission electron microscopy (Philips CM200, FEI Company) was performed on each type of MNP for sizing. SQUID (superconducting quantum interference device) magnetometry (1T MPMS, Quantum Design, Inc.) showed the bare MNPs exhibited a volume magnetic susceptibility χ = 4.1, saturation magnetization Msat = 93 emu/g Fe, and remanence of 7 emu/g Fe. In comparison, the COOH-MNPs exhibited a χ = 2.5, Msat = 105 emu/g Fe, and remanence of 0.3 emu/g Fe. We expect the coercive field to be small because the MNPs are on the order of a single domain size; lacking significant remanance, they can be approximated as superparamagnetic. Inductively-coupled plasma mass spectrometry (OES Optima 200 DV, Perkin Elmer) revealed that, due to their polymer coating, the COOH-MNPs consist of 34% Fe by weight, compared to 72% Fe for the bare MNPs.
Silicone tissue phantoms which have comparable optical and mechanical properties to tissue were prepared exactly in the same way as reported previously [18
], except we note a typographical error in [18
] where the concentration of TiO2
used is actually 4 mg/g. Briefly, a mixture of crosslinking and non-crosslinking polymers is used to provide a tissue-like viscoelastic medium, and TiO2
microparticles are added to qualitatively match the OCT signal achieved from 2% intralipid (~40 cm−1
scattering coefficient). Varying concentrations of bare MNPs are added and the samples are homogenized via sonication before crosslinking in an oven. SQUID magnetometry was also performed on the tissue phantom medium without added MNPs, resulting in a measured magnetic susceptibility of χ
. In comparison, human tissues are known to have a susceptibility typically within 20% of that of pure water, χ = 9×10−6
Mammary tumors were induced in a Wistar-Furth female inbred rat (The Jackson Laboratory, Bar Harbor, ME) via intraperitoneal injection of a carcinogen (N
-nitrosourea) according to a protocol described in detail previously [25
]. After euthanasia, a tumor of 1.4 × 1.6 × 0.6 cm dimensions was harvested from the right groin area and stored at −80°C before imaging. MMOCT imaging was performed immediately after thawing the tumor. The tumor was subsequently immersed in a saline solution with ~4 mg/g COOH-MNPs for 15 minutes at room temperature, rinsed vigorously in pure saline for ~1 minute, and imaged using MMOCT. (The relatively large MNP concentration was chosen to ensure positive results for this single tumor). Again, the tumor was immersed in the MNP solution and the process of rinsing and imaging repeated to collect data for cumulative immersion times of 30, 60, 90, and 120 minutes. Every effort was made to image the same tumor surface area, however, exact registration between successive images was not maintained.