Amit S. Paranjape

Small multifunctional nanoclusters (Nanoroses) for targeted cellular imaging and therapy

The ability of 20-50 nm nanoparticles to target and modulate the biology of specific types of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. Herein we report approximately 30 nm stable uniformly sized near-infrared (NIR) active, superparamagnetic nanoclusters formed by kinetically controlled self-assembly of gold-coated iron oxide nanoparticles. The controlled assembly of nanocomposite particles into clusters with small primary particle spacings produces collective responses of the electrons that shift the absorbance into the NIR region. The nanoclusters of approximately 70 iron oxide primary particles with thin gold coatings display intense NIR (700-850 nm) absorbance with a cross section of approximately 10(-14) m(2). Because of the thin gold shells with an average thickness of only 2 nm, the r(2) spin-spin magnetic relaxivity is 219 mM(-1) s(-1), an order of magnitude larger than observed for typical iron oxide particles with thicker gold shells. Despite only 12% by weight polymeric stabilizer, the particle size and NIR absorbance change very little in deionized water over 8 months. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast in dark field and hyperspectral microscopy, both in cell culture and an in vivo rabbit model of atherosclerosis. Small nanoclusters with optical, magnetic, and therapeutic functionality, designed by assembly of nanoparticle building blocks, offer broad opportunities for targeted cellular imaging, therapy, and combined imaging and therapy.

Depth resolved photothermal OCT detection of macrophages in tissue using nanorose

Application of photothermal Optical Coherence Tomography (OCT) to detect macrophages in ex vivo rabbit arteries which have engulfed nanoclusters of gold coated iron oxide (nanorose) is reported. Nanorose engulfed by macrophages associated with atherosclerotic lesions in rabbit arteries absorb incident laser (800nm) energy and cause optical pathlength (OP) variation which is measured using photothermal OCT. OP variation in polydimethyl siloxane tissue phantoms containing varying concentrations of nanorose match values predicted from nanoparticle and material properties. Measurement of OP variation in rabbit arteries in response to laser excitation provides an estimate of nanorose concentration in atherosclerotic lesions of 2.5x109 particles/ml. OP variation in atherosclerotic lesions containing macrophages taking up nanorose has a different magnitude and profile from that observed in control thoracic aorta without macrophages and is consistent with macrophage presence as identified with RAM-11 histology staining. Our results suggest that tissue regions with macrophages taking up nanorose can be detected using photothermal OCT.

Comparison of pulsed photothermal radiometry, optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non-invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three-dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 μm-1.7 mm and 120-910 μm respectively) but poor axial resolution (75 and 120 μm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14-450 μm) but highest axial resolution (14 μm). The Monte Carlo simulation and three-dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (>400 μm), while OCT is better to measure thin melanoma lesions (<400 μm).

Optimized Retinal Nerve Fiber Layer Segmentation Based On Optical Reflectivity And Birefringence For Polarization-Sensitive Optical Coherence Tomography

Segmentation of the retinal nerve fiber layer (RNFL) from swept source polarization-sensitive optical coherence tomography (SS-PSOCT) images is required to determine RNFL thickness and calculate birefringence. Traditional RNFL segmentation methods based on image processing and boundary detection algorithms utilize only optical reflectivity contrast information, which is strongly affected by speckle noise. We present a novel approach to segment the retinal nerve fiber layer (RNFL) using SS-PSOCT images including both optical reflectivity and phase retardation information. The RNFL anterior boundary is detected based on optical reflectivity change due to refractive index difference between the vitreous and inner limiting membrane. The posterior boundary of the RNFL is a transition zone composed of birefringent axons extending from retinal ganglion cells and may be detected by a change in birefringence. A posterior boundary detection method is presented that segments the RNFL by minimizing the uncertainty of RNFL birefringence determined by a Levenberg-Marquardt nonlinear fitting algorithm. Clinical results from a healthy volunteer show that the proposed segmentation method estimates RNFL birefringence and phase retardation with lower uncertainty and higher continuity than traditional intensity-based approaches.

Fast Low-Noise Brillouin Spectroscopy Measurements of Elasticity for Corneal Crosslinking

The Brillouin scattering spectra of biological systems have shown to be inherently related to the intrinsic elasticity and molecular constants of tissues involved. Our approach of combining confocal microscopy and high-resolution Brillouin spectroscopy via a virtual imaging phase array enabled 10-microsecond single-pixel acquisition time without dedicated spatial filtering. Such an approach is adapted via a single-frequency fiber-coupled 780-nm wavelength laser, frequency stabilized by Rb-D2 absorption line, polarization extinction scheme, ASE filtering, heated Rb-vapor Rayleigh-scattering absorbent, and spectroscopic EMCCD camera, unified as CMS-VIPA: confocal virtual-imaging phase array microscopespectrometer. Steady strengthening of corneal bulk modulus was observed via spectral shifts of Brillouin scattering from 5.0-5.2 GHz in untreated porcine eyes to 5.7-5.9 GHz in ones cross-linked in riboflavin plus UV-A light  at 0.7-0.9 GHz level of enhancement. The cross-linking depths reaching 300400 microns were measured, as predicted by modeling. A noncontact Brillouin spectroscopic microscopy system for in-vivo corneal elasticity measurement is under development.

