In this paper, the features of the intensity-based Doppler variance (IBDV) method were analyzed systemically with a flow phantom. The effects of beam scanning density, flow rate and the time interval between neighboring A-lines on the performance of this method were investigated. The IBDV method can be used to quantify the flow rate and its sensitivity can be improved by increasing the time interval between the neighboring A-lines. A higher sensitivity IBDV method that applies the algorithm along the slower scan direction was proposed. In comparison to laser speckle imaging maps of blood flow, we demonstrated the ability of the method to identify vessels with altered blood flow. In clinical measurements, we demonstrated the ability of the method to image vascular networks with exquisite spatial resolution and at depths up to 1.2 mm in human skin. These results collectively demonstrated the potential of the method to monitor the microvasculature during disease progression and in response to therapeutic intervention.

Functional measurement is important for retinal study and disease diagnosis. Transient intrinsic optical signal (IOS) response, tightly correlated with functional stimulation, has been previously detected in normal retinas. In this paper, comparative IOS imaging of wild-type (WT) and rod-degenerated mutant mouse retinas is reported. Both 2-month and 1-year-old mice were measured. In 2-month-old mutant mice, time course and peak value of the stimulus-evoked IOS were significantly delayed (relative to stimulus onset) and reduced, respectively, compared to age matched WT mice. In 1-year-old mutant mice, stimulus-evoked IOS was totally absent. However, enhanced spontaneous IOS responses, which might reflect inner neural remodeling in diseased retina, were observed in both 2-month and 1-year-old mutant retinas. Our experiments demonstrate the potential of using IOS imaging for noninvasive and high resolution identification of disease-associated retinal dysfunctions. Moreover, high spatiotemporal resolution IOS imaging may also lead to advanced understanding of disease-associated neural remodeling in the retina.

The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.

We present a phase derivative microscopy technique referred to as gradient field microscopy (GFM), which provides the first-order derivatives of the phase associated with an optical field passing through a transparent specimen. GFM utilizes spatial light modulation at the Fourier plane of a bright field microscope to optically obtain the derivatives of the phase and increase the contrast of the final image. The controllable spatial modulation pattern allows us to obtain both one component of the field gradient (derivative along one direction) and the gradient intensity, which offers some advantages over the regular differential interference contrast (DIC) microscopy. Most importantly, unlike DIC, GFM does not use polarizing optics and, thus, it is applicable to birefringent samples. We demonstrate these features of GFM with studies of static and dynamic biological cells (HeLa cells and red blood cells). We show that GFM is capable of qualitatively providing information about cell membrane fluctuations. Specifically, we captured the disappearance of the bending mode of fluctuations in osmotically swollen red blood cells.

Measurements of Cherenkov emission in tissue during radiation therapy are shown to enable estimation of hemoglobin oxygen saturation non-invasively, through spectral fitting of the spontaneous emissions from the treated tissue. Tissue oxygenation plays a critical role in the efficacy of radiation therapy to kill tumor tissue. Yet in-vivo measurement of this has remained elusive in routine use because of the complexity of oxygen measurement techniques. There is a spectrally broad emission of Cherenkov light that is induced during the time of irradiation, and as this travels through tissue from the point of the radiation deposition, the tissue absorption and scatter impart spectral changes. These changes can be quantified by diffuse spectral fitting of the signal. Thus Cherenkov emission spectroscopy is demonstrated for the first time quantitatively in vitro and qualitatively in vivo, and has potential for real-time online tracking of tissue oxygen during radiation therapy when fully characterized and developed.

