2011 was another record year for JBO. The number of manuscript submissions reached nearly 750. The impact factor leaped to 3.188, with the 5-year combined impact factor at 3.659. The publication frequency changed to monthly starting in January. The manuscript review cycles have been shortened considerably without sacrificing quality of review. JBO is deeply indebted to the authors, reviewers, and editorial board members for their contributions to such marvelous achievements.

Submicron-resolution photoacoustic microscopy (PAM) currently exists only in transmission mode, due to the technical difficulties of combining high numerical-aperture (NA) optical illumination with high NA acoustic detection. The lateral resolution of reflection-mode PAM has not reached <2  μm in the visible light range. Here we develop the first reflection-mode submicron-resolution PAM system with a new compact design. By using a parabolic mirror to focus and reflect the photoacoustic waves, sufficient signals were collected for good sensitivity without distorting the optical focusing. By imaging nanospheres and a resolution test chart, the lateral resolution was measured to be ∼ 0.5  μm with an optical wavelength of 532 nm, an optical NA of 0.63. The axial resolution was measured at 15 μm. Here the axial resolution was measured by a different experiment with the lateral resolution measurement. But we didn't describe the details of axial resolution measurement due to space limit. The maximum penetration was measured at ∼ 0.42  mm in optical-scattering soft tissue. As a comparison, both the submicron-resolution PAM and a 2.4 μm-resolution PAM were used to image a mouse ear in vivo with the same optical wavelength and similar pulse energy. Capillaries were resolved better by the submicron-resolution PAM. Therefore, the submicron-resolution PAM is suitable for in vivo high-resolution imaging, or even subcellular imaging, of optical absorption.

An introduction to the special section by the guest editors.

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5  frames/sec with a field of view of 362×212  μm² and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

Gastrointestinal cancers are heterogeneous and can overexpress several protein targets that can be imaged simultaneously on endoscopy using multiple molecular probes. We aim to demonstrate a multispectral scanning fiber endoscope for wide-field fluorescence detection of colonic dysplasia. Excitation at 440, 532, and 635 nm is delivered into a single spiral scanning fiber, and fluorescence is collected by a ring of light-collecting optical fibers placed around the instrument periphery. Specific-binding peptides are selected with phage display technology using the CPC;Apc mouse model of spontaneous colonic dysplasia. Validation of peptide specificity is performed on flow cytometry and in vivo endoscopy. The peptides KCCFPAQ, AKPGYLS, and LTTHYKL are selected and labeled with 7-diethylaminocoumarin-3-carboxylic acid (DEAC), 5-carboxytetramethylrhodamine (TAMRA), and CF633, respectively. Separate droplets of KCCFPAQ-DEAC, AKPGYLS-TAMRA, and LTTHYKL-CF633 are distinguished at concentrations of 100 and 1 μM. Separate application of the fluorescent-labeled peptides demonstrate specific binding to colonic adenomas. The average target/background ratios are 1.71±0.19 and 1.67±0.12 for KCCFPAQ-DEAC and AKPGYLS-TAMRA, respectively. Administration of these two peptides together results in distinct binding patterns in the blue and green channels. Specific binding of two or more peptides can be distinguished in vivo using a novel multispectral endoscope to localize colonic dysplasia on real-time wide-field imaging.

We present a foveated miniature endoscopic lens implemented by amplifying the optical distortion of the lens. The resulting system provides a high-resolution region in the central field of view and low resolution in the outer fields, such that a standard imaging fiber bundle can provide both the high resolution needed to determine tissue health and the wide field of view needed to determine the location within the inspected organ. Our proof of concept device achieves 7 ∼ 8  μm resolution in the fovea and an overall field of view of 4.6 mm. Example images and videos show the foveated lens' capabilities.

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

Micro-optical probes, including gradient index (GRIN) lenses and microprisms, have expanded the range of in vivo multiphoton microscopy to reach previously inaccessible deep brain structures such as deep cortical layers and the underlying hippocampus in mice. Yet imaging with GRIN lenses has been fundamentally limited by large amounts of spherical aberration and the need to construct compound lenses that limit the field-of-view. Here, we demonstrate the use of 0.5-mm-diameter, 1.7-mm-long GRIN lens singlets with 0.6 numerical aperture in conjunction with a cover glass and a conventional microscope objective correction collar to balance spherical aberrations. The resulting system achieves a lateral resolution of 618 nm and an axial resolution of 5.5 μm, compared to lateral and axial resolutions of ∼ 1  μm and ∼ 15  μm, respectively, for compound GRIN lenses of similar diameter. Furthermore, the GRIN lens singlets display fields-of-view in excess of 150 μm, compared with a few tens of microns for compound GRIN lenses. The GRIN lens/cover glass combination presented here is easy to assemble and inexpensive enough for use as a disposable device, enabling ready adoption by the neuroscience community.