Near infrared femtosecond laser ablation of urinary calculi in water

Pulsed light emitted from a near infrared (λ=800nm) femtosecond laser is capable of plasma induced photodisruption of various materials. We used femtosecond laser pulses to ablate human urinary calculi. Femtosecond pulsed laser interaction with urinary calculi was investigated with various stone compositions, different incident fluences and number of applied pulses. Spectral-domain optical coherence tomography was used to image cross sections of ablation craters on the surface of urinary calculi. Our results indicate that femtosecond laser pulses can ablate various calculi compositions. Crater diameter and depth varies from tens of microns to several hundred microns when up to 1000 pulses were applied. Future studies are required to determine if pulsed near infrared femtosecond laser pulses can be applied clinically for lithotripsy of urinary calculi.

Melanoma thickness measurement in two-layer tissue phantoms using pulsed photothermal radiometry (PPTR)

Melanoma is a malignant tumor of melanocytes which are found predominantly in skin. Melanoma is one of the rarer types of skin cancer but causes the majority of skin cancer related deaths. The staging of malignant melanoma using Breslow thickness is important because of the relationship to survival rate after five years. Pulsed photothermal radiometry (PPTR) is based on the time-resolved acquisition of infrared (IR) emission from a sample after pulsed laser exposure. PPTR can be used to investigate the relationship between melanoma thickness and detected radiometric temperature using two-layer tissue phantoms. We used a Monte Carlo simulation to mimic light transport in melanoma and employed a three-dimensional heat transfer model to obtain simulated radiometric temperature increase and, in comparison, we also conducted PPTR experiments to confirm our simulation results. Simulation and experimental results show similar trends: thicker absorbing layers corresponding to deeper lesions produce slower radiometric temperature decays. A quantitative relationship exists between PPTR radiometric temperature decay time and thickness of the absorbing layer in tissue phantoms.

Automated method for RNFL segmentation in spectral domain OCT

We introduce a method based on optical reflectivity changes to segment the retinal nerve fiber layer (RNFL) in images recorded using swept source spectral domain optical coherence tomography (OCT). The segmented image is used to determine the RNFL thickness. Simple filtering followed by edge detecting techniques can successfully be applied to segment the RNFL from recorded images and estimate RNFL thickness. The method is computationally more efficient than previously reported approaches. Higher computational efficiency allows faster segmentation and provides the ophthalmologist segmented retinal images that better utilize advantages of spectral domain OCT instrumentation. OCT B-scan and fundus images of the retina are recorded for 5 patients. The segmentation method is applied on B-scan images recorded from all patients. An expert ophthalmologist separately demarcates the RNFL layer in the OCT images from the same patients in each B-scan image. Results from automated image processing software are compared to the boundary demarcated by the expert ophthalmologist. The absolute error between the boundaries demarcated by the expert and the algorithm is expressed in terms of area and is used as an error metric. Ability of the algorithm to accurately segment the RNFL in comparison with an expert ophthalmologist is reported.

Quality assessment for spectral domain optical coherence tomography (OCT) images

Retinal nerve fiber layer (RNFL) thickness, a measure of glaucoma progression, can be measured in images acquired by spectral domain optical coherence tomography (OCT). The accuracy of RNFL thickness estimation, however, is affected by the quality of the OCT images. In this paper, a new parameter, signal deviation (SD), which is based on the standard deviation of the intensities in OCT images, is introduced for objective assessment of OCT image quality. Two other objective assessment parameters, signal to noise ratio (SNR) and signal strength (SS), are also calculated for each OCT image. The results of the objective assessment are compared with subjective assessment. In the subjective assessment, one OCT expert graded the image quality according to a three-level scale (good, fair, and poor). The OCT B-scan images of the retina from six subjects are evaluated by both objective and subjective assessment. From the comparison, we demonstrate that the objective assessment successfully differentiates between the acceptable quality images (good and fair images) and poor quality OCT images as graded by OCT experts. We evaluate the performance of the objective assessment under different quality assessment parameters and demonstrate that SD is the best at distinguishing between fair and good quality images. The accuracy of RNFL thickness estimation is improved significantly after poor quality OCT images are rejected by automated objective assessment using the SD, SNR, and SS.

Quality assessment for spectral domain optical coherence tomography (OCT) images.

Retinal nerve fiber layer (RNFL) thickness, a measure of glaucoma progression, can be measured in images acquired by spectral domain optical coherence tomography (OCT). The accuracy of RNFL thickness estimation, however, is affected by the quality of the OCT images. In this paper, a new parameter, signal deviation (SD), which is based on the standard deviation of the intensities in OCT images, is introduced for objective assessment of OCT image quality. Two other objective assessment parameters, signal to noise ratio (SNR) and signal strength (SS), are also calculated for each OCT image. The results of the objective assessment are compared with subjective assessment. In the subjective assessment, one OCT expert graded the image quality according to a three-level scale (good, fair, and poor). The OCT B-scan images of the retina from six subjects are evaluated by both objective and subjective assessment. From the comparison, we demonstrate that the objective assessment successfully differentiates between the acceptable quality images (good and fair images) and poor quality OCT images as graded by OCT experts. We evaluate the performance of the objective assessment under different quality assessment parameters and demonstrate that SD is the best at distinguishing between fair and good quality images. The accuracy of RNFL thickness estimation is improved significantly after poor quality OCT images are rejected by automated objective assessment using the SD, SNR, and SS.