An important problem in minimally invasive photoacoustic (PA) imaging of brachytherapy seeds is reflection artifacts caused by the high signal from the optical fiber/needle tip reflecting off the seed. The presence of these artifacts confounds interpretation of images. In this letter, we demonstrate a recently developed concept called photoacoustic-guided focused ultrasound (PAFUSion) for the first time in the context of interstitial illumination PA imaging to identify and remove reflection artifacts. In this method, ultrasound (US) from the transducer is focused on the region of the optical fiber/needle tip identified in a first step using PA imaging. The image developed from the US diverging from the focus zone at the tip region visualizes only the reflections from seeds and other acoustic inhomogeneities, allowing identification of the reflection artifacts of the first step. These artifacts can then be removed from the PA image. Using PAFUSion, we demonstrate reduction of reflection artifacts and thereby improved interstitial PA visualization of brachytherapy seeds in phantom and ex vivo measurements on porcine tissue.

High resolution is important for sensitive detection of subtle distortions of retinal morphology at an early stage of eye diseases. We demonstrate virtually structured detection (VSD) as a feasible method to achieve in vivo super-resolution ophthalmoscopy. A line-scanning strategy was employed to achieve a super-resolution imaging speed up to 127  frames/s with a frame size of 512×512  pixels. The proof-of-concept experiment was performed on anesthetized frogs. VSD-based super-resolution images reveal individual photoreceptors and nerve fiber bundles unambiguously. Both image contrast and signal-to-noise ratio are significantly improved due to the VSD implementation.

This PDF file contains the editorial “Special Section Guest Editorial:Adaptive Optics and Wavefront Control for Biological Systems” for JBO Vol. 21 Issue 12

One of the prime limiting factors of optical imaging in biological applications is the diffusion of light by tissue, which prevents focusing at depths greater than the optical diffusion limit (typically ∼1  mm). To overcome this challenge, wavefront shaping techniques that use a spatial light modulator (SLM) to correct the phase of the incident wavefront have recently been developed. These techniques are able to focus light through scattering media beyond the optical diffusion limit. However, the low speeds of typically used liquid crystal SLMs limit the focusing speed. Here, we present a method using a digital micromirror device (DMD) and an electro-optic modulator (EOM) to measure the scattering-induced aberrations, and using a liquid crystal SLM to apply the correction to the illuminating wavefront. By combining phase modulation from an EOM with the DMD’s ability to provide selective illumination, we exploit the DMD’s higher refresh rate for phase measurement. We achieved focusing through scattering media in less than 8 ms, which is sufficiently short for certain in vivo applications, as it is comparable to the speckle correlation time of living tissue.

All aberrations produced inside a biospecimen can degrade the quality of a three-dimensional image in two-photon excitation laser scanning microscopy. Previously, we developed a transmissive liquid-crystal device to correct spherical aberrations that improved the image quality of a fixed-mouse-brain slice treated with an optical clearing reagent. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism. The motivation for this study is that asymmetric aberration can be induced by the shape of a biospecimen and/or by a complicated refractive-index distribution in a sample; this can considerably degrade optical performance even near the sample surface. The device’s performance was evaluated by observing fluorescence beads. The device was inserted between the objective lens and microscope revolver and succeeded in improving the spatial resolution and fluorescence signal of a bead image that was originally degraded by asymmetric aberration. Finally, we implemented the device for observing a fixed whole mouse brain with a sloping surface shape and complicated internal refractive-index distribution. The correction with the device improved the spatial resolution and increased the fluorescence signal by ∼2.4×. The device can provide a simple approach to acquiring higher-quality images of biospecimens.

We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation.

We describe a simple and compact full-field optical coherence tomography (FFOCT) setup coupled to a transmissive liquid crystal spatial light modulator (LCSLM) to induce or correct aberrations. To reduce the system complexity, strict pupil conjugation was abandoned because low-order aberrations are often dominant. We experimentally confirmed a recent theoretical and experimental demonstration that the image resolution was almost insensitive to aberrations that mostly induce a reduction of the signal level. As a consequence, an image-based algorithm was applied for the optimization process by using the FFOCT image intensity as the metric. Aberration corrections were demonstrated with both an USAF resolution target and biological samples for LCSLM-induced and sample-induced wavefront distortions.

We take stock of the progress that has been made into developing ultrathin endoscopes assisted by wave front shaping. We focus our review on multicore fiber-based lensless endoscopes intended for multiphoton imaging applications. We put the work into perspective by comparing with alternative approaches and by outlining the challenges that lie ahead.

Active optics such as deformable mirrors can be used to control both focal depth and aberrations during scanning laser microscopy. If the focal depth can be changed dynamically during scanning, then imaging of oblique surfaces becomes possible. If aberrations can be corrected dynamically during scanning, an image can be optimized throughout the field of view. Here, we characterize the speed and dynamic precision of a Boston Micromachines Corporation Multi-DM 140 element aberration correction mirror and a Revibro Optics 4-zone focus control mirror to assess suitability for use in an active and adaptive two-photon microscope. Tests for the multi-DM include both step response and sinusoidal frequency sweeps of specific Zernike modes (defocus, spherical aberration, coma, astigmatism, and trefoil). We find wavefront error settling times for mode amplitude steps as large as 400 nm to be less than 52  μs, with 3 dB frequencies ranging from 6.5 to 10 kHz. The Revibro Optics mirror was tested for step response only, with wavefront error settling time less than 80  μs for defocus steps up to 3000 nm, and less than 45  μs for spherical aberration steps up to 600 nm. These response speeds are sufficient for intrascan correction at scan rates typical of two-photon microscopy.