Cerebral palsy (CP) is the most common motor disorder in children. Currently available neuroimaging techniques require complete body confinement and steadiness and thus are extremely difficult for pediatric patients. Here, we report the use and quantification of functional near infrared spectroscopy (fNIRS) to investigate the functional reorganization of the sensorimotor cortex in children with hemiparetic CP. Ten of sixteen children with congenital hemiparesis were measured during finger tapping tasks and compared with eight of sixteen age-matched healthy children, with an overall measurement success rate of 60%. Spatiotemporal analysis was introduced to quantify the motor activation and brain laterality. Such a quantitative approach reveals a consistent, contralateral motor activation in healthy children at 7 years of age or older. In sharp contrast, children with congenital hemiparesis exhibit all three of contralateral, bilateral and ipsilateral motor activations, depending on specific ages of the pediatric subjects. This study clearly demonstrates the feasibility of fNIRS to be utilized for investigating cortical reorganization in children with CP or other cortical disorders.

Amplitude decorrelation measurement is sensitive to transverse flow and immune to phase noise in comparison to Doppler and other phase-based approaches. However, the high axial resolution of OCT makes it very sensitive to the pulsatile bulk motion noise in the axial direction. To overcome this limitation, we developed split-spectrum amplitude-decorrelation angiography (SSADA) to improve the signal-to-noise ratio (SNR) of flow detection. The full OCT spectrum was split into several narrower bands. Inter-B-scan decorrelation was computed using the spectral bands separately and then averaged. The SSADA algorithm was tested on in vivo images of the human macula and optic nerve head. It significantly improved both SNR for flow detection and connectivity of microvascular network when compared to other amplitude-decorrelation algorithms.

Monitoring (currently invasive) of cerebral venous blood oxygenation is a key to avoiding hypoxia-induced brain injury resulting in death or severe disability. Noninvasive, optoacoustic monitoring of cerebral venous blood oxygenation can potentially replace existing invasive methods. To the best of our knowledge, we report for the first time noninvasive monitoring of cerebral venous blood oxygenation through intact scalp that was validated with invasive, “gold standard” measurements. We performed an in vivo study in the sheep superior sagittal sinus (SSS), a large midline cerebral vein, using our novel, multi-wavelength optoacoustic system. The study results demonstrated that: 1) the optoacoustic signal from the sheep SSS is detectable through the thick, intact scalp and skull; 2) the SSS signal amplitude correlated well with wavelength and actual SSS blood oxygenation measured invasively using SSS catheterization, blood sampling, and measurement with “gold standard” CO-Oximeter; 3) the optoacoustically predicted oxygenation strongly correlated with that measured with the CO-Oximeter. Our results indicate that monitoring of cerebral venous blood oxygenation may be performed in humans noninvasively and accurately through the intact scalp using optoacoustic systems because the sheep scalp and skull thickness is comparable to that of humans whereas the sheep SSS is much smaller than that of humans.

Despite the extensive use of polycapillary x-ray optics for focusing and collimating applications, there remains a significant need for characterization of the coherence properties of the output wavefield. In this work, we present the first quantitative computational method for calculation of the spatial coherence effects of polycapillary x-ray optical devices. This method employs the coherent mode decomposition of an extended x-ray source, geometric optical propagation of individual wavefield modes through a polycapillary device, output wavefield calculation by ray data resampling onto a uniform grid, and the calculation of spatial coherence properties by way of the spectral degree of coherence.

Lensfree in-line holographic microscopy offers sub-micron resolution over a large field-of-view (e.g., ~24 mm2) with a cost-effective and compact design suitable for field use. However, it is limited to relatively low-density samples. To mitigate this limitation, we demonstrate an on-chip imaging approach based on pixel super-resolution and phase recovery, which iterates among multiple lensfree intensity measurements, each having a slightly different sample-to-sensor distance. By digitally aligning and registering these lensfree intensity measurements, phase and amplitude images of dense and connected specimens can be iteratively reconstructed over a large field-of-view of ~24 mm2 without the use of any spatial masks. We demonstrate the success of this multi-height in-line holographic approach by imaging dense Papanicolaou smears (i.e., Pap smears) and blood samples.

