Molecular Imaging
Spectroscopic OCT
Spectroscopic OCT is an extension of OCT technology where not only the structural information, but also the spectroscopic information is retrieved. It is based on the principle that the bandwidth of a light source used in OCT is broad; therefore by using appropriate time-frequency analysis, a depth-resolved spectroscopy study can be performed. Spectroscopic OCT has at least two imaging targets: imaging spectral absorption and spectral scattering. These can be used to detect either endogenous molecules or exogenous agents.
OCT Spectroscopic OCT has many different contrast contributions, e.g., spectral absorption and spectral scattering. It is important to separate them so that more accurate tissue properties can be retrieved. This is possible because different contrast mechanisms often have different spectral or range properties. For example, a least-squares algorithm can separate the attenuation due to absorption and scattering based on spectral properties.
Targeted imaging
Functionalization of agents for specific targeting to molecular sites is an important tool for molecular specific imaging. Along with the synthesis of these nanoprobes, the chemistry of bioconjugation has also contributed significantly to the development of targeted probes that can be conjugated to different biomarkers, proteins, peptides, or oligonucleotides. Development of functionalized nanoprobes for in vivo targeting is extremely challenging and involves multidisciplinary skills. A number of factors determine the clinical usefulness of the nanoparticles. Their physical and functional biokinetic properties, clearance profiles, biodistribution, biocompatibility, and long-term toxicity need to be carefully considered for their potential use in in vivo applications.
RGD targeted protein microspheres
Air-filled microbubbles and perfluorocarbon-filled protein microspheres that are commercially available as ultrasound contrast agents have been reported in the past as scattering contrast agents in OCT. Protein microspheres with different cores can be fabricated using high frequency ultrasound. A wide variety of materials like gases, lipids, and water can be selected for the core region of these protein-shell microspheres [Figure]. The encapsulation process is mediated through sonication using high frequency ultrasound. The protein shell is crosslinked through the reduction of disulphide bonds by the radicals formed during the sonication process. The sizes of microspheres can be controlled from hundreds of nanometers to a few tens of microns by the addition of surfactants to change the surface tension of the protein mixture.
Antibody conjugated Iron Oxide Nanoparticles
More figures on in vivo MM-OCT imaging /MRI
Accuracy of retrieval of dye concentration from SOCT signal using different time-frequency analyses
| Xu C, Kamalabadi F, Boppart SA. Comparative performance analysis of time-frequency distributions for spectroscopic optical coherence tomography. Applied Optics, 44:1813-1822, 2005. | PubMed Abstract |
Wavelength-dependent spectral scattering The spectral-scattering information contained within the SOCT signal can be used diagnostically to assess scatterer size and spatial distribution in cells and tissues. Dominant scatterers include nuclei and mitochondria. This technique is similar to Light Scattering Spectroscopy, except SOCT can perform analysis in depth, and in three-dimensions.
Spectral-scattering analysis of SOCT signals can differentiate scatterer size.
| Xu C, Carney PS, Boppart SA. Wavelength-dependent scattering in spectroscopic optical coherence tomography. Optics Express, 13:5450-5462, 2005. | n/a |
Spectroscopic spectral-domain optical coherence microscopy of adipose and muscle tissue. Spectral analysis of scatterer size enhances contrast
| Xu C, Vinegoni C, Ralston TS, Luo W, Tan W, Boppart SA. Spectroscopic spectral-domain optical coherence microscopy. Opt. Lett., 31:1079-1081, 2006. | PubMed Abstract |
Contrast Agents
Novel molecularly-targeted contrast agents are being developed which enhance the diagnostic imaging capabilities of OCT and other non-fluorescent or non-bioluminescent imaging techniques. These include scattering microspheres as contrast and drug-delivery agents, magnetomotive nanoparticles, plasmon-resonant nanoparticles, and absorbing near-infrared dyes. In addition, there are a large number of optical properties and characteristics from contrast agents that can be leveraged to enable optical molecular imaging of cells and tissues.