Our ability to see fine detail at depth in tissues is limited by scattering and other refractive characteristics of the tissue. For fixed tissue, we can limit scattering with a variety of clearing protocols. This allows us to see deeper but not necessarily clearer. Refractive aberrations caused by the bulk index of refraction of the tissue and its variations continue to limit our ability to see fine detail. Refractive aberrations are made up of spherical and other Zernike modes, which can be significant at depth. Spherical aberration that is common across the imaging field can be corrected using an objective correcting collar, although this can require manual intervention. Other aberrations may vary across the imaging field and can only be effectively corrected using adaptive optics. Adaptive optics can also correct other aberrations simultaneously with the spherical aberration, eliminating manual intervention and speeding imaging. We use an adaptive optics two-photon microscope to examine the impact of the spherical and higher order aberrations on imaging and contrast the effect of compensating only for spherical aberration against compensating for the first 22 Zernike aberrations in two tissue types. Increase in image intensity by 1.6× and reduction of root mean square error by 3× are demonstrated.

The possibility to adopt biological matter as photonic optical elements can open scenarios in biophotonics research. Recently, it has been demonstrated that a red blood cell (RBC) can be seen as an optofluidic microlens by showing its imaging capability as well as its focal tunability. Moreover, correlation between an RBC’s morphology and its behavior as a refractive optical element has been established and its exploitation for biomedical diagnostic purposes has been foreseen. In fact, any deviation from the healthy RBC morphology can be seen as additional aberration in the optical wavefront passing through the cell. By this concept, accurate localization of focal spots of RBCs can become very useful in the blood disorders identification. We investigate the three-dimensional positioning of such focal spots over time for samples with two different osmolarity conditions, i.e., when they assume discocyte and spherical shapes, respectively. We also demonstrate that a temporal variation of an RBC’s focal points along the optical axis is correlated to the temporal fluctuations in the RBC’s thickness maps. Furthermore, we show a sort of synchronization of the whole erythrocytes ensemble.

We demonstrate that simultaneous application of optical clearing agents (OCAs) and complex wavefront shaping in optical coherence tomography (OCT) can provide significant enhancement of penetration depth and imaging quality. OCA reduces optical inhomogeneity of a highly scattering sample, and the wavefront shaping of illumination light controls multiple scattering, resulting in an enhancement of the penetration depth and signal-to-noise ratio. A tissue phantom study shows that concurrent applications of OCA and wavefront shaping successfully operate in OCT imaging. The penetration depth enhancement is further demonstrated for ex vivo mouse ears, revealing hidden structures inaccessible with conventional OCT imaging.

Rapidly rising levels of myopia, particularly in the developing world, have led to an increased need for inexpensive and automated approaches to optometry. A simple and robust technique is provided for estimating major ophthalmic aberrations using a gradient-based wavefront sensor. The approach is based on the use of numerical calculations to produce diverse combinations of phase components, followed by Fourier transforms to calculate the coefficients. The approach does not utilize phase unwrapping nor iterative solution of inverse problems. This makes the method very fast and tolerant to image artifacts, which do not need to be detected and masked or interpolated as is needed in other techniques. These features make it a promising algorithm on which to base low-cost devices for applications that may have limited access to expert maintenance and operation.

While fs-lasers are clinically established for surgery in the anterior eye, their use in the posterior eye is impeded by aberrations and focus position errors. We implemented a laboratory system to investigate whether fs-laser surgery in the posterior eye is made more feasible by aberration correction and tomographic image guidance. Aberration correction is obtained by adaptive optics (AO) and the image guidance is accomplished by optical coherence tomography (OCT). System characteristic measurements and cutting experiments were performed inside an eye model. By aberration correction, wavefront errors were reduced from 270 nm root-mean-square (rms) to 64 nm rms, ignoring Zernike terms for tilts and focus. The Strehl ratio of the assigned point spread function is improved from 0.11 to 0.78. The threshold pulse energy of laser-induced optical breakdown in water is lowered from about 3.0 to about 1.3  μJ measured at the eye model entrance. After laser cutting of a synthetic foil placed 300  μm in front of porcine retinal tissue with the corrected system, postoperative three-dimensional OCT imaging showed no lesions in the tissue. Our results corroborate that AO and OCT will be two essential assistive components for possible clinical systems for fs-laser–based surgery in the posterior eye.

Despite widespread government and public interest, there are significant barriers to translating basic science discoveries into clinical practice. Biophotonics and biomedical optics technologies can be used to overcome many of these hurdles, due, in part, to offering new portable, bedside, and accessible devices. The current JBO special issue highlights promising activities and examples of translational biophotonics from leading laboratories around the world. We identify common essential features of successful clinical translation by examining the origins and activities of three major international academic affiliated centers with beginnings traceable to the mid-late 1970s: The Wellman Center for Photomedicine (Mass General Hospital, USA), the Beckman Laser Institute and Medical Clinic (University of California, Irvine, USA), and the Medical Laser Center Lübeck at the University of Lübeck, Germany. Major factors driving the success of these programs include visionary founders and leadership, multidisciplinary research and training activities in light-based therapies and diagnostics, diverse funding portfolios, and a thriving entrepreneurial culture that tolerates risk. We provide a brief review of how these three programs emerged and highlight critical phases and lessons learned. Based on these observations, we identify pathways for encouraging the growth and formation of similar programs in order to more rapidly and effectively expand the impact of biophotonics and biomedical optics on human health.