Bio-mechanism investigations demand single particle tracking with high spatial and temporal resolutions which require single fluorophore 3D localization measurements with matching precision and speed. Although the precision for lateral-localization measurements is well described by an analytical expression, for the axial direction, it is often obtained by repeating location measurements or by estimating a lower bound. Here, we report a precision expression for an axial-localization method that analyzes the standard deviations of single fluorophores’ intensity profiles. Like the lateral-localization precision, this expression includes all relevant experimental effects measurable from a Gaussian intensity profile of the fluorophore. This expression completes the precision analysis for single-image 3D localization of individual fluorophores and lifts the temporal resolution to the typical exposure timescales of milliseconds.

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

In vivo optical microscopic imaging techniques have recently emerged as important tools for the study of neurobiological development and pathophysiology. In particular, two-photon microscopy has proved to be a robust and highly flexible method for in vivo imaging in highly scattering tissue. However, two-photon imaging typically requires extrinsic dyes or contrast agents, and imaging depths are limited to a few hundred microns. Here we demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of neuronal cell bodies and cortical myelination up to depths of ~1.3 mm in the rat neocortex. Imaging does not require the administration of exogenous dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, using OCM we demonstrate in vivo, quantitative measurements of optical properties (index of refraction and attenuation coefficient) in the cortex, and correlate these properties with laminar cellular architecture determined from the images. Lastly, we show that OCM enables direct visualization of cellular changes during cell depolarization and may therefore provide novel optical markers of cell viability.

A flexible curled optical cord is useful for a common-path optical coherence tomography (OCT) system because a bending-insensitive arbitrary length can be chosen for the endoscopic imaging probe. However, there has been a critical problem that the partial reflector needs to be placed in between the sample and the objective lens. It limits the structure design of optical probe and leads to a low transverse resolution OCT imaging. Instead of a conventional single common-path interferometer, we propose a novel double common-path interferometer configuration in order to generate an interference signal that is independent of the optical distance between the partial reflector and sample. Due to the limitless tuning of the objective distance, an objective lens with a high numerical aperture (NA) up to 0.85 can be successfully used for phase-sensitive optical coherence tomography to achieve a 3-dimensional profile image of a transverse resolution of 0.7 μm. The intensity and phase terms of the interference signal can be obtained simultaneously from a Fourier-domain mode locked swept laser source for fast data acquisition with a phase stability of 979 pm.

Dispersion-flattened dispersion-decreased all-normal dispersion (DFDD-ANDi) photonic crystal fibers have been identified as promising candidates for high-spectral-power coherent supercontinuum (SC) generation. However, the effects of the unintentional birefringence of the fibers on the SC generation have been ignored. This birefringence is widely present in nonlinear non-polarization maintaining fibers with a typical core size of 2 μm, presumably due to the structural symmetry breaks introduced in the fiber drawing process. We find that an intrinsic form-birefringence on the order of 10−5 profoundly affects the SC generation in a DFDD-ANDi photonic crystal fiber. Conventional simulations based on the scalar generalized nonlinear Schrödinger equation (GNLSE) fail to reproduce the prominent observed features of the SC generation in a short piece (9-cm) of this fiber. However, these features can be qualitatively or semi-quantitatively understood by the coupled GNLSE that takes into account the form-birefringence. The nonlinear polarization effects induced by the birefringence significantly distort the otherwise simple spectrotemporal field of the SC pulses. We therefore propose the fabrication of polarization-maintaining DFDD-ANDi fibers to avoid these adverse effects in pursuing a practical coherent fiber SC laser.

A flexible curled optical cord is useful for a common-path optical coherence tomography (OCT) system because a bending-insensitive arbitrary length can be chosen for the endoscopic imaging probe. However, there has been a critical problem that the partial reflector needs to be placed in between the sample and the objective lens. It limits the structure design of optical probe and leads to a low transverse resolution OCT imaging. Instead of a conventional single common-path interferometer, we propose a novel double common-path interferometer configuration in order to generate an interference signal that is independent of the optical distance between the partial reflector and sample. Due to the limitless tuning of the objective distance, an objective lens with a high numerical aperture (NA) up to 0.85 can be successfully used for phase-sensitive optical coherence tomography to achieve a 3-dimensional profile image of a transverse resolution of 0.7 μm. The intensity and phase terms of the interference signal can be obtained simultaneously from a Fourier-domain mode locked swept laser source for fast data acquisition with a phase stability of 979 pm.

