Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment.

INTRODUCTION: KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA). PATIENTS AND METHODS: Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations. RESULTS: Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004). CONCLUSION: Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Evaluating multi-exposure speckle imaging estimates of absolute autocorrelation times.

Multi-exposure speckle imaging (MESI) is a camera-based flow-imaging technique for quantitative blood-flow monitoring by mapping the speckle-contrast dependence on camera exposure duration. The ability of laser speckle contrast imaging to measure the temporal dynamics of backscattered and interfering coherent fields, in terms of the accuracy of autocorrelation measurements, is a major unresolved issue in quantitative speckle flowmetry. MESI fits for a number of parameters including an estimate of the electric field autocorrelation decay time from the imaged speckles. We compare the MESI-determined correlation times in vitro and in vivo with accepted true values from direct temporal measurements acquired with a photon-counting photon-multiplier tube and an autocorrelator board. The correlation times estimated by MESI in vivo remain on average within 14±11% of those obtained from direct temporal autocorrelation measurements, demonstrating that MESI yields highly comparable statistics of the time-varying fields that can be useful for applications seeking not only quantitative blood flow dynamics but also absolute perfusion.

Narrow band imaging of squamous cell carcinoma tumors using topically delivered anti-EGFR antibody conjugated gold nanorods.

BACKGROUND: Nanoparticles have recently gained interest as exogenous contrast agents in a variety of biomedical applications related to cancer detection and treatment. The objective of this study was to determine the potential of topically administered antibody conjugated gold nanorods (GNRs) for imaging squamous cell carcinomas (SCCs) of the skin using near-infrared narrowband imaging (NBI). Near-infrared (NIR) NBI images narrow wavelength bands to enhance contrast from plasmonic particles in a wide field portable and noncontact device that is clinically compatible for real-time tumor imaging and tumor margin demarcation. STUDY DESIGN: We conjugated GNRs to Cetuximab, a clinically approved humanized antibody that targets the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of many tumor cells, especially SCCs. We excised subcutaneous xenografts of SCCs (A431) from Swiss nu/nu mice and divided the tumors into two groups: (1) the targeted group (Cetuximab conjugated GNRs) and (2) the control group (polyethylene glycol-conjugated GNRs). After topical application of particles and incubation for 30 minutes, the tumors were washed and imaged using NBI. In addition, we performed two-photon imaging to quantify the binding of EGFR targeted GNRs in tumors and their depth profile. RESULTS: The NBI images showed a visual increase in contrast from tumors after topical administration of targeted GNR. Targeted GNR tumors showed increased contrast compared to tumors administered with the control GNR. There was a statistically significant increase in mean pixel intensity (∼2.5×) from targeted GNR tumors (n = 6). Two-photon microscopy images of targeted GNRs confirmed their binding affinity to the EGF receptors over expressed in the A431 tumors. CONCLUSION: We have demonstrated that a topical application of gold nanorods targeted specifically to tumor growth factor receptors results in a significantly higher image contrast compared to nontargeted gold nanorods. These results demonstrate the feasibility of near-infrared NBI to image and demarcate tumor margins during surgical resection using topical administration of targeted GNR.

Combined two-photon luminescence microscopy and OCT for macrophage detection in the hypercholesterolemic rabbit aorta using plasmonic gold nanorose.

BACKGROUND AND OBJECTIVES: The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent. STUDY DESIGN/MATERIALS AND METHODS: Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 × 8 × 2 mm(3) ) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy. RESULTS: Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta. CONCLUSIONS: Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions.

Pheochromocytomas, PASS, and immunohistochemistry.