This PDF file contains the editorial “Special Section Guest Editorial:Translational biophotonics” for JBO Vol. 21 Issue 12

The aim of this study was to use microchannels drilled by an Er:YAG laser into a human tooth through the enamel into the dentin for direct injection of hydrogen peroxide (HP) to produce a minimally invasive, rapid, tooth bleaching effect. The experiments were conducted in vitro. Five microchannels with a diameter of ∼200  μm and a depth of ∼2  mm were drilled through the palatal side of a human tooth crown using the microbeam of an Er:YAG-laser with a wavelength of 2.94  μm. After injection of an aqueous solution of 31%-HP through the microchannels, the tooth color was evaluated using a VITA shade guide and International Commission on Illumination L*ab color parameters. A tooth model used for the evaluation of the distribution of HP concentration was created and the amount of HP which can be injected into tooth dentin to bleach it safely was estimated. Injection of 1.5±0.1  mm³ of 31%-HP into the tooth led to noticeable bleaching within 3 h and significant improvement of tooth color within 24 h.

In the confocal Raman spectra of skin dermis, the band area in the spectral region of proline and hydroxyproline varies according to the age and health condition of the volunteers, classified as healthy young women, healthy elderly women, and diabetic elderly women. Another observation refers to the intensity variation and negative Raman shift of the amide I band. To understand these effects, we adopted a model system using the DFT/B3LYP:3-21G procedure, considering the amino acid chain formed by glycine, hydroxyproline, proline, and alanine, which interacts with two and six water molecules. Through these systems, polarizability variations were analyzed to correlate its values with the observed Raman intensities of the three groups of volunteers and to assign the vibrational spectra of the skin dermis. As a way to correlate other experimental trends, we propose a model of chemical reaction of water interchange between the bonding amino acids, in which water molecules are attached with glucose by hydrogen bonds. The theoretical results are in accordance with the observed experimental trends.

Surface-enhanced Raman spectroscopy (SERS) was employed to detect deoxyribose nucleic acid (DNA) variations associated with the development of nasopharyngeal carcinoma (NPC). Significant SERS spectral differences between the DNA extracted from early NPC, advanced NPC, and normal nasopharyngeal tissue specimens were observed at 678, 729, 788, 1337, 1421, 1506, and 1573  cm⁻¹, which reflects the genetic variations in NPC. Principal component analysis combined with discriminant function analysis for early NPC discrimination yielded a diagnostic accuracy of 86.8%, 92.3%, and 87.9% for early NPC, advanced NPC, and normal nasopharyngeal tissue DNA, respectively. In this exploratory study, we demonstrated the potential of SERS for early detection of NPC based on the DNA molecular study of biopsy tissues.

We performed the feasibility study using speckle variance optical coherence tomography (SvOCT) to monitor the thermally induced protein denaturation and coagulation process as a function of temperature and depth. SvOCT provided the depth-resolved image of protein denaturation and coagulation with microscale resolution. This study was conducted using egg white. During the heating process, as the temperature increased, increases in the speckle variance signal was observed as the egg white proteins coagulated. Additionally, by calculating the cross-correlation coefficient in specific areas, denaturized egg white conditions were successfully estimated. These results indicate that SvOCT could be used to monitor the denaturation process of various proteins.

We propose in this work a model for describing visual acuity (V) as a function of defocus and pupil diameter. Although the model is mainly based on geometrical optics, it also incorporates nongeometrical effects phenomenologically. Compared to similar visual acuity models, the proposed one considers the effect of astigmatism and the variability of best corrected V among individuals; it also takes into account the accommodation and the “tolerance to defocus,” the latter through a phenomenological parameter. We have fitted the model to the V data provided in the works of Holladay et al. and Peters, showing the ability of this model to accurately describe the variation of V against blur and pupil diameter. We have also performed a comparison between the proposed model and others previously published in the literature. The model is mainly intended for use in the design of ophthalmic compensations, but it can also be useful in other fields such as visual ergonomics, design of visual tests, and optical instrumentation.

Patients with brain tumor or refractory epilepsy may be candidates for neurosurgery. Presurgical evaluation often includes language investigation to prevent or reduce the risk of postsurgical language deficits. Current techniques involve significant limitations with pediatric populations. Recently, near-infrared spectroscopy (NIRS) has been shown to be a valuable neuroimaging technique for language localization in children. However, it typically requires the child to perform a task (task-based NIRS), which may constitute a significant limitation. Resting-state functional connectivity NIRS (fcNIRS) is an approach that can be used to identify language networks at rest. This study aims to assess the utility of fcNIRS in children by comparing fcNIRS to more conventional task-based NIRS for language mapping in 33 healthy participants: 25 children (ages 3 to 16) and 8 adults. Data were acquired at rest and during a language task. Results show very good concordance between both approaches for language localization (Dice similarity coefficient=0.81±0.13) and hemispheric language dominance (kappa=0.86, p<0.006). The fcNIRS technique may be a valuable tool for language mapping in clinical populations, including children and patients with cognitive and behavioral impairments.

The structural and chemical modifications induced in dentin by ultrafast laser ablation were studied. The laser experiments were performed with a Yb:KYW chirped-pulse-regenerative amplification laser system (560-fs pulse duration, 1030-nm radiation wavelength), fluences in the range 2 to 14  J/cm2, 1-kHz pulse repetition rate, and 5-mm/s scanning speed. The ablation surfaces were characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The ablation surfaces produced with 2  J/cm2 presented an irregular morphology with exposed dentinal tubules and no evidence of thermal effects. For 7 and 14  J/cm2, the ablation surfaces were covered by a layer of redeposited ablation debris, consisting mainly of amorphous calcium phosphate. This layer is weakly adherent to the underlying tissue and can be easily removed by ultrasonication, revealing a surface with a morphology similar to the one obtained with 2  J/cm2. The constitution of the dentin ablation surfaces is similar to the constitution of pristine dentin, showing that, within this fluence range, the laser treatment does not significantly modify the structure and constitution of dentin. The results achieved suggest an ablation mechanism where collagen is preferentially decomposed by the laser radiation, reducing the tissue cohesive strength and leading, ultimately, to its ablation.

The availability of reproducible, convenient, and inexpensive model organisms able to generate predictable levels of endogenous porphyrins, including protoporphyrin IX (PpIX), is essential in photomedicine research. Saccharomyces cerevisiae produces endogenous PpIX and was used as a model organism for this study with the aim to maximize endogenous PpIX fluorescence intensity. It was found that PpIX fluorescence was significantly enhanced by administration of 5-aminolevulinic acid (ALA) and 2,2′-bipyridyl. Fluorescence intensity and spectroscopy of PpIX produced endogenously were measured in diluted yeast solutions under various conditions. The optimal protocol was: 5  μM ALA and 1 mM 2,2′-bipyridyl administered synchronously at 32°C. After 3 h, PpIX in yeast demonstrated similar steady-state and time-resolved spectroscopy as that of PpIX in DMSO. Moreover, under hypoxic conditions, the reciprocal lifetime of PpIX delayed fluorescence measured in real time was correlated to the partial pressure of oxygen (pO2) measured concomitantly with a commercially available pO2 probe. These data show that yeast can, in optimal conditions, reproducibly generate PpIX. This is of interest in various fields such as photodiagnosis, photodynamic therapy, and photobiomodulation. Use of this model organism focuses on essential mechanisms, without the complexity of a multicellular organism.

A method for the depth-resolved detection of fluorescent radiation based on imaging of an interference pattern of two intersecting beams and shearing interferometry is presented. The illumination setup provides the local addressing of the excitation of fluorescence and a coarse confinement of the excitation volume in axial and lateral directions. The reconstruction of the depth relies on the measurement of the phase of the fluorescent wave fronts. Their curvature is directly related to the distance of a source to the focus of the imaging system. Access to the phase information is enabled by a lateral shearing interferometer based on a Michelson setup. This allows the evaluation of interference signals even for spatially and temporally incoherent light such as emitted by fluorophors. An analytical signal model is presented and the relations for obtaining the depth information are derived. Measurements of reference samples with different concentrations and spatial distributions of fluorophors and scatterers prove the experimental feasibility of the method. In a setup optimized for flexibility and operating in the visible range, sufficiently large interference signals are recorded for scatterers placed in depths in the range of hundred micrometers below the surface in a material with scattering properties comparable to dental enamel.

This study introduces a single-channel optical video stereomicroscope based on a transparent rotating deflector (TRD) for high-resolution and high-magnification stereomicroscopy. The existing stereomicroscopes have some limitations, such as limited resolution and magnification, fixed optical channel, and the necessity to use higher quality and higher cost optical channel components compared with the conventional optical microscopes. The goal of this study was to develop a method for improved stereo imaging and stereovision for optical microscopy. Here, we demonstrate the generation of stereo video images of left and right pairs by the refraction of light passing through a motorized TRD. We estimated the corresponding rotation angles for human stereovision and the required torque. In addition, we evaluated the image quality stability under the TRD rotation.

Noninvasive and nonradioactive imaging modality to track and image apoptosis during chemotherapy of triple negative breast cancer is much needed for an effective treatment plan. Phosphatidylserine (PS) is a biomarker transiently exposed on the outer surface of the cells during apoptosis. Its externalization occurs within a few hours of an apoptotic stimulus by a chemotherapy drug and leads to presentation of millions of phospholipid molecules per apoptotic cell on the cell surface. This makes PS an abundant and accessible target for apoptosis imaging. In the current work, we show that PS monoclonal antibody tagged with indocyanine green (ICG) can help to track and image apoptosis using multispectral optoacoustic tomography in vivo. When compared to saline control, the doxorubicin treated group showed a significant increase in uptake of ICG-PS monoclonal antibody in triple negative breast tumor xenografted in NCr nude female mice. Day 5 posttreatment had the highest optoacoustic signal in the tumor region, indicating maximum apoptosis and the tumor subsequently shrank. Since multispectral optoacoustic imaging does not involve the use of radioactivity, the longer the circulatory time of the PS antibody can be exploited to monitor apoptosis over a period of time without multiple injections of commonly used imaging probes such as Tc-99m Annexin V or F-18 ML10. The proposed apoptosis imaging technique involving multispectral optoacoustic tomography, monoclonal antibody, and near-infrared absorbing fluorescent marker can be an effective tool for imaging apoptosis and treatment planning.

Transport of intensity equation (TIE) is a powerful tool for phase retrieval and quantitative phase imaging, which requires intensity measurements only at axially closely spaced planes without a separate reference beam. It does not require coherent illumination and works well on conventional bright-field microscopes. The quantitative phase reconstructed by TIE gives valuable information that has been encoded in the complex wave field by passage through a sample of interest. Such information may provide tremendous flexibility to emulate various microscopy modalities computationally without requiring specialized hardware components. We develop a requisite theory to describe such a hybrid computational multimodal imaging system, which yields quantitative phase, Zernike phase contrast, differential interference contrast, and light field moment imaging, simultaneously. It makes the various observations for biomedical samples easy. Then we give the experimental demonstration of these ideas by time-lapse imaging of live HeLa cell mitosis. Experimental results verify that a tunable lens-based TIE system, combined with the appropriate postprocessing algorithm, can achieve a variety of promising imaging modalities in parallel with the quantitative phase images for the dynamic study of cellular processes.