Dispersion-flattened dispersion-decreased all-normal dispersion (DFDD-ANDi) photonic crystal fibers have been identified as promising candidates for high-spectral-power coherent supercontinuum (SC) generation. However, the effects of the unintentional birefringence of the fibers on the SC generation have been ignored. This birefringence is widely present in nonlinear non-polarization maintaining fibers with a typical core size of 2 µm, presumably due to the structural symmetry breaks introduced in the fiber drawing process. We find that an intrinsic form-birefringence on the order of 10−5 profoundly affects the SC generation in a DFDD-ANDi photonic crystal fiber. Conventional simulations based on the scalar generalized nonlinear Schrödinger equation (GNLSE) fail to reproduce the prominent observed features of the SC generation in a short piece (9-cm) of this fiber. However, these features can be qualitatively or semi-quantitatively understood by the coupled GNLSE that takes into account the form-birefringence. The nonlinear polarization effects induced by the birefringence significantly distort the otherwise simple spectrotemporal field of the SC pulses. We therefore propose the fabrication of polarization-maintaining DFDD-ANDi fibers to avoid these adverse effects in pursuing a practical coherent fiber SC laser.

An optical coherence tomography (OCT) system employing a microelectromechanical system (MEMS) mirror was used to measure the refractive index (RI) of anatomically different regions in acute brain tissue slices, in which viability was maintained. RI was measured in white-matter and grey-matter regions, including the cerebral cortex, putamen, hippocampus, thalamus and corpus callosum. The RI in the corpus callosum was found to be ~4% higher than the RIs in other regions. Changes in RI with tissue deformation were also measured in the cerebral cortex and corpus callosum under uniform compression (20-80% strain). For 80% strain, measured RIs increased nonlinearly by up to 70% and 90% in the cerebral cortex and corpus callosum respectively. Knowledge of RI in heterogeneous tissues can be used to correct distorted optical images caused by RI variations between different regions. Also deformation-dependent changes in RI can be applied to OCT elastography or to mechanical tests based on optical imaging such as indentation tests.

We report a method of assessing the contribution of whole cell body and its nucleus to the clinically most relevant backward light scattering. We first construct an experimental system that can measure forward scattering and use the system to precisely extract the optical properties of a specimen such as the refractive index contrast, size distribution, and their density. A system that can simultaneously detect the backscattered light is installed to collect the backscattering for the same specimen. By comparing the measured backscattering spectrum with that estimated from the parameters determined by the forward scattering experiment, the contribution of cell body and nucleus to the backward light scattering is quantitatively assessed. For the HeLa cells in suspension, we found that the cell body contributes less than 10% and cell nucleus on the order of 0.1% to the total backscattering signal. Quantitative determination of the origin of backscattered light may help design a system that aims for detecting particular structure of biological tissues.

Structured illumination (SI) has long been regarded as a nonquantitative technique for obtaining sectioned microscopic images. Its lack of quantitative results has restricted the use of SI sectioning to qualitative imaging experiments, and has also limited researchers’ ability to compare SI against competing sectioning methods such as confocal microscopy. We show how to modify the standard SI sectioning algorithm to make the technique quantitative, and provide formulas for calculating the noise in the sectioned images. The results indicate that, for an illumination source providing the same spatially-integrated photon flux at the object plane, and for the same effective slice thicknesses, SI sectioning can provide higher SNR images than confocal microscopy for an equivalent setup when the modulation contrast exceeds about 0.09.