In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.

We compare the illumination uniformity and the associated effects of the spiral and Lissajous scanning patterns that are commonly used in an endomicroscope. Theoretical analyses and numerical simulations were first performed to quantitatively investigate the area illumination density in the spiral scanning pattern. The results revealed the potential problem of manifest photodamage due to the very high illumination density in the center of the spiral scan. Similar analyses of the Lissajous scanning pattern, which can be conveniently implemented on the same endomicroscope with no hardware modifications, showed a more uniform illumination density with about an 80-fold reduction in the peak illumination density. To underscore the benefit offered by the improved illumination uniformity, we conducted in vitro two-photon fluorescence imaging of cultured cells stained with a LIVE/DEAD viability assay using our home-built, fiber-optic, two-channel endomicroscopy system. Both the spiral and the Lissajous scans were implemented. Our experimental results showed that cells near the spiral scan center experienced obvious photodamage, whereas cells remained alive over the entire region under the Lissajous beam scanning, confirming the predicted advantage offered by the Lissajous scan over this spiral scan in an endomicroscopy setting.

Direct measurements of aggregation forces in piconewton range between two red blood cells in pair rouleau are performed under physiological conditions using double trap optical tweezers. Aggregation and disaggregation properties of healthy and pathologic (system lupus erythematosis) blood samples are analyzed. Strong difference in aggregation speed and behavior is revealed using the offered method which is proposed to be a promising tool for SLE monitoring at single cell level.

Dental caries is the leading cause of tooth loss, which can promptly be prevented if detected in early stages of progression. Unfortunately, conventional diagnostic modalities currently used in dentistry lack the sensitivity to detect early caries. The authors' intention is to compare the ability of polarized Raman spectroscopy and thermophotonic imaging to make early caries diagnosis. Extracted human teeth with no visible stain or defects were artificially demineralized in accordance to a well-known protocol in dentistry for simulated early caries development at several demineralization stages. Samples were then inspected using polarized Raman spectroscopy and thermophotonic imaging. The sensitivities of these two diagnostic modalities are compared, and the results are verified using transverse micro-radiography. It was found that compared to polarized Raman spectroscopy, thermophotonic imaging exhibits superior sensitivity to very early stages of demineralization.

The inverse relationship between two-photon excited fluorescence (TPEF) and laser pulse duration suggests that two-photon microscopy (TPM) performance may be improved by decreasing pulse duration. However, for ultrashort pulses of sub-10 femtosecond (fs) in duration, its spectrum contains the effective gain bandwidth of Ti:Sapphire and its central wavelength is no longer tunable. An experimental study was performed to explore this apparent tradeoff between untuned sub-10 fs transform-limited pulse (TLP) and tunable 140 fs pulse for TPEF. Enhancement factors of 1.6, 6.7, and 5.2 are measured for Indo-1, FITC, and TRITC excited by sub-10 fs TLP compared with 140 fs pulse tuned to the two-photon excitation (TPE) maxima at 730 nm, 800 nm, and 840 nm, respectively. Both degenerate (v₁ = v₂) and nondegenerate (v₁ ≠ v₂) mixing of sub-10 fs TLP spectral components result in its broad second-harmonic (SH) power spectrum and high spectral density, which can effectively compensate for the lack of central wavelength tuning and lead to large overlap with dye TPE spectra for TPEF enhancements. These pulse properties were also exploited for demonstrating its potential applications in multicolor imaging with TPM.

Purpose: Investigations of foveal aberrations assume circular pupils. However, the pupil becomes increasingly elliptical with increase in visual field eccentricity. We address this and other issues concerning peripheral aberration specification. Methods: One approach uses an elliptical pupil similar to the actual pupil shape, stretched along its minor axis to become a circle so that Zernike circular aberration polynomials may be used. Another approach uses a circular pupil whose diameter matches either the larger or smaller dimension of the elliptical pupil. Pictorial presentation of aberrations, influence of wavelength on aberrations, sign differences between aberrations for fellow eyes, and referencing position to either the visual field or the retina are considered. Results: Examples show differences between the two approaches. Each has its advantages and disadvantages, but there are ways to compensate for most disadvantages. Two representations of data are pupil aberration maps at each position in the visual field and maps showing the variation in individual aberration coefficients across the field. Conclusions: Based on simplicity of use, adequacy of approximation, possible departures of off-axis pupils from ellipticity, and ease of understanding by clinicians, the circular pupil approach is preferable to the stretched elliptical approach for studies involving field angles up to 30 deg.