Forward and adjoint Monte Carlo (MC) models of radiance are proposed for use in model-based quantitative photoacoustic tomography. A two-dimensional (2-D) radiance MC model using a harmonic angular basis is introduced and validated against analytic solutions for the radiance in heterogeneous media. A gradient-based optimization scheme is then used to recover 2-D absorption and scattering coefficients distributions from simulated photoacoustic measurements. It is shown that the functional gradients, which are a challenge to compute efficiently using MC models, can be calculated directly from the coefficients of the harmonic angular basis used in the forward and adjoint models. This work establishes a framework for transport-based quantitative photoacoustic tomography that can fully exploit emerging highly parallel computing architectures.

Intravascular optical coherence tomography (IVOCT) is an imaging technique that is used to analyze the underlying cause of cardiovascular disease. Because a catheter is used during imaging, the intensities can be affected by the catheter position. This work aims to analyze the effect of the catheter position on IVOCT image intensities and to propose a compensation method to minimize this effect in order to improve the visualization and the automatic analysis of IVOCT images. The effect of catheter position is modeled with respect to the distance between the catheter and the arterial wall (distance-dependent factor) and the incident angle onto the arterial wall (angle-dependent factor). A light transmission model incorporating both factors is introduced. On the basis of this model, the interaction effect of both factors is estimated with a hierarchical multivariant linear regression model. Statistical analysis shows that IVOCT intensities are significantly affected by both factors with p<0.001, as either aspect increases the intensity decreases. This effect differs for different pullbacks. The regression results were used to compensate for this effect. Experiments show that the proposed compensation method can improve the performance of the automatic bioresorbable vascular scaffold strut detection.

Measurement of photon penetration in biological tissues is a central theme in optical imaging. A great number of endogenous tissue factors such as absorption, scattering, and anisotropy affect the path of photons in tissue, making it difficult to predict the penetration depth at different wavelengths. Traditional studies evaluating photon penetration at different wavelengths are focused on tissue spectroscopy that does not take into account the heterogeneity within the sample. This is especially critical in shortwave infrared where the individual vibration-based absorption properties of the tissue molecules are affected by nearby tissue components. We have explored the depth penetration in biological tissues from 900 to 1650 nm using Monte–Carlo simulation and a hyperspectral imaging system with Michelson spatial contrast as a metric of light penetration. Chromatic aberration-free hyperspectral images in transmission and reflection geometries were collected with a spectral resolution of 5.27 nm and a total acquisition time of 3 min. Relatively short recording time minimized artifacts from sample drying. Results from both transmission and reflection geometries consistently revealed that the highest spatial contrast in the wavelength range for deep tissue lies within 1300 to 1375 nm; however, in heavily pigmented tissue such as the liver, the range 1550 to 1600 nm is also prominent.

We propose two automatic methods for detecting bleeding in wireless capsule endoscopy videos of the small intestine. The first one uses solely the color information, whereas the second one incorporates the assumptions about the blood spot shape and size. The original idea is namely the definition of a new color space that provides good separability of blood pixels and intestinal wall. Both methods can be applied either individually or their results can be fused together for the final decision. We evaluate their individual performance and various fusion rules on real data, manually annotated by an endoscopist.

Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology. However, the current major limitation of light sheet microscopy arises from optical aberrations, with the main drawback being the defocusing introduced by refractive index variations that accompany clearing techniques. Here, we propose an inexpensive and easy to build light sheet based instrumentation to overcome this limitation by optomechanically decoupling the sample scanning movement from the detection step. Our solution is relatively simple to implement and also provides increased modularity by using a swappable excitation arm. This expands the range of samples we can image on a single system, from high resolution for single cells at μm spatial resolution, to tissues with mm spatial resolution. We demonstrate our approach, using the system to image iDISCO cleared embryos and sciatic nerves, and provide the full three-dimensional reconstruction of these objects in minutes.

In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, selective plane illumination microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single-photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (∼10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly.

A combination of approaches to the image analysis in cross-polarization optical coherence tomography (CP OCT) and high-resolution imaging by nonlinear microscopy and atomic force microscopy (AFM) at the different stages of atherosclerotic plaque development is studied. This combination allowed us to qualitatively and quantitatively assess the disorganization of collagen in the atherosclerotic arterial tissue (reduction and increase of CP backscatter), at the fiber (change of the geometric distribution of fibers in the second-harmonic generation microscopy images) and fibrillar (violation of packing and different nature of a basket-weave network of fibrils in the AFM images) organization levels. The calculated CP channel-related parameters are shown to have a statistically significant difference between stable and unstable (also called vulnerable) plaques, and hence, CP OCT could be a potentially powerful, minimally invasive method for vulnerable plaques detection.

Current diagnostic capabilities and limitations of fluorescence endomicroscopy in the cervix are assessed by qualitative and quantitative image analysis. Four cervical tissue types are investigated: normal columnar epithelium, normal and precancerous squamous epithelium, and stromal tissue. This study focuses on the perceived variability within and the subtle differences between the four tissue groups in the context of endomicroscopic in vivo pathology. Conclusions are drawn on the general ability to distinguish and diagnose tissue types, on the need for imaging depth control to enhance differentiation, and on the possible risks for diagnostic misinterpretations.