Optically resonant devices are promising as label-free biomolecular sensors due to their ability to concentrate electromagnetic energy into small mode volumes and their capacity for multiplexed detection. A fundamental limitation of current optical biosensor technology is that the biomolecular interactions are limited to the surface of the resonant device, while the highest intensity of electromagnetic energy is trapped within the core. In this paper, we present nanoporous polymer optofluidic devices consisting of ring resonators coupled to bus waveguides. We report a 40% increase in polymer device sensitivity attributed to the addition of core energy- bioanalyte interactions.

A forward-imaging needle-type optical coherence tomography (OCT) probe with Doppler OCT (DOCT) capability has the potential to solve critical challenges in interventional procedures. A case in point is stereotactic neurosurgery where probes are advanced into the brain based on predetermined coordinates. Laceration of blood vessels in front of the advancing probe is an unavoidable complication with current methods. Moreover, cerebrospinal fluid (CSF) leakage during surgery can shift the brain rendering the predetermined coordinates unreliable. In order to address these challenges, we developed a forward-imaging OCT probe (740 μm O.D.) using a gradient-index (GRIN) rod lens that can provide real-time imaging feedback for avoiding at-risk vessels (8 frames/s with 1024 A-scans per frame for OCT/DOCT dual imaging) and guiding the instrument to specific targets with 12 μm axial resolution (100 frames/s with 160 A-scans per frame for OCT imaging only). The high signal-to-background characteristic of DOCT provides exceptional sensitivity in detecting and quantifying the blood flow within the sheep brain parenchyma in real time. The OCT/DOCT dual imaging also demonstrated its capability to differentiate the vessel type (artery/vein) on rat’s femoral vessels. We also demonstrated in ex vivo human brain that the location of the tip of the OCT probe can be inferred from micro-anatomical landmarks in OCT images. These findings demonstrate the suitability of OCT guidance during stereotactic procedures in the brain and its potential for reducing the risk of cerebral hemorrhage.

This paper proposes an automatic algorithm for the montage of OCT data sets, which produces a composite 3D OCT image over a large field of view out of several separate, partially overlapping OCT data sets. First the OCT fundus images (OFIs) are registered, using blood vessel ridges as the feature of interest and a two step iterative procedure to minimize the distance between all matching point pairs over the set of OFIs. Then the OCT data sets are merged to form a full 3D montage using cross-correlation. The algorithm was tested using an imaging protocol consisting of 8 OCT images for each eye, overlapping to cover a total retinal region of approximately 50x35 degrees. The results for 3 normal eyes and 3 eyes with retinal degeneration are analyzed, showing registration errors of 1.5±0.3 and 2.0±0.8 pixels respectively.

With the advent of Talbot-Lau interferometers for x-ray phase-contrast imaging, oblique and grazing incidence configurations are now used in the pursuit of sub-micron grating periods and high sensitivity. Here we address the question whether interferometers having oblique incident beams behave in the same way as the well-understood normal incidence ones, particularly when the grating planes are non-parallel. We derive the normal incidence equivalence of oblique incidence geometries from wave propagation modeling. Based on the theory, we propose a practical method to correct for non-parallelism of the grating planes, and demonstrate its effectiveness with a polychromatic hard x-ray reflective interferometer.

Super-resolution techniques like PALM and STORM require accurate localization of single fluorophores detected using a CCD. Popular localization algorithms inefficiently assume each photon registered by a pixel can only come from an area in the specimen corresponding to that pixel (not from neighboring areas), before iteratively (slowly) fitting a Gaussian to pixel intensity; they fail with noisy images. We present an alternative; a probability distribution extending over many pixels is assigned to each photon, and independent distributions are joined to describe emitter location. We compare algorithms, and recommend which serves best under different conditions. At low signal-to-noise ratios, ours is 2-fold more precise than others, and 2 orders of magnitude faster; at high ratios, it closely approximates the maximum likelihood estimate.