Differentiation between malignant and benign pheochromocytomas of the adrenal gland traditionally relies on the presence of clinically detectable metastases. The PASS system for differentiating between benign and malignant pheochromocytomas is based on defined morphological criteria, of which some are related to tumour cell proliferation and survival. Immunohistochemical markers for important events in the cell cycle were explored in order to characterise differences in apoptosis, G1 checkpoints, and S phase in more detail. A panel consisting of p53, tenascin, bcl-2, pRb, cyclin D1, mcm2, and p27 was employed. Only for pRb a statistically significant difference between PASS 3 and less and PASS 4+ tumours was detected, indicating qualitative differences in the mitotic cycle, probably immediately before early S phase. These results are discussed in relation to similar studies in recent literature.

In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure.

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 µm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm.

Comparison of N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid in the regulation of retinoid receptor-mediated gene expression in human breast cancer cell lines.

The activities of N-(4-hydroxyphenyl)retinamide [(4-HPR), Fenretinide] and all-trans-retinoic acid (RA) were determined for (a) the inhibition of cell proliferation; (b) the activation of human retinoid receptor-mediated target gene expression; (c) the inhibition of estradiol- and progesterone-induced gene activation in breast cancer cell lines; and (d) the regulation of the expression of tumor suppressor retinoblastoma protein. Similar to RA, both 4-HPR and its active metabolite N-(4-methoxyphenyl)retinamide (4-MPR) effectively impeded the growth of MCF7 and T-47D human breast cancer cell lines, except that 4-HPR also inhibited the proliferation of RA-resistant BT-20 cells. However, when tested in human recombinant retinoic acid receptor (RAR-alpha, RAR-beta, and RAR-gamma)-induced reporter gene assays, RA was much more potent (>100-fold) than either 4-HPR or 4-MPR. 4-HPR induced transcriptional activation through all three RAR subtypes at 1-10microM, while RA showed comparable activity at 10-100microM. Despite the apparent weak interaction at the RAR level, 4-HPR was comparable to RA in the inhibition of both estrogen receptor- and progesterone receptor-mediated transcriptional activation in MCF7 and T-47D cells, respectively. Moreover, similar to RA, 4-HPR and 4-MPR caused marked up-regulation of tumor suppressor retinoblastoma protein in both MCF7 and T-47D cells. Since RA and 4-HPR showed comparable activity in the inhibition of estrogen recptor- and progesterone receptor-induced gene transcription and in the stimulation of retinoblastoma protein expression in MCF7 and T-47D cells, the reduced RAR activation by 4-HPR may result in the lack of hepatic toxicity and therefore the improved therapeutic efficacy relative to RA.

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids.

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D2 receptor and should prove useful in studies on functional reconstitution of the receptor.

Expanding applications, accuracy, and interpretation of laser speckle contrast imaging of cerebral blood flow.

Laser speckle contrast imaging (LSCI) provides a rapid characterization of cortical flow dynamics for functional monitoring of the microcirculation. The technique stems from interactions of laser light with moving particles. These interactions encode the encountered Doppler phenomena within a random interference pattern imaged in widefield, known as laser speckle. Studies of neurovascular function and coupling with LSCI have benefited from the real-time characterization of functional dynamics in the laboratory setting through quantification of perfusion dynamics. While the technique has largely been relegated to acute small animal imaging, its scalability is being assessed and characterized for both chronic and clinical neurovascular imaging.

Chronic monitoring of vascular progression after ischemic stroke using multiexposure speckle imaging and two-photon fluorescence microscopy.

Monitoring the progression of the vascular structure and cerebral blood flow (CBF) after brain injury is vital to understand the neurovascular recovery process. Multiexposure speckle imaging (MESI) provides a quantitatively accurate technique for chronically measuring the postocclusion CBF perfusion of the infarct and peri-infarct regions in rodent stroke models, while multiphoton microscopy offers direct visualization of the microvascular structure. In this paper, we present imaging outcomes extending 35 days after photo-thrombotic occlusion, tracking the progression of the vasculature throughout this period. We compare MESI flow estimates within the unresolvable parenchyma with subsurface microvascular volume fractions taken with two-photon microscopy in the same regions to assess how the vascular density influences the surface-integrated MESI flow values. The MESI flow measurements and volume fractions are shown to have high correlations (r=0.90) within areas of recovering vasculature in the peri-infarct region. We also observe vascular reorientation occurring within the microvascular structure throughout the 35-day postocclusion period. With the combination of a chronic mouse model and relatively noninvasive optical imaging techniques, we present an imaging protocol for monitoring long-term vascular progression after photo-thrombotic occlusion with the potential to test the efficacy of rehabilitation and pharmacological therapies.