Metaphorical diagram of a contrast agent showing the wide range of substrates, optical properties, and applications.
| Boppart SA, Oldenburg AL, Xu C, Marks DL. Optical probes and techniques for molecular contrast enhancement in coherence imaging. Article and Cover Figure. J Biomedical Optics, 10:041208, 2005. | PubMed Abstract |
TEM of gold microspheres.
(a) OCT image of an in vivo mouse liver before contrast agents were introduced. (b) OCT image of an in vivo mouse liver after injection of gold microspheres into the mouse tail vein. Liver sinusoids are now apparent.
| Lee TM, Toublan FJ, Sitafalwalla S, Oldenburg AL, Suslick KS, Boppart SA. Engineered microsphere contrast agents for optical coherence tomography. Optics Letters, 28(17): 1546-1548, Sept 2003. | PubMed Abstract |
Magnetomotive OCT of single cells dispersed in a 3-D gel. Cells containing magnetite are clearly identified.
| Oldenburg AL, Gunther JR, Boppart SA. Imaging magnetically labeled cells with magnetomotive optical coherence tomography. Optics Letters, 30:747-749, 2005. | PubMed Abstract |
In vivo magnetomotive OCT of magnetic nanoparticles in a Xenopus (African frog) tadpole model.
| Oldenburg AL, Toublan FJ, Suslick KS, Wei A, Boppart SA. Magnetomotive contrast for in vivo optical coherence tomography. Optics Express, 13:6597-6614, 2005. | n/a |
Plasmon-resonant gold nanorods exhibit specific longitudinal (L) and transverse (T) resonances and can function as highly absorbing and scattering contrast agents.
| Oldenburg AL, Hansen MN, Zweifel DA, Wei A, Boppart SA. Plasmon-resonant gold nanorods as low backscattering albedo contrast agents for optical coherence tomography. Opt. Express 14(15):6724-6738, 2006. | n/a |
Tumor cell targeting. RGD-peptide conjugated microspheres containing fluorescent Nile Red dye are shown targeting to HT29 tumor cells.
| Toublan FJJ, Boppart SA, Suslick KS. Tumor targeting by surface modified microspheres. J. Am. Chem. Soc., 128:3472-3473, 2006. | PubMed Abstract |
In this study, we demonstrate the use of MM-OCT for quantitative measurement of magnetic iron-oxide nanoparticle transport and concentration in ex vivo muscle, lung and liver tissues. The effect of temperature on the dynamics of these nanoparticles is also analyzed. We observe that the rate of transport of nanoparticles in tissues is directly related to the elasticity of tissues, and describe how the origin of the MM-OCT signal is associated with nanoparticle binding. These results improve our understanding of how iron-oxide nanoparticles behave dynamically in biological tissues, which has direct implications for medical and biological applications of targeted nanoparticles for contrast enhancement and therapy.
The paper reports on the method's use in imaging mammary tumor tissue but Boppart said that the technology has many potential applications.
Boppart said the technique uses magnetic forces to move nanoparticles bound to proteins, cells, tissue; phase-sensitive OCT then measures changes in light scattering from the movement. The method's uniqueness comes from the ability to target the nanoparticles to a specific site, such as a tumor, where their multifunctionality can then be utilized.
The nanoparticles can provide information on contrast (especially valuable when it comes, for example, to distinguishing between healthy cells and cancerous cells) due to the fact that the MNPs are magnetic while the rest of the tissue is not. Another use is to record biomechanical tissue measurements (such as cell elasticity and viscosity, important factors in disease detection). It works by using a magnetic field to make the nanoparticles vibrate, thereby providing signals from the tissue through the rate and frequency of the movements.
Another use enabled by the technology is therapeutic. By turning up the frequency of the magnetic field, the nanoparticles move so quickly they begin to heat up, providing a possible treatment tool.
"So now we have a platform where we can target these to a tumor, find them with contrast, measure the mechanical properties, and treat it right there," Boppart said.
The magnetic nanoparticles in this project were antibody functionalized to target the human epidermal growth factor receptor 2 (HER2 neu) protein, a receptor which is over expressed in breast cancer. The fabrication of the antibody-directed functionalized nanoparticles was also rather unique for biomedicine: conjugating the antibody to the nanoparticle in a specific way to provide the highest target specificity to the receptor.