The purpose of this study is to analyze human sperm motility and energetics in media with different viscosities. Multiple experiments were performed to collect motility parameters using customized computer tracking software that measures the curvilinear velocity (VCL) and the minimum laser power (Pesc) necessary to hold an individual sperm in an optical trap. The Pesc was measured by using a 1064 nm Nd:YVO4 continuous wave laser that optically traps motile sperm at a power of 450 mW in the focused trap spot. The VCL was measured frame by frame before trapping. In order to study sperm energetics under different viscous conditions sperm were labeled with the fluorescent dye DiOC₆(3) to measure membrane potentials of mitochondria in the sperm midpiece. Fluorescence intensity was measured before and during trapping. The results demonstrate a decrease in VCL but an increase in Pesc with increasing viscosity. Fluorescent intensity is the same regardless of the viscosity level indicating no change in sperm energetics. The results suggest that, under the conditions tested, viscosity physically affects the mechanical properties of sperm motility rather than the chemical pathways associated with energetics.

A decoupled analytical technique based on the Mueller matrix method and the Stokes parameters is proposed for extracting effective parameters of anisotropic optical materials in linear birefringence (LB), linear dichroism (LD), circular birefrinegence (CB), and circular dichroism (CD) properties. This technique is essential in determining the optical properties of opto-electric or biomedical materials for the development of advanced inspection and/or diagnostic applications. The error and resolution analysis of the proposed approach is demonstrated by extracting the effective parameters given an assumption of errors ranging ±0.005 in the values of the output Stokes parameters. The results confirm the ability of the proposed method to yield full-range measurements of all the optical parameters. The decoupled nature of the analytical model yields several important advantages, including an improved accuracy and the ability to extract the parameters of optical samples with only LB, CB, LD, or CD property without using compensation technique or pretreatment. Moreover, by decoupling the extraction process, the "multiple solutions" problem inherent in previous models presented by the current group is avoided.

An open-loop adaptive optics (AO) system for retinal imaging was constructed using a liquid crystal spatial light modulator (LC-SLM) as the wavefront compensator. Due to the dispersion of the LC-SLM, there was only one illumination source for both aberration detection and retinal imaging in this system. To increase the field of view (FOV) for retinal imaging, a modified mechanical shutter was integrated into the illumination channel to control the size of the illumination spot on the fundus. The AO loop was operated in a pulsing mode, and the fundus was illuminated twice by two laser impulses in a single AO correction loop. As a result, the FOV for retinal imaging was increased to 1.7-deg without compromising the aberration detection accuracy. The correction precision of the open-loop AO system was evaluated in a closed-loop configuration; the residual error is approximately 0.0909λ (root-mean-square, RMS), and the Strehl ratio ranges to 0.7217. Two subjects with differing rates of myopia (-3D and -5D) were tested. High-resolution images of capillaries and photoreceptors were obtained.

Optimization is an important but relatively unexplored aspect of contrast-enhanced fluorescence imaging, since minimizing contrast agent usage reduces the associated cost and potential toxicity. In a previous study, the authors developed a quantitative experimental approach to optimize quantum dot (QD)-based imaging using homogenized liver as a model tissue. In this follow-up study, the authors further extend and validate the approach using eight different tissues and five QDs emission wavelengths, and introduce quantitative imaging performance metrics, namely the threshold QD concentration and wavelength optimization gain. These metrics allow quantification of the improvements through spectral optimization in terms of reduced QD dose and identify the conditions that make the optimization process worthwhile. The authors show that, for most tissues, the most important parameter to optimize is the emission wavelength, yielding improvements of up to four orders of magnitude, followed by the excitation wavelength (up to 20-fold improvement) and the excitation filter bandwidth (up to 50% improvement). The authors also observe, by means of the optimization gain metric, that tissues exhibiting both high autofluorescence and strong pigmentation are generally better candidates for excitation wavelength optimization. This work contributes to the development of robust and quantitative dosimetry for QD-based fluorescence imaging near to the tissue surface.

We implement differential interference contrast (DIC) microscopy using high-speed synthetic aperture imaging that expands the passband of coherent imaging by a factor of 2.2. For an aperture synthesized coherent image, we apply for the numerical post-processing and obtain a high-contrast DIC image for arbitrary shearing direction and bias retardation. In addition, we obtain images at different depths without a scanning objective lens by numerically propagating the acquired coherent images. Our method achieves high-resolution and high-contrast 3-D DIC imaging of live biological cells. The proposed method will be useful for monitoring 3-D dynamics of intracellular particles.

Recently, the miniaturized multiphoton microscopy (MPM) and multiphoton probe allