Single-shot 4-step phase-shifting multispectral fringe projection profilometry.

Phase-shifting profilometry (PSP) is considered to be the most accurate technique for phase retrieval with fringe projection profilometry (FPP) systems. However, PSP requires that multiple phase-shifted fringe patterns be acquired, usually sequentially, which has limited PSP to static or quasi-static imaging. In this paper, we introduce multispectral 4-step phase-shifting FPP that provides 3D imaging using a single acquisition. The method enables real-time profilometry applications. A single frame provides all four phase-shifted fringe patterns needed for the PSP phase retrieval algorithm. The multispectral nature of the system ensures that light does not leak between the spectral bands, which is a common problem in simultaneous phase-shifting with color cameras. With the use of this new concept, custom composite patterns containing multiple patterns can be acquired with a single acquisition.

Surface plasmon resonance sensing properties of a 3D nanostructure consisting of aligned nanohole and nanocone arrays.

Molecular surface plasmon resonance (SPR) sensing is one of the most common applications of an array of periodic nanoholes in a metal film. However, metallic nanohole arrays (NHAs) with low-hole count have lower resolution and SPR sensing performance compared to NHAs with high-hole count. In this paper, we present a compact three-dimensional (3D) plasmonic nanostructure with extraordinary optical transmission properties benefiting from surface plasmon matching and enhanced localized surface plasmon coupling. The 3D nanostructure consisted of a gold film containing a NHA with an underlying cavity and a gold nanocone array (NCA) at the bottom of the cavity. Each nanocone was aligned with the nanohole above and the truncated apex of each nanocone was in close proximity (100 nm) to the gold film. The NHA-NCA structures outperformed conventional NHA structures in terms of bulk sensitivity and Figure of Merit (FOM). Furthermore, the NHA-NCA structure with 525 nm periodicity was capable of sensing streptavidin down to 2 nM exhibiting a 10-fold increase in streptavidin sensitivity compared to conventional NHA structures. The sensitivity and performance of the 3D nanostructure can be further improved by exploiting multiplexing methods in combination with stable light sources and detection systems.

Radial angular filter arrays for angle-resolved scattering spectroscopy.

The radial angular filter array (RAFA) consists of a series of radially-distributed micro-machined channels, where the long axes of the channels converge at a focal point. The high aspect ratio of each channel provides a means to reject photons with trajectories outside the acceptance angle of the channel. The output of the RAFA represents the angular distribution of photons emitted from the focal point. A series of RAFAs were designed, fabricated, and tested to evaluate the impact of device geometry, inter-channel cross talk, achromaticity, and channel leakage on device performance. As an application example, an RAFA was used together with an imaging spectrometer to capture angle-resolved spectra of turbid samples.

Photoacoustic tomography as a method to estimate the optical fluence distribution in turbid media.

Currently, there are no non-invasive experimental methods available for measuring optical fluence distributions in tissue. We present photoacoustic tomography (PAT) as a method to approximate the relative optical fluence distribution in a homogeneous optically scattering medium. Three-dimensional photoacoustic images were captured with a near-full view PAT scanner in phantoms with known optical absorption and scatter properties. Resultant 3D PAT images were compared to the expected optical fluence distributions from Monte Carlo simulations and diffusion theory using volumetric and shape analysis. Volumetric analysis of PAT images compared well with the optical fluence distributions from simulation. Dice similarity coefficients ranged from 51 to 82%. The reduced scattering coefficient estimated from PAT images compared well to estimates from simulations for values below 0.5 mm-1. Near full-view PAT has been found to be useful for estimating the optical fluence distribution in an optically scattering medium. Further development is needed to extend the measurement range.

Effect of surface plasmon cross-talk on optical properties of closely packed nano-hole arrays.

The integration and miniaturization of nanostructure-based optical devices based on interaction with surface plasmons requires the fabrication of patterns of multiple nanostructures with tight spacing. The effect of surface plasmon energy interchange (cross-talk) across large grids of nanostructures and its effect on the optical characteristics of individual nanostructures have not been investigated. In this paper, we experimentally fabricated a large grid of individual nano-hole arrays of various hole diameter, hole spacing, and inter-array spacing. The spectral optical transmission of each nano-hole array was measured and the effect of inter-array spacing on the transmission spectra and resonance wavelength was determined.