Nonlinear Interferometric Vibrational Imaging
NIVI is a CARS-based imaging technique that combines the broadband, high spectral resolution of spontaneous Raman spectroscopy with the fast acquisition rates of CARS microscopy. It uses broadband excitation fields and takes advantage of the coherent properties of CARS to perform heterodyne detection, which fully suppresses the background, allowing the acquisition of broadband, high resolution vibrational spectra.
The original concept of NIVI included the use of two broadband pulses chirped at different rates, the superposition of which would result in a field whose beating frequency is itself chirped. This way, the vibrational levels would be populated sequentially. This method favors an adiabatic transition of the bonds from the ground to the excited states.
The current NIVI system uses a different pulse design. Here, a transform limited broadband Stokes, and a chirped broadband pump are used. This approach is simpler because it does not require shaping the incident optical fields. Furthermore, the current scheme effectively avoids the generation of background.
| Marks DL, Boppart SA. Nonlinear interferometric vibrational imaging. Phys. Rev. Lett., 92:123905, 2004. | PubMed Abstract |
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| Marks DL, Vinegoni C, Bredfeldt JS, Boppart SA. Interferometric differentiation between resonant coherent anti-Stokes Raman scattering and nonresonant four-wave-mixing processes. Applied Phys. Lett., 85:5787-5789, 2004. | n/a | |
| Jones GW, Marks DL, Vinegoni C, Boppart SA. High-spectral-resolution coherent anti-Stokes Raman scattering with interferometrically-detected broadband chirped pulses. Opt. Lett., 31(10):1543-1545, 2006. | PubMed Abstract |
Principles of Operation
An important characteristic of 
for CARS is its exclusive dependence on the difference 
. This allows separating this four-wave-mixing process in two steps:
where Ep is the pump field, Es is the Stokes field, 
are the optical frequencies and 
is the third order susceptibility written as a function of the vibrational frequencies 
. In the first step, corresponding to Equation (1), the molecules in the ground vibrational state interact with two photons-the pump and the Stokes-causing a transition to an excited state. The totality of these excited states is represented by 
, a function that can be understood as a truncated and weighted version of that is activated by the cross correlation of the pump and Stokes fields.
In the second step, corresponding to Equation (2), the field interacts with to induce the nonlinear polarization 
that radiates the anti-Stokes field. A schematic of these steps is shown in the figure.
Schematic (not to scale) of the pulses incident to the sample in NIVI. TL=transform limited (constant spectral phase).
Microscopy Setup
The schematic of the microscopy setup used in our experiment is shown in the figure below. Pulses out of the compressor at 250 kHz repetition rate with 808 nm center wavelength and 25 nm bandwidth (FWHM) are used as the pump pulses for the CARS process, as well as a seed for the second-harmonic-generation optical parametric amplifier (OPA). The division of the pulses is done with a 90/10 beam splitter (10% of power for the CARS pump and 90% for the OPA). The OPA, generates an idler to be used as the Stokes pulses and a signal to be used as the reference for the heterodyne detection of the molecular anti-Stokes field. When transform limited, the duration of the pulses out of the compressor is ~100 fs. These pulses are chirped by passing them through 85 cm of BK7 glass, after which the duration of the pulses is ~6 ps. The pump and the Stokes beams are combined and focused onto the sample, which is placed on a translation stage for raster scanning. A delay is included in the pump path to temporally overlap the beginning of the pump and Stokes pulses at the sample. The resulting anti-Stokes signal is collected in the forward direction and is separated from the incident beams with a high pass filter. The anti-Stokes pulses are combined with the reference pulses from the OPA, diffracted by a grating focused to a line scan camera that acts as a spectrometer. A second delay is included in the reference path to assure that the interference fringes in the line camera are within the resolution limits of the camera.
Schematic of the experimental setup. Verdi-6, seed laser for Mira (6 W of power); Verdi-10, seed laser for Reg A (10 W of power); Reg A, Regenerative amplifier; OPA, optical parametric amplifier; BS, beam splitter; DM, dichroic mirror; HPF, high-pass filter; SF, spatial filter with telescope.