Fluorescence probes have distinct yields and lifetimes when located in different environments, which makes the reconstruction of fluorescence molecular lifetime tomography (FMLT) challenging. To enhance the reconstruction performance of time-domain (TD) FMLT with heterogeneous targets, a self-guided L1 regularization projected steepest descent (SGL1PSD) algorithm is proposed. Different from other algorithms performed in time domain, SGL1PSD introduces a time-resolved strategy into fluorescence yield reconstruction. The algorithm consists of four steps. Step 1 reconstructs the initial yield map with full time gate strategy; steps 2–4 reconstruct the inverse lifetime map, the yield map, and the inverse lifetime map again with time-resolved strategy, respectively. The reconstruction result of each step is used as a priori for the reconstruction of the next step. Projected iterated Tikhonov regularization algorithm is adopted for the yield map reconstructions in steps 1 and 3 to provide a solution with iterative refinement and nonnegative constraint. The inverse lifetime map reconstructions in steps 2 and 4 are based on L1 regularization projected steepest descent algorithm, which employ the L1 regularization to reduce the ill-posedness of the high-dimensional nonlinear problem. Phantom experiments with heterogeneous targets at different edge-to-edge distances demonstrate that SGL1PSD can provide high resolution and quantification accuracy for TD FMLT.

Optical coherence tomography (OCT) can map the stiffness of biological tissue by imaging mechanical perturbations (shear waves) propagating in the tissue. Most shear wave elastography (SWE) techniques rely on active shear sources to generate controlled displacements that are tracked at ultrafast imaging rates. Here, we propose a noise-correlation approach to retrieve stiffness information from the imaging of diffuse displacement fields using low-frame rate spectral-domain OCT. We demonstrated the method on tissue-mimicking phantoms and validated the results by comparison with classic ultrafast SWE. Then we investigated the in vivo feasibility on the eye of an anesthetized rat by applying noise correlation to naturally occurring displacements. The results suggest a great potential for passive elastography based on the detection of natural pulsatile motions using conventional spectral-domain OCT systems. This would facilitate the transfer of OCT-elastography to clinical practice, in particular, in ophthalmology or dermatology.

Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration.

The classification of erythrocytes plays an important role in the field of hematological diagnosis, specifically blood disorders. Since the biconcave shape of red blood cell (RBC) is altered during the different stages of hematological disorders, we believe that the three-dimensional (3-D) morphological features of erythrocyte provide better classification results than conventional two-dimensional (2-D) features. Therefore, we introduce a set of 3-D features related to the morphological and chemical properties of RBC profile and try to evaluate the discrimination power of these features against 2-D features with a neural network classifier. The 3-D features include erythrocyte surface area, volume, average cell thickness, sphericity index, sphericity coefficient and functionality factor, MCH and MCHSD, and two newly introduced features extracted from the ring section of RBC at the single-cell level. In contrast, the 2-D features are RBC projected surface area, perimeter, radius, elongation, and projected surface area to perimeter ratio. All features are obtained from images visualized by off-axis digital holographic microscopy with a numerical reconstruction algorithm, and four categories of biconcave (doughnut shape), flat-disc, stomatocyte, and echinospherocyte RBCs are interested. Our experimental results demonstrate that the 3-D features can be more useful in RBC classification than the 2-D features. Finally, we choose the best feature set of the 2-D and 3-D features by sequential forward feature selection technique, which yields better discrimination results. We believe that the final feature set evaluated with a neural network classification strategy can improve the RBC classification accuracy.

Lateral in-homogeneities in lipid compositions cause microdomains formation and change in the physical properties of biological membranes. With the presence of cholesterol and mixed species of lipids, phospholipid membranes segregate into lateral domains of liquid-ordered and liquid-disordered phases. Coupling of two-dimensional intralayer phase separations and interlayer liquid-crystalline ordering in multicomponent membranes has been previously demonstrated. By the use of digital holographic microscopy (DHMicroscopy), we quantitatively analyzed the volumetric dynamical behavior of such membranes. The specimens are lipid mixtures composed of sphingomyelin, cholesterol, and unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine. DHMicroscopy in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes, three-dimensional information on the morphology variation of lipid stacks at arbitrary time scales is obtained. Moreover, the thickness distribution of the object at demanded axial planes can be obtained by numerical focusing. Our results show that the volume evolution of lipid domains follows approximately the same universal growth law of previously reported area evolution. However, the thickness of the domains does not alter significantly by time; therefore, the volume evolution is mostly attributed to the changes in area dynamics. These results might be useful in the field of membrane-based functional materials.

Choroid thickness and volume estimated from optical coherence tomography (OCT) images have emerged as important metrics in disease management. This paper presents an automated three-dimensional (3-D) method for segmenting the choroid from 1-μm wide-view swept source OCT image volumes, including the Bruch’s membrane (BM) and the choroidal–scleral interface (CSI) segmentation. Two auxiliary boundaries are first detected by modified Canny operators and then the optical nerve head is detected and removed. The BM and the initial CSI segmentation are achieved by 3-D multiresolution graph search with gradient-based cost. The CSI is further refined by adding a regional cost, calculated from the wavelet-based gradual intensity distance. The segmentation accuracy is quantitatively evaluated on 32 normal eyes by comparing with manual segmentation and by reproducibility test. The mean choroid thickness difference from the manual segmentation is 19.16±4.32  μm, the mean Dice similarity coefficient is 93.17±1.30%, and the correlation coefficients between fovea-centered volumes obtained on repeated scans are larger than 0.97.

Laser Doppler flowmetry (LDF) and laser speckle contrast analysis (LASCA) both utilize the spatiotemporal properties of laser speckle patterns to assess microcirculatory blood flow in tissue. Although the techniques analyze the speckle pattern differently, there is a close relationship between them. We present a theoretical overview describing how the LDF power spectrum and the LASCA contrast can be calculated from each other, and how both these can be calculated from an optical Doppler spectrum containing various degrees of Doppler shifted light. The theoretical relationships are further demonstrated using time-resolved speckle simulations. A wide range of Monte Carlo simulated tissue models is then used to show how perfusion estimates for LDF and LASCA are affected by changes in blood concentration and speed distribution, as well as by geometrical and optical properties. We conclude that perfusion estimates from conventional single exposure time LASCA are in general more sensitive to changes in optical and geometrical properties and are less accurate in the prediction of real perfusion changes, especially speed changes. Since there is a theoretical one-to-one relationship between Doppler power spectrum and contrast, one can conclude that those drawbacks with the LASCA technique can be overcome using a multiple exposure time setup.

We present a phase-shifting quantitative phase imaging technique providing high temporal and spatial phase stability and high acquisition speed. A piezoelectric microfabricated phase modulator allows tunable modulation frequencies up to the kHz range. After assessing the quantitative phase accuracy with technical samples, we demonstrate the high acquisition rate while monitoring cellular processes at temporal scales ranging from milliseconds to hours.

Multiple reference optical coherence tomography (MR-OCT) is a technology ideally suited to low-cost, compact OCT imaging. This modality is an extension of time-domain OCT with the addition of a partial mirror in front of the reference mirror. This enables extended, simultaneous depth scanning with the relatively short scan range of a miniature voice coil motor on which the scanning mirror is mounted. This work details early stage development of the first iteration of a miniature MR-OCT device. This iteration utilizes a fiber-coupled input from an off-board superluminescent diode. The dimensions of the module are 40×57  mm. Off-the-shelf miniature optical components, voice coil motors, and photodetectors are used, with the complexity of design depending on the specific application. The photonic module can be configured as either polarized or nonpolarized and can include balanced detection. The results shown in this work are from the nonpolarized device. The system was characterized through measurement of the input spectrum, axial resolution, and signal-to-noise ratio. Typical B-scans of static and in vivo samples are shown, which illustrate the potential applications for such a technology.

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

A phase variance optical coherence microscope (pvOCM) has been created to image blood flow in the microvasculature of zebrafish embryos, without the use of exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2.8  μm in tissue and imaging depth of more than 100  μm. Images of 2 to 5 days postfertilization zebrafish embryos identified the detailed anatomical structure based on OCM intensity contrast. Phase variance contrast offered visualization of blood flow in the arteries, veins, and capillaries. The pvOCM images of the vasculature were confirmed by direct comparisons with fluorescence microscopy images of transgenic embryos in which the vascular endothelium is labeled with green fluorescent protein. The ability of pvOCM to capture activities of regional blood flow permits it to reveal functional information that is of great utility for the study of vascular development.

We investigate an intensity and wavelength modulation combined plasmon resonance-based fiber-optic sensor technology. Composed of gold nanoparticles (GNPs) and sandwich configuration of Au/indium tin oxide (ITO)/Au film, two sensing regions are fabricated separately along with unclad portions of the fiber-optic probe. It can simultaneously monitor both the light intensity from the Au NP channel and the wavelength from the Au/ITO/Au film channel with a single detector. As the refractive index (RI) of the external environment changes, the transmission intensity and resonance wavelength in the two channels are modified, which provides an interrogation of intensity and wavelength modulation. The sandwich film structure is formed using magnetron sputtering technology, and the GNPs functioning as localized surface plasmon resonators are coated on a multimode optical fiber via the layer-by-layer method. The experimental results reveal that the RI sensitivities of the two sensing channels are 334.1% RIU⁻¹ and 1963.2  nm/RIU, respectively. Based on the above sensing design, we conduct real-time and label-free monitoring of IgG/anti-IgG and Con A/RNase B biomolecular interaction. The resonant dips excited by different sensing modes make it more attractive as a multichannel surface plasmon resonance analysis technology, which is valuable in biological and life sciences research and rapid diagnostics.

This paper explores the potential of optical coherence microscopy (OCM) for the in situ monitoring of biofilm growth. The quantitative imaging of the early developmental biology of a representative biofilm, Klebsiella pneumonia (KP-1), was performed using a swept source-based Fourier domain OCM system. The growth dynamics of the KP-1 biofilms and their transient response under perturbation was investigated using the enface visualization of microcolonies and their spatial localization. Furthermore, the optical density (OD) and planar density of the biofilms are calculated using an OCM technique and compared with OD and colony forming units measured using standard procedures via the sampling of the flow-cell effluent.

A fast surface plasmon resonance (SPR) imaging biosensor system based on wavelength interrogation using a liquid crystal tunable filter (LCTF) is presented. The system combines the merits of wide-dynamic detection range offered by the spectral approach and multiplexed high-throughput data collection with a two-dimensional (2-D) biosensor array. The key feature of the reported scheme is a feedback loop that drives the LCTF to achieve fast tracking of the SPR dip movement caused by the binding of target molecules to the sensor surface. Experimental results show that the system is capable of completing an SPR dip measurement within 4 s. Based on using a spectral window of about 100 nm, the experimental dynamic detection range and refractive index resolution are 4.63×10⁻²  RIU and 5.87×10⁻⁶  RIU, respectively. As also demonstrated herein using 2-D microsensor arrays, among the spectral SPR sensors, the reported system is most suitable for multiplexed label-free biosensing applications.

Microscale optical devices enabled by wireless power harvesting and telemetry facilitate manipulation and testing of localized biological environments (e.g., neural recording and stimulation, targeted delivery to cancer cells). Design of integrated microsystems utilizing optical power harvesting and telemetry will enable complex in vivo applications like actuating a single nerve, without the difficult requirement of extreme optical focusing or use of nanoparticles. Silicon-on-insulator (SOI)-based platforms provide a very powerful architecture for such miniaturized platforms as these can be used to fabricate both optoelectronic and microelectronic devices on the same substrate. Near-infrared biomedical optics can be effectively utilized for optical power harvesting to generate optimal results compared with other methods (e.g., RF and acoustic) at submillimeter size scales intended for such designs. We present design and integration techniques of optical power harvesting structures with complementary metal oxide semiconductor platforms using SOI technologies along with monolithically integrated electronics. Such platforms can become the basis of optoelectronic biomedical systems including implants and lab-on-chip systems.

Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid’s optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic “target” and magnetic “probe” nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1×5 array of 50  μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ∼25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology’s potential to translate magnetic nanoparticle assays into a user-free optical analys

A clear correlation has been observed between the resonance Raman (RR) spectra of plaques in the aortic tunica intimal wall of a human corpse and three states of plaque evolution: fibrolipid plaques, calcified and ossified plaques, and vulnerable atherosclerotic plaques (VPs). These three states of atherosclerotic plaque lesions demonstrated unique RR molecular fingerprints from key molecules, rendering their spectra unique with respect to one another. The vibrational modes of lipids, cholesterol, carotenoids, tryptophan and heme proteins, the amide I, II, III bands, and methyl/methylene groups from the intrinsic atherosclerotic VPs in tissues were studied. The salient outcome of the investigation was demonstrating the correlation between RR measurements of VPs and the thickness measurements of fibrous caps on VPs using standard histopathology methods, an important metric in evaluating the stability of a VP. The RR results show that VPs undergo a structural change when their caps thin to 66  μm, very close to the 65-μm empirical medical definition of a thin cap fibroatheroma plaque, the most unstable type of VP.

The concentration of the biologically active molecular oxygen gas is of crucial importance for fruits in the metabolic respiration, maturation, and ripening processes. In our study, oxygen content and oxygen transport in fruits, exemplified by apples and guavas, were studied noninvasively by gas in scattering media absorption spectroscopy. The technique is based on the fact that free gases typically have 10,000 times narrower absorption features than the bulk material. The technique was demonstrated in studies of the influence of the fruit skin in regulating the internal oxygen balance, by observing the signal response of the internal oxygen gas to a transient change in the ambient gas concentration on peeled and unpeeled fruits. In addition, the gas exchange rate at different ripening stages was also studied in intact guavas.

Pelvic organ prolapse (POP) occurs when changes to the pelvic organ support structures cause descent or herniation of the pelvic organs into the vagina. Clinical evaluation of POP is a series of manual measurements known as the pelvic organ prolapse quantification (POP-Q) score. However, it fails to identify the mechanism causing POP and relies on the skills of the practitioner. We report on a modified vaginal speculum incorporating a double-helix fiber-Bragg grating structure for distributed pressure measurements along the length of the vagina and include preliminary data in an ovine model of prolapse. Vaginal pressure profiles were recorded at 10 Hz as the speculum was dilated incrementally up to 20 mm. At 10-mm dilation, nulliparous sheep showed higher mean pressures (102±46  mmHg) than parous sheep (39±23  mmHg) (P=0.02), attributable largely to the proximal (cervical) end of the vagina. In addition to overall pressure variations, we observed a difference in the distribution of pressure that related to POP-Q measurements adapted for the ovine anatomy, showing increased tissue laxity in the upper anterior vagina for parous ewes. We demonstrate the utility of the fiber-optic instrumented speculum for rapid distributed measurement of vaginal support.

Several medical procedures involve the use of needles. The advent of robotic and robot assisted procedures requires dynamic estimation of the needle tip location during insertion for use in both assistive systems as well as for automatic control. Most prior studies have focused on the maneuvering of solid flexible needles using external force measurements at the base of the needle holder. However, hollow needles are used in several procedures and measurements of forces in proximity of such needles can eliminate the need for estimating frictional forces that have high variations. These measurements are also significant for endoscopic procedures in which measurement of forces at the needle holder base is difficult. Fiber Bragg grating sensors, due to their small size, inert nature, and multiplexing capability, provide a good option for this purpose. Force measurements have been undertaken during needle insertion into tissue mimicking phantoms made of polydimethylsiloxane as well as chicken tissue using an 18-G needle instrumented with FBG sensors. The results obtained show that it is possible to estimate the different stages of needle penetration including partial rupture, which is significant for procedures in which precise estimation of needle tip position inside the organ or tissue is required.

Although TiO₂ can be used to effectively generate reactive oxygen species (ROS) for photodynamic application, its absorption in the ultraviolet range makes the excitation harmful to tissue. Based on the concept of a sensitized solar cell, TiO₂ nanoparticles (NPs) are sensitized by linking with the photosensitizer, HMME, to form HMME-TiO₂ nanocomposites (NCs) for demonstrating the photodynamic effects under the illumination of white light. The HMME-TiO₂ NCs of different composition ratios are prepared for maximizing the generation of ROS and optimizing the inactivation effect of KB cells. The material characteristics and the ROS generation capability of the HMME-TiO₂ NCs with the optimized combination ratio show their merits in a photodynamic process under white light irradiation. The application of such NCs to KB cell experiments results in a higher inactivation efficiency when compared to pure HMME of the same concentration.