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Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.

In vivo imaging of self-illuminating bio-and chemiluminescent reporters is used to observe the physiology of small animals. However, strong light scattering by biological tissues results in poor spatial resolution of the optical imaging, which also degrades the quantitative accuracy. To overcome this challenging problem, focused ultrasound is used to modulate the light from the reporter at the ultrasound frequency. This produces an ultrasound switchable light ‘beacon’ that reduces the influence of light scattering in order to improve spatial resolution. The experimental results demonstrate that apart from light modulation at the ultrasound frequency (AC signal at 3.5 MHz), ultrasound also increases the DC intensity of the reporters. This is shown to be due to a temperature rise caused by insonification that was minimized to be within acceptable mammalian tissue safety thresholds by adjusting the duty cycle of the ultrasound. Line scans of bio-and chemiluminescent objects embedded within a scattering medium were obtained using ultrasound modulated (AC) and ultrasound enhanced (DC) signals. Lateral resolution is improved by a factor of 12 and 7 respectively, as compared to conventional CCD imaging. Two chemiluminescent sources separated by ~10 mm at ~20 mm deep inside a 50 mm thick chicken breast have been successfully resolved with an average signal-to-noise ratio of approximately 8-10 dB.

A visible light spectral domain optical coherence microscopy system was developed. A high axial resolution of 0.88 μm in tissue was achieved using a broad visible light spectrum (425 – 685 nm). Healthy human brain tissue was imaged to quantify the difference between white (WM) and grey matter (GM) in intensity and attenuation. The high axial resolution enables the investigation of amyloid-beta plaques of various sizes in human brain tissue and animal models of Alzheimer’s disease (AD). By performing a spectroscopic analysis of the OCM data, differences in the characteristics for WM, GM, and neuritic amyloid-beta plaques were found. To gain additional contrast, Congo red stained AD brain tissue was investigated. A first effort was made to investigate optically cleared mouse brain tissue to increase the penetration depth and visualize hyperscattering structures in deeper cortical regions.

The state-of-the-art laparoscope lacks the ability to capture high-magnification and wide-angle images simultaneously, which introduces challenges when both close- up views for details and wide-angle overviews for orientation are required in clinical practice. A multi-resolution foveated laparoscope (MRFL) which can provide the surgeon both high-magnification close-up and wide-angle images was proposed to address the limitations of the state-of-art surgical laparoscopes. In this paper, we present the overall system design from both clinical and optical system perspectives along with a set of experiments to characterize the optical performances of our prototype system and describe our preliminary in-vivo evaluation of the prototype with a pig model. The experimental results demonstrate that at the optimum working distance of 120mm, the high-magnification probe has a resolution of 6.35lp/mm and image a surgical area of 53 × 40mm(2); the wide-angle probe provides a surgical area coverage of 160 × 120mm(2) with a resolution of 2.83lp/mm. The in-vivo evaluation demonstrates that MRFL has great potential in clinical applications for improving the safety and efficiency of the laparoscopic surgery.

Near-infrared (NIR) fluorescence imaging systems have been developed for image guided surgery in recent years. However, current systems are typically bulky and work only when surgical light in the operating room (OR) is off. We propose a single camera imaging system that is capable of capturing NIR fluorescence and color images under normal surgical lighting illumination. Using a new RGB-NIR sensor and synchronized NIR excitation illumination, we have demonstrated that the system can acquire both color information and fluorescence signal with high sensitivity under normal surgical lighting illumination. The experimental results show that ICG sample with concentration of 0.13 μM can be detected when the excitation irradiance is 3.92 mW/cm(2) at an exposure time of 10 ms.

A photoacoustic tomograph based on optical ultrasound detection is demonstrated, which is capable of high resolution real-time projection imaging and fast three-dimensional (3D) imaging. Snapshots of the pressure field outside the imaged object are taken at defined delay times after photoacoustic excitation by use of a charge coupled device (CCD) camera in combination with an optical phase contrast method. From the obtained wave patterns photoacoustic projection images are reconstructed using a back propagation Fourier domain reconstruction algorithm. Applying the inverse Radon transform to a set of projections recorded over a half rotation of the sample provides 3D photoacoustic tomography images in less than one minute with a resolution below 100 µm. The sensitivity of the device was experimentally determined to be 5.1 kPa over a projection length of 1 mm. In vivo images of the vasculature of a mouse demonstrate the potential of the developed method for biomedical applications.