Experimental Demonstration
The pattern for the expected anti-Stokes pulses (schematic seen above) was observed in various organic samples. In the figure, an example is show for acetone, where its is clear to see that the resonant tail in the time resolved signal vanishes out of resonance.
Interferogram of four-wave-mixing in acetone at various vibrational excitation frequencies.
Applications
Chemical imaging of material samples and mammary tissue
NIVI has been proven successful in imaging and spectroscopically differentiating highly scattering material samples (top figure) and mammary tissue (bottom figure). Notice that spectral information is obtained at each pixel that closely resembles that of the gold standard Raman spectra.
Reconstruction of the spectra of silicone in the material sample. (a)Interference pattern at spectrometer. (b) Time evolution of the anti-Stokes signal (for both silicone and sodium silicate). (c) Phase and (d) magnitude of the complex vibrational spectrum (3) . (e) Obtained Im(3) and spontaneous Raman spectra. Dotted lines indicate the Stokes and reference spectral profiles. Images of sample (f) out of resonance at 2850 cm-1 and at resonances (g) 2906 cm-1 and (h) 2965 cm-1.
Vibrational image of mammary tissue. (a) Temporal evolution of the anti-Stokes beam. (b) Phase and (c) magnitude of the retrieved susceptibility. (d) Imaginary component of the susceptibility in comparison with spontaneous Raman spectrum. (e) NIVI hyperspectral cube image at 2855 cm-1 showing adipocytes, parenchyma, and connective tissue. (f) Stained histological (hematoxylin & eosin) image of sample in similar region. (g) Profile of NIVI image intensity along white line in (e). Dotted red lines delineate connective tissue, extracellular matrix, and parenchyma from adipocytes. Arrows indicate extracellular space between cells.
| Benalcazar WA, Chowdary PD, Jiang Z, Marks DL, Chaney EJ, Gruebele M, Boppart SA. High-speed nonlinear interferometric vibrational imaging of biological tissue with comparison to Raman microscopy. IEEE J. Sel. Topics Quantum Elect., 16:824-832, 2010. | n/a | n/a |
Fast spectroscopy for the differentiation of saturation levels in lipids
The high spectral resolution also allows for fast spectroscopic analysis of the levels of saturation in closely related oils. The fidelity in the degree of saturation was about the same that spontaneous Raman spectroscopy would obtain. This chemical differentiation can easily extended to the differentiation in other chemical domains in tissues, such as in the case of lipids and collagen in skin, as shown in the figure.
NIVI chemical imaging of an unstained skin section (interface between pig dermis and subcutaneous adipose tissue). Left: Total resonant power. Middle: Spatial map of lipid content (from black to green as fraction of maximum). Right: Spatial map of protein content (from black to blue as fraction of maximum). The section was 50 um thick. The insets shows average NIVI spectra corresponding to the green and blue color-coded regions, with vertical red lines indicating 2855 and 2935 cm-1 (CH2 and CH3 stretches).
| Chowdary PD, Benalcazar WA, Jiang Z, Marks DL, Boppart SA, Gruebele M. High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition. Analytical Chemistry, 82:3812-3818, 2010. | n/a | n/a |
Molecular histopathology with NIVI
Recently, we have concentrated efforts into chemically differentiating normal and tumor mammary tissue, as shown in the figure. Besides the intensity images showed to the left, each pixel contains a vibrational spectrum, the average of which are showed to the right for each of the four images. Notice that, besides observing disruption of the regular adiposite structures in tumors when compared to normal tissue, their chemical spectra also shows differences. In particular, the vibrational profiles for tumor are proper of collagen, while the spectral profile for normal tissue corresponds to lipids.
Spatial mapping of CH groups (present in different proportions in lipid and protein domains) in mammary tissue. (A) Endogenous-contrast NIVI images (color scale: blue to red, as percentage of CH vibrations) of one normal and three developed mammary tumors of various sizes. (B) Vibrational spectra for images in (A). Images and spectra reveal disruptions of lipid and collagen domains in adipose and tumor tissues, with a predominance of collagen-based structures in tumors.