Flux or speed? Examining speckle contrast imaging of vascular flows.

Speckle contrast imaging enables rapid mapping of relative blood flow distributions using camera detection of back-scattered laser light. However, speckle derived flow measures deviate from direct measurements of erythrocyte speeds by 47 ± 15% (n = 13 mice) in vessels of various calibers. Alternatively, deviations with estimates of volumetric flux are on average 91 ± 43%. We highlight and attempt to alleviate this discrepancy by accounting for the effects of multiple dynamic scattering with speckle imaging of microfluidic channels of varying sizes and then with red blood cell (RBC) tracking correlated speckle imaging of vascular flows in the cerebral cortex. By revisiting the governing dynamic light scattering models, we test the ability to predict the degree of multiple dynamic scattering across vessels in order to correct for the observed discrepancies between relative RBC speeds and multi-exposure speckle imaging estimates of inverse correlation times. The analysis reveals that traditional speckle contrast imagery of vascular flows is neither a measure of volumetric flux nor particle speed, but rather the product of speed and vessel diameter. The corrected speckle estimates of the relative RBC speeds have an average 10 ± 3% deviation in vivo with those obtained from RBC tracking.

Differential regulation of human progesterone receptor A and B form-mediated trans-activation by phosphorylation.

Hormone-dependent phosphorylation of progesterone receptors (PRs) plays a functional role in their transcriptional activity. However, hormone-independent phosphorylation has also been shown to modulate the chicken PR-mediated trans-activation in the presence of phosphorylating agents. The present study was designed to investigate the effects of protein kinase A- and protein kinase C-mediated signal transduction pathways on the regulation of the activity of the two forms of human PR (hPRA and hPRB). Similar to chicken PR, hPR was activated by 8-bromo-cAMP (8-Br-cAMP) in the absence of ligand, whereas 8-Br-cAMP synergized with the progestin agonist R5020 to amplify hPRA- and hPRB-mediated reporter activity. Interestingly, the effect of 8-Br-cAMP was much more pronounced on hPRA-induced trans-activation than on hPRB. This differential regulation by 8-Br-cAMP could also be mimicked by okadaic acid. Both mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyl transferase and progesterone response element-thymidine kinase-chloramphenicol acetyl transferase showed a similar response to 8-Br-cAMP in the presence of R5020. Protein kinase C, on the other hand, did not discriminate between hPRA- and hPRB-mediated trans-activation. Unlike 8-Br-cAMP, phorbol 12-myristate 13-acetate did not cause marked ligand-independent trans-activation through either of the two receptor forms. RU486, an antagonist of progestin, preferentially blocked R5020-induced trans-activation compared to R5020 + 8-Br-cAMP synergism. As expected, H-89, a specific inhibitor of protein kinase A was more effective in inhibiting ligand-independent activity. Western analysis of transfected receptors suggested that 8-Br-cAMP and 8-Br-cAMP + R5020 but not R5020 alone down-regulated the level of hPRB in COS-1 cells. Only marginal modulation of hPRA levels was observed with R5020 treatment in the presence and absence of 8-Br-cAMP. These data suggest that R5020 and 8-Br-cAMP mediate PR-dependent transactivation through distinct pathways, and that phosphorylation can differentially regulate the activity of hPRA and hPRB forms, an observation which may be important for selective target gene activation in vivo by progestins.

Effect of 6-aminonicotinamide on monoamine oxidase and Na+K+ATPase activity in different regions of rat brain.

The monoamine oxidase activity in the cerebral hemispheres decreased significantly after 2, 4, 8 and 16 hr of 6-amino-nicotinamide (35 mg/kg body weight, i.p.) administration. In the cerebellum, the MAO activity was not affected significantly. In the brain stem, however, a significant increase was observed after 2 hr of drug administration followed by a gradual decrease at later time intervals. The Na+K+ATPase activity in the cerebral hemispheres was increased significantly at 2 and 4 hr of 6-aminonicotinamide administration. This was followed by a gradual decrease at later time intervals. In the cerebellum, like monoamine oxidase, the Na+K+ATPase did not change significantly. The brain stem showed a decrease at 2 hr of drug administration, followed by a significant increase at 4 hr and then a gradual decrease to near control values.

Novel intracellular signaling function of prostaglandin H synthase-1 in NF-kappa B activation.

Many potent nonsteroidal antiinflammatory drugs (NSAIDs) exert their effects by inhibiting the cyclooxygenase activity of prostaglandin H synthase-1 (PGHS1, thus disrupting prostaglandin biosynthesis. However, these drugs do not block the activation of NF-kappa B, an inducible transcription factor which regulates numerous inflammation-related genes. Here we demonstrate that PGHS1 peroxidase, a NSAID-insensitive activity of PGHS1, mediates NF-kappa B activation through an intracellular reactive oxygen signaling pathway. Overexpression of PGHS1 strongly potentiated NF-kappa B activation by phorbol esters and dramatically elevated the generation of intracellular reactive oxygen species (ROS) in response to low concentrations of t-butyl peroxide. Both functions were dependent on PGHS1 peroxidase activity and could be suppressed by the potent antioxidant pyrrolidine dithiocarbamate. In contrast, elimination of PGHS1 cyclooxygenase activity by NSAIDs or site-directed mutagenesis failed to block ROS production or NF-kappa B activation. Thus, PGHS1 peroxidase serves an intracellular signaling function leading to NF-kappa B activation, separable from its role in prostaglandin synthesis.

Optimization of camera exposure durations for multi-exposure speckle imaging of the microcirculation.

Improved Laser Speckle Contrast Imaging (LSCI) blood flow analyses that incorporate inverse models of the underlying laser-tissue interaction have been used to develop more quantitative implementations of speckle flowmetry such as Multi-Exposure Speckle Imaging (MESI). In this paper, we determine the optimal camera exposure durations required for obtaining flow information with comparable accuracy with the prevailing MESI implementation utilized in recent in vivo rodent studies. A looping leave-one-out (LOO) algorithm was used to identify exposure subsets which were analyzed for accuracy against flows obtained from analysis with the original full exposure set over 9 animals comprising n = 314 regional flow measurements. From the 15 original exposures, 6 exposures were found using the LOO process to provide comparable accuracy, defined as being no more than 10% deviant, with the original flow measurements. The optimal subset of exposures provides a basis set of camera durations for speckle flowmetry studies of the microcirculation and confers a two-fold faster acquisition rate and a 28% reduction in processing time without sacrificing accuracy. Additionally, the optimization process can be used to identify further reductions in the exposure subsets for tailoring imaging over less expansive flow distributions to enable even faster imaging.

Imaging depth and multiple scattering in laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is a powerful and simple method for full field imaging of blood flow. However, the depth dependence and the degree of multiple scattering have not been thoroughly investigated. We employ three-dimensional Monte Carlo simulations of photon propagation combined with high resolution vascular anatomy to investigate these two issues. We found that 95% of the detected signal comes from the top 700 µm of tissue. Additionally, we observed that single-intravascular scattering is an accurate description of photon sampling dynamics, but that regions of interest (ROIs) in areas free of obvious surface vessels had fewer intravascular scattering events than ROI over resolved surface vessels. Furthermore, we observed that the local vascular anatomy can strongly affect the depth dependence of LSCI. We performed simulations over a wide range of intravascular and extravascular scattering properties to confirm the applicability of these results to LSCI imaging over a wide range of visible and near-infrared wavelengths.

Low-cost laser speckle contrast imaging of blood flow using a webcam.

Laser speckle contrast imaging has become a widely used tool for dynamic imaging of blood flow, both in animal models and in the clinic. Typically, laser speckle contrast imaging is performed using scientific-grade instrumentation. However, due to recent advances in camera technology, these expensive components may not be necessary to produce accurate images. In this paper, we demonstrate that a consumer-grade webcam can be used to visualize changes in flow, both in a microfluidic flow phantom and in vivo in a mouse model. A two-camera setup was used to simultaneously image with a high performance monochrome CCD camera and the webcam for direct comparison. The webcam was also tested with inexpensive aspheric lenses and a laser pointer for a complete low-cost, compact setup ($90, 5.6 cm length, 25 g). The CCD and webcam showed excellent agreement with the two-camera setup, and the inexpensive setup was used to image dynamic blood flow changes before and after a targeted cerebral occlusion.

Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion.

Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique's utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.

Comparison of indocyanine green angiography and laser speckle contrast imaging for the assessment of vasculature perfusion.

BACKGROUND: Assessment of the vasculature is critical for overall success in cranial vascular neurological surgery procedures. Although several methods of monitoring cortical perfusion intraoperatively are available, not all are appropriate or convenient in a surgical environment. Recently, 2 optical methods of care have emerged that are able to obtain high spatial resolution images with easily implemented instrumentation: indocyanine green (ICG) angiography and laser speckle contrast imaging (LSCI). OBJECTIVE: To evaluate the usefulness of ICG and LSCI in measuring vessel perfusion. METHODS: An experimental setup was developed that simultaneously collects measurements of ICG fluorescence and LSCI in a rodent model. A 785-nm laser diode was used for both excitation of the ICG dye and the LSCI illumination. A photothrombotic clot model was used to occlude specific vessels within the field of view to enable comparison of the 2 methods for monitoring vessel perfusion. RESULTS: The induced blood flow change demonstrated that ICG is an excellent method for visualizing the volume and type of vessel at a single point in time; however, it is not always an accurate representation of blood flow. In contrast, LSCI provides a continuous and accurate measurement of blood flow changes without the need of an external contrast agent. CONCLUSION: These 2 methods should be used together to obtain a complete understanding of tissue perfusion.

Dual-wavelength multifrequency photothermal wave imaging combined with optical coherence tomography for macrophage and lipid detection in atherosclerotic plaques using gold nanoparticles.

The objective of this study was to assess the ability of combined photothermal wave (PTW) imaging and optical coherence tomography (OCT) to detect, and further characterize the distribution of macrophages (having taken up plasmonic gold nanorose as a contrast agent) and lipid deposits in atherosclerotic plaques. Aortas with atherosclerotic plaques were harvested from nine male New Zealand white rabbits divided into nanorose- and saline-injected groups and were imaged by dual-wavelength (800 and 1210 nm) multifrequency (0.1, 1 and 4 Hz) PTW imaging in combination with OCT. Amplitude PTW images suggest that lateral and depth distribution of nanorose-loaded macrophages (confirmed by two-photon luminescence microscopy and RAM-11 macrophage stain) and lipid deposits can be identified at selected modulation frequencies. Radiometric temperature increase and modulation amplitude of superficial nanoroses in response to 4 Hz laser irradiation (800 nm) were significantly higher than native plaque (P<0.001). Amplitude PTW images (4 Hz) were merged into a coregistered OCT image, suggesting that superficial nanorose-loaded macrophages are distributed at shoulders on the upstream side of atherosclerotic plaques (P<0.001) at edges of lipid deposits. Results suggest that combined PTW-OCT imaging can simultaneously reveal plaque structure and composition, permitting characterization of nanorose-loaded macrophages and lipid deposits in atherosclerotic plaques.