Fixational eye movements remain a major cause of artifacts in optical coherence tomography (OCT) images despite the increases in acquisition speeds. One approach to eliminate the eye motion is to stabilize the ophthalmic imaging system in real-time. This paper describes and quantifies the performance of a tracking OCT system, which combines a phase-stabilized optical frequency domain imaging (OFDI) system and an eye tracking scanning laser ophthalmoscope (TSLO). We show that active eye tracking minimizes artifacts caused by eye drift and micro saccades. The remaining tracking lock failures caused by blinks and large saccades generate a trigger signal which signals the OCT system to rescan corrupted B-scans. Residual motion artifacts in the OCT B-scans are reduced to 0.32 minutes of arc (~1.6 μm) in an in vivo human eye enabling acquisition of high quality images from the optic nerve head and lamina cribrosa pore structure. © 2012 Optical Society of America.

Intracoronary optical frequency domain imaging (OFDI) provides high resolution, three-dimensional views of coronary artery microstructure, but requires a non-occlusive saline/contrast purge to displace blood for clear artery views. Recent studies utilized manual pullback initiation/termination based on real-time image observation. Automated pullback initiation/termination by real-time OFDI signal analysis would enable more efficient data acquisition. We evaluate the use of simple imaging parameters to automatically and robustly differentiate between diagnostic-quality clear artery wall (CAW) versus blood-obstructed fields (BOF). Algorithms are tested using intracoronary OCT human data retrospectively and intracoronary OFDI swine and human data prospectively. In prospective analysis of OFDI swine data, the sensitivity and specificity of the ratio of second and first moments (contrast parameter) were 99.6% and 97.2%, respectively. In prospective analysis of OFDI clinical data, the contrast parameter yielded 96.0% sensitivity and 94.5% specificity. Accuracy improved further by analyzing sequential frames. These results indicate the algorithm may be utilized with intracoronary OFDI for initiating and terminating automated pullback and digital data recording.

In polarization-sensitive optical coherence tomography (PSOCT) the use of single-mode fibers causes unpredictable polarization distortions which can result in increased noise levels and erroneous changes in calculated polarization parameters. In the current paper this problem is addressed by a new Jones matrix analysis method that measures and corrects system polarization distortions as a function of wavenumber by spectral analysis of the sample surface polarization state and deeper located birefringent tissue structures. This method was implemented on a passivecomponent depth-multiplexed swept-source PS-OCT system at 1040 nm which was theoretically modeled using Jones matrix calculus. Highresolution B-scan images are presented of the double-pass phase retardation, diattenuation, and relative optic axis orientation to show the benefits of the new analysis method for in vivo imaging of the human retina. The correction of system polarization distortions yielded reduced phase retardation noise, and better estimates of the diattenuation and the relative optic axis orientation in weakly birefringent tissues. The clinical potential of the system is shown by en face visualization of the phase retardation and optic axis orientation of the retinal nerve fiber layer in a healthy volunteer and a glaucoma patient with nerve fiber loss. © 2014 Optical Society of America.

We demonstrate the phase fluctuation introduced by oscillation of scattering centers in the focal volume of an ultrasound transducer in an optical tomography experiment has a nonzero mean. The conditions to be met for the above are: (i) the frequency of the ultrasound should be in the vicinity of the most dominant natural frequency of vibration of the ultrasound focal volume, (ii) the corresponding acoustic wavelength should be much larger than [Formula: see text], a modified transport mean-free-path applicable for phase decorrelation and (iii) the focal volume of the ultrasound transducer should not be larger than 4 - 5 times [Formula: see text]. We demonstrate through simulations that as the ratio of the ultrasound focal volume to [Formula: see text] increases, the average of the phase fluctuation decreases and becomes zero when the focal volume becomes greater than around [Formula: see text]; and through simulations and experiments that as the acoustic frequency increases from 100 Hz to 1 MHz, the average phase decreases to zero. Through experiments done in chicken breast we show that the average phase increases from around 110° to 130° when the background medium is changed from water to glycerol, indicating that the average of the phase fluctuation can be used to sense changes in refractive index deep within tissue.

A noninvasive, multimodal photoacoustic and optical coherence tomography (PAT/OCT) scanner for three-dimensional in vivo (3D) skin imaging is described. The system employs an integrated, all optical detection scheme for both modalities in backward mode utilizing a shared 2D optical scanner with a field-of-view of ~13 × 13 mm(2). The photoacoustic waves were detected using a Fabry Perot polymer film ultrasound sensor placed on the surface of the skin. The sensor is transparent in the spectral range 590-1200 nm. This permits the photoacoustic excitation beam (670-680 nm) and the OCT probe beam (1050 nm) to be transmitted through the sensor head and into the underlying tissue thus providing a backward mode imaging configuration. The respective OCT and PAT axial resolutions were 8 and 20 µm and the lateral resolutions were 18 and 50-100 µm. The system provides greater penetration depth than previous combined PA/OCT devices due to the longer wavelength of the OCT beam (1050 nm rather than 829-870 nm) and by operating in the tomographic rather than the optical resolution mode of photoacoustic imaging. Three-dimensional in vivo images of the vasculature and the surrounding tissue micro-morphology in murine and human skin were acquired. These studies demonstrated the complementary contrast and tissue information provided by each modality for high-resolution 3D imaging of vascular structures to depths of up to 5 mm. Potential applications include characterizing skin conditions such as tumors, vascular lesions, soft tissue damage such as burns and wounds, inflammatory conditions such as dermatitis and other superficial tissue abnormalities.

We explore the use of fiber optic microendoscopy to image and quantify bacterial infection in the skin and lungs using an animal model. The contact probe fiber bundle fluorescence microendoscope has a 4 µm resolution, a 750 µm field of view, and a 1 mm outer diameter. Subcutaneous and intra-tracheal infections of fluorescent Mycobacterium bovis Bacillus Calmette-Guérin (BCG) bacteria were detected in situ from inocula down to 10(4) and 10(7) colony forming units, respectively.

In phase-resolved OCT angiography blood flow is detected from phase changes in between A-scans that are obtained from the same location. In ophthalmology, this technique is vulnerable to eye motion. We address this problem by combining inter-B-scan phase-resolved OCT angiography with real-time eye tracking. A tracking scanning laser ophthalmoscope (TSLO) at 840 nm provided eye tracking functionality and was combined with a phase-stabilized optical frequency domain imaging (OFDI) system at 1040 nm. Real-time eye tracking corrected eye drift and prevented discontinuity artifacts from (micro)saccadic eye motion in OCT angiograms. This improved the OCT spot stability on the retina and consequently reduced the phase-noise, thereby enabling the detection of slower blood flows by extending the inter-B-scan time interval. In addition, eye tracking enabled the easy compounding of multiple data sets from the fovea of a healthy volunteer to create high-quality eye motion artifact-free angiograms. High-quality images are presented of two distinct layers of vasculature in the retina and the dense vasculature of the choroid. Additionally we present, for the first time, a phase-resolved OCT angiogram of the mesh-like network of the choriocapillaris containing typical pore openings. © 2012 Optical Society of America.

The editors introduce the Biomedical Optics Express feature issue on "Phantoms for the Performance Evaluation and Validation of Optical Medical Imaging Devices." This topic was the focus of a technical workshop that was held on November 7-8, 2011, in Washington, D.C. The feature issue includes 13 contributions from workshop attendees.

Understanding the optical properties of clustered quantum dots (QDs) is essential to the design of QD-based optical phantoms for molecular imaging. Single and clustered core/shell colloidal QDs of dimers, trimers, and tetramers are self-assembled, separated, and preferentially collected using electrospray differential mobility analysis (ES-DMA) with electrostatic deposition. Multimodal optical characterization and analysis of their dynamical photoluminescence (PL) properties enables the long-term evaluation of the physicochemical and optical properties of QDs in a single or a clustered state. A multimodal time-correlated spectroscopic confocal microscope capable of simultaneously measuring the time evolution of PL intensity fluctuation, PL lifetime, and emission spectra reveals the long-term dynamic optical properties of interacting QDs in individual dimeric clusters of QDs. This new method will benefit research into the quantitative interpretation of fluorescence intensity and lifetime results in QD-based molecular imaging techniques. The process of photooxidation leads to coupling of the QDs in a dimer, leading to unique optical properties when compared to an isolated QD. These results guide the design and evaluation of QD-based phantom materials for the validation of the PL measurements for quantitative molecular imaging of biological samples labeled with QD probes.