Optical resonance transmission properties of nano-hole arrays in a gold film: effect of adhesion layer.

In this paper, we present a systematic study on the influence of composition of the adhesion layer between gold and a Pyrex substrate on the optical resonance transmission properties of nano-hole arrays in an optically thick gold film. Large nano-hole arrays with different hole periodicities in a square lattice arrangement were fabricated using Electron Beam Lithography using different adhesion layers (chromium, titanium, or etched adhesion layer). The fabricated nano-hole arrays were optically characterized using transmission spectroscopy. The optical performance of each nano-hole array was numerically simulated using a Finite Difference Time Domain (FDTD) method. The experiments and simulations revealed that the optical resonance transmission properties (i.e. the resonance wavelength, the spectral transmission modulation ratio, and the resonance bandwidth) of the nano-hole arrays depended highly on the composition and the thickness of the adhesion layer. The optical resonance bandwidths were larger for the nano-hole arrays with chromium or titanium adhesion layers. Also, a red-shift of the optical resonance peak was observed for nano-hole arrays with a metal adhesion layer compared to the corresponding nano-hole arrays with an etched adhesion layer, but the red-shift was greatest for the nano-hole array with the titanium adhesion layer. For adhesion layers of greater thickness, the optical resonance peaks were reduced in magnitude. Finally, nano-hole arrays with an etched adhesion layer had a significant blue-shift in the optical resonance peak and a narrower optical resonance bandwidth compared to nano-hole arrays with a titanium or a chromium adhesion layer. Consequently, a narrow optical resonance bandwidth characteristic of a nano-hole array with an etched adhesion layer can potentially enhance the spectral selectivity and offer improved optical performance.

Singular value decomposition analysis of a photoacoustic imaging system and 3D imaging at 0.7 FPS.

Photoacoustic imaging is a non-ionizing imaging modality that provides contrast consistent with optical imaging techniques while the resolution and penetration depth is similar to ultrasound techniques. In a previous publication [Opt. Express 18, 11406 (2010)], a technique was introduced to experimentally acquire the imaging operator for a photoacoustic imaging system. While this was an important foundation for future work, we have recently improved the experimental procedure allowing for a more densely populated imaging operator to be acquired. Subsets of the imaging operator were produced by varying the transducer count as well as the measurement space temporal sampling rate. Examination of the matrix rank and the effect of contributing object space singular vectors to image reconstruction were performed. For a PAI system collecting only limited data projections, matrix rank increased linearly with transducer count and measurement space temporal sampling rate. Image reconstruction using a regularized pseudoinverse of the imaging operator was performed on photoacoustic signals from a point source, line source, and an array of point sources derived from the imaging operator. As expected, image quality increased for each object with increasing transducer count and measurement space temporal sampling rate. Using the same approach, but on experimentally sampled photoacoustic signals from a moving point-like source, acquisition, data transfer, reconstruction and image display took 1.4 s using one laser pulse per 3D frame. With relatively simple hardware improvements to data transfer and computation speed, our current imaging results imply that acquisition and display of 3D photoacoustic images at laser repetition rates of 10Hz is easily achieved.

Effect of glutathione depletion, hyperthermia, and a 100-mT static magnetic field on an hsp70/luc reporter system.

Heat shock proteins, in particular hsp70, are induced under conditions of cellular stress. It has been reported that environmental stimuli such as hyperthermia, oxidative stress, and exposure to magnetic fields increase levels of hsp70. It has also been reported that hyperthermia in combination with magnetic field exposure results in a synergistic increase in hsp70 production. We tested the hypothesis that oxidative stress induced by glutathione (GSH) depletion in combination with static magnetic field (SMF) exposure will produce a similar synergistic increase in hsp70 production. We exposed cells to heat, SMF, and diethylmaleate (DEM), which depletes GSH levels alone and in combination with each other, and measured hsp70 production using an hsp70/luciferase reporter and mRNA levels using PCR. We found that treatment with DEM significantly reduced the rate of luciferase bioluminescence production, particularly in the presence of heat. There was no significant effect of a 100-mT SMF exposure either alone or in combination with heat, DEM, or both on bioluminescence, however there was a significant interaction between SMF and DEM on hsp70 mRNA levels. Therefore, under our exposure conditions, GSH depletion reduced hsp70 levels but a synergistic effect of combining this stress with other external stimuli was only observed at the level of mRNA.

Single-shot detection of 8 unique monochrome fringe patterns representing 4 distinct directions via multispectral fringe projection profilometry.

Spatial resolution in three-dimensional fringe projection profilometry is determined in large part by the number and spacing of fringes projected onto an object. Due to the intensity-based nature of fringe projection profilometry, fringe patterns must be generated in succession, which is time-consuming. As a result, the surface features of highly dynamic objects are difficult to measure. Here, we introduce multispectral fringe projection profilometry, a novel method that utilizes multispectral illumination to project a multispectral fringe pattern onto an object combined with a multispectral camera to detect the deformation of the fringe patterns due to the object. The multispectral camera enables the detection of 8 unique monochrome fringe patterns representing 4 distinct directions in a single snapshot. Furthermore, for each direction, the camera detects two π-phase shifted fringe patterns. Each pair of fringe patterns can be differenced to generate a differential fringe pattern that corrects for illumination offsets and mitigates the effects of glare from highly reflective surfaces. The new multispectral method solves many practical problems related to conventional fringe projection profilometry and doubles the effective spatial resolution. The method is suitable for high-quality fast 3D profilometry at video frame rates.

Multispectral intravital microscopy for simultaneous bright-field and fluorescence imaging of the microvasculature.

Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which combines bright-field and fluorescence microscopy in a standard light microscope. The technique enables simultaneous real-time observation of fluorescently-labeled structures in relation to their direct physical surroundings. The advancement provides context for the orientation, movement, and function of labeled structures in the microcirculation.

Experimental and numerical analysis on the optical resonance transmission properties of nano-hole arrays.

In this paper, we present experimental and numerical analysis on Extraordinary Optical Transmission (EOT) or optical resonance transmission through various nano-hole arrays constructed from an optically thick metal film within the visible and near infra-red spectrum. Nano-hole arrays with different geometrical parameters (hole size, hole shape, and hole periodicity) having their EOT properties in the visible and near-infrared regime were simulated based on Finite Difference Time Domain (FDTD). Large nano-hole arrays with geometric properties similar to the simulated arrays were fabricated using Electron Beam Lithography (EBL). The optical resonance transmission properties (resonance position, transmission efficiency, and spectral bandwidth of resonance peak) of the fabricated nano-hole arrays were characterized. Finally, the experimental and numerical results were analyzed to determine the dependencies and discrepancies between optical resonance transmission properties for various nano-hole arrays.

Contrast and resolution analysis of iterative angular domain optical projection tomography.

In Angular Domain Imaging, image contrast and resolution are position dependent. The objective of this work was to characterize the contrast and resolution of an ADI system at a multitude of locations within the imaging plane, then compare the reconstructions of different targets using filtered back projection and iterative reconstruction algorithms. Contrast varied significantly with depth and minimally with lateral position, while resolution varied significantly with lateral position and minimally with depth. The iterative reconstruction algorithm was robust against ring and streak artifacts. The back projection reconstructions suffered from artifacts related to a lack of projection data.

Angular domain fluorescence lifetime imaging: a tissue-like phantom study.

We describe a fluorescence lifetime imaging technique employing the collimation detection capabilities of an angular filter array (AFA). The AFA accepts minimally scattered photons emitted from fluorophores up to 2 mm deep within turbid media. The technique, referred to as Angular Domain Fluorescence Lifetime Imaging (ADFLI), is described and its performance evaluated in comparison to a conventional (lens and pinhole) system. Results from a tissue-mimicking phantom demonstrated that ADFLI provides better spatial resolution and image contrast for fluorescent probes at greater depths compared to a lens and pinhole system.

Analysis of a photoacoustic imaging system by the crosstalk matrix and singular value decomposition.

Photoacoustic imaging is a hybrid imaging modality capable of producing contrast similar to optical imaging techniques but with increased penetration depth and resolution in turbid media by encoding the information as acoustic waves. In general, it is important to characterize the performance of a photoacoustic imaging system by parameters such as sensitivity, resolution, and contrast. However, system characterization can extend beyond these metrics by implementing advanced analysis via the crosstalk matrix and singular value decomposition. A method was developed to experimentally measure a matrix that represented the imaging operator for a photoacoustic imaging system. Computations to produce the crosstalk matrix were completed to provide insight into the spatially dependent sensitivity and aliasing for the photoacoustic imaging system. Further analysis of the imaging operator was done via singular value decomposition to estimate the capability of the imaging system to reconstruct objects and the inherent sensitivity to those objects. The results provided by singular value decomposition were compared to SVD results from a de-noised imaging operator to estimate the number of measurable singular vectors for the system. These characterization techniques can be broadly applied to any photoacoustic system and, with regards to the studied system, could be used as a basis for improvements to future iterations.

Localization of spherical lesions in tumor-mimicking phantoms by 3D sparse array photoacoustic imaging.

PURPOSE: The authors have developed a sparse-array photoacoustic imaging (SPAI) system that is capable of mapping 3D distributions of optical absorption using a small number of laser pulses with no mechanical scanning needed. In previous studies, the authors have shown the localization accuracy and the high frame-rate image acquisition on simple phantoms with limited medical relevance. The purpose of this study was to test the imaging capabilities of SPAI in the context of breast tumor detection and localization. METHODS: The authors constructed an array of phantoms that include spherical lesions of sizes 1.5-9 mm, buried in highly scattering tissue phantoms at depths of 3-30 mm. The authors investigated both homogeneous lesions made of blood at varying concentrations and heterogeneous lesions containing vessel-like structures. Volumetric images of the deeply buried lesions were taken at increasingly shallower depths and image-based localization was compared to measured depth. RESULTS: The authors were able to detect and accurately localize homogeneous lesions having a realistic absorption coefficient of 0.2 cm(-1) down to depths of 9-20 mm, and heterogeneous lesions containing 0.5 mm diameter vessel-like structures down to depths of 13-20 mm. Image acquisition required 2.5 s for each volumetric lesion image. CONCLUSIONS: These results suggest that 3D SPAI can detect highly vascularized lesions well below 1 cm in diameter and can overcome optical scatter of tissue to depths of 1-2 cm. With further improvement in the sensitivity and noise characteristics of the imaging system, similar imaging depths should be within reach in real breast tissue. The method, due to its optical contrast, 3D imaging, and fast acquisition, may prove useful in the clinic as an adjunct to existing breast screening tools.

Structured-light surface scanning system to evaluate breast morphology in standing and supine positions.

Breast shapes are affected by gravitational loads and deformities. Measurements obtained in the standing position may not correlate well with measurements in the supine position, which is more representative of patient position during breast surgery. A dual color 3D surface imaging system capable of scanning patients in both supine and standing positions was developed to evaluate the effect of changes in body posture on breast morphology. The system was evaluated with  breast phantoms to assess accuracy, then tested on ten subjects in three body postures to assess its effectiveness as a clinical tool. The accuracy of the system was within 0.4 mm on average across the model. For the human study, there was no effect of body posture on breast volumes (p value > 0.05), but we observed an effect of completeness of breast scans on body posture (p value < 0.05). Post-hoc tests showed that the supine position and the standing position with hands at the waist differed significantly (p value < 0.05). This study shows that the system can quantitatively evaluate the effect of subject postures, and thereby has the potential to be used to investigate peri-operative changes in breast morphology.

Lipid-weighted intraoperative photoacoustic tomography of breast tumors: Volumetric comparison to preoperative MRI.

With a lifetime risk of 1 in 8, breast cancer continues to be a major concern for women and their physicians. The optimal treatment of the disease depends on the stage of the cancer at diagnosis, which is typically assessed using medical imaging. However, currently employed imaging systems for breast tumor measurement rarely agree perfectly. Our group developed an Intraoperative Photoacoustic Screening (iPAS) soft tissue scanner featuring high bulk tissue sensitivity, a clinically compatible scan-time of 6 min, imaging depths greater than 2 cm and the capability to visualize whole breast tumors based on their lipid, rather than hemoglobin, profile. Here, we report on the first clinical experience with breast cancer patients by comparing tumor-measurement using iPAS, preoperative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and gold-standard pathology. Tumor size was measured volumetrically for iPAS and DCE-MRI, and separately using maximum diameters for pathology, DCE-MRI and iPAS. Comparisons were performed using Pearson's correlation coefficients, and the non-parametric Wilcoxon signed-rank test. Twelve consecutive patients were included in the study, contingent on pathologically documented invasive carcinoma. iPAS volumetric tumor size was positively correlated to DCE-MRI (Pearson's r = 0.78, p = 0.003) and not significantly different (Wilcoxon, p = 0.97). In comparison to pathology, tumor diameters given by iPAS were positively correlated (Pearson's r = 0.87, p = 0.0002) and significantly different (Wilcoxon, p = 0.0015). The results indicated that volumetric-measurement of invasive breast tumors with iPAS is similar to that of DCE-MRI. On the other hand, tumor diameter measurements were less reliable. Beyond enhancing surgical specimen examination, an extension of this technology to diagnostic imaging promises a new perspective on tumor assessment, potentially improving our current understanding and treatment of breast cancer.

Perfusion and Metabolic Neuromonitoring during Ventricular Taps in Infants with Post-Hemorrhagic Ventricular Dilatation.

Post-hemorrhagic ventricular dilatation (PHVD) is characterized by a build-up of cerebral spinal fluid (CSF) in the ventricles, which increases intracranial pressure and compresses brain tissue. Clinical interventions (i.e., ventricular taps, VT) work to mitigate these complications through CSF drainage; however, the timing of these procedures remains imprecise. This study presents Neonatal NeuroMonitor (NNeMo), a portable optical device that combines broadband near-infrared spectroscopy (B-NIRS) and diffuse correlation spectroscopy (DCS) to provide simultaneous assessments of cerebral blood flow (CBF), tissue saturation (StO2), and the oxidation state of cytochrome c oxidase (oxCCO). In this study, NNeMo was used to monitor cerebral hemodynamics and metabolism in PHVD patients selected for a VT. Across multiple VTs in four patients, no significant changes were found in any of the three parameters: CBF increased by 14.6 ± 37.6% (p = 0.09), StO2 by 1.9 ± 4.9% (p = 0.2), and oxCCO by 0.4 ± 0.6 µM (p = 0.09). However, removing outliers resulted in significant, but small, increases in CBF (6.0 ± 7.7%) and oxCCO (0.1 ± 0.1 µM). The results of this study demonstrate NNeMo's ability to provide safe, non-invasive measurements of cerebral perfusion and metabolism for neuromonitoring applications in the neonatal intensive care unit.

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system.

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real-time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real-time. Two heat parameters were considered in combination with sham- and 100 mT-exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult.

Development and characterization of an omnidirectional photoacoustic point source for calibration of a staring 3D photoacoustic imaging system.

Photoacoustic imaging is a modality which makes use of the contrast provided by optical imaging techniques and the spatial resolution and penetration depth similar to acoustic imaging modalities. We have developed a method for fast 3D photoacoustic imaging using a sparse hemispherical array of transducers. Such a system requires characterization of the transducer's response to an ideal point source in order to accurately reconstruct objects in the imaging volume. First, an attempt was made to design an ideal photoacoustic point source via a combination of liquids which would appropriately scatter and absorb the light such that a spherical distribution was achieved. Methylene blue (MB(+)) was used as the primary optical absorber while Intralipid (IL) was used as the liquid responsible for the optical scatter. A multitude of combinations were tested and the signal uniformity was characterized. The combination of 200 microM MB(+) and 0.09% IL was found to produce the most uniform signal over the range of transducers in the hemispherical array. The liquid source was then characterized over a broader range of azimuthal and zenith angles where it was shown the azimuthal consistency was much greater than the stability seen in different zenith elevations. The source was then used in a calibration scan for an imaging volume of 40 x 40 x 40 mm(3). At 216 points evenly spaced in the imaging volume, parameters were recorded for signal amplitude, width, and time-of-flight. These calibration parameters could then be applied to an iterative reconstruction algorithm in an attempt to more accurately produce images.