Current treatment of unilateral breast cancer-related lymphedema (BCRL) is only directed to the afflicted arm. Near-infrared fluorescent imaging (NIRF) of arm lymphatic vessel architecture and function in BCRL and control subjects revealed a trend of increased lymphatic abnormalities in both the afflicted and unafflicted arms with increasing time after lymphedema onset. These pilot results show that BCRL may progress to affect the clinically “normal” arm, and suggest that cancer-related lymphedema may become a systemic, rather than local, malady. These findings support further study to understand the etiology of cancer-related lymphedema and lead to better diagnostics and therapeutics directed to the systemic lymphatic system.

The sensitivity to surface profile of non-contact optical imaging, such as spatial frequency domain imaging, may lead to incorrect measurements of optical properties and consequently erroneous extrapolation of physiological parameters of interest. Previous correction methods have focused on calibration-based, model-based, and computation-based approached. We propose an experimental method to correct the effect of surface profile on spectral images. Three-dimensional (3D) phantoms were built with acrylonitrile butadiene styrene (ABS) plastic using an accurate 3D imaging and an emergent 3D printing technique. In this study, our method was utilized for the correction of optical properties (absorption coefficient μ(a) and reduced scattering coefficient μ(s)') of objects obtained with a spatial frequency domain imaging system. The correction method was verified on three objects with simple to complex shapes. Incorrect optical properties due to surface with minimum 4 mm variation in height and 80 degree in slope were detected and improved, particularly for the absorption coefficients. The 3D phantom-based correction method is applicable for a wide range of purposes. The advantages and drawbacks of the 3D phantom-based correction methods are discussed in details.

Biofilms – communities of microorganisms attached to surfaces – are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far. Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.

A new methodology describing the effects of aperiodic and multiplexed gratings in volume holographic imaging systems (VHIS) is presented. The aperiodic gratings are treated as an ensemble of localized planar gratings using coupled wave methods in conjunction with sequential and non-sequential ray-tracing techniques to accurately predict volumetric diffraction effects in VHIS. Our approach can be applied to aperiodic, multiplexed gratings and used to theoretically predict the performance of multiplexed volume holographic gratings within a volume hologram for VHIS. We present simulation and experimental results for the aperiodic and multiplexed imaging gratings formed in PQ-PMMA at 488 nm and probed with a spherical wave at 633 nm. Simulation results based on our approach that can be easily implemented in ray-tracing packages such as Zemax® are confirmed with experiments and show proof of consistency and usefulness of the proposed models.

Using a hand-held photoacoustic probe integrated with a clinical ultrasound array system, we successfully imaged objects deeply positioned in biological tissues. The optical contrasts were enhanced by methylene blue with a concentration of ~30 mM. The penetration depth reached ~5.2 cm in chicken breast tissue by using 650-nm wavelength, which is ~4.7 times the 1/e optical penetration depth. This imaging depth was achieved using a laser fluence on the tissue surface of only 3 mJ/cm(2), which is 1/7 of the American National Standards Institute (ANSI) safety limit (20 mJ/cm(2)). The noise equivalent sensitivity at this depth was ~11 mM. Further, after intradermal injection of methylene blue in a rat, a sentinel lymph node was easily detected in vivo, beneath a 2-cm thick layer of chicken breast. Also, blood located 3.5 cm deep in the rat was clearly imaged with intrinsic contrast. We have photoacoustically guided insertion of a needle into a rat sentinel lymph node with accumulated methylene blue. These results highlight the clinical potential of photoacoustic image-guided identification and needle biopsy of sentinel lymph nodes for axillary staging in breast cancer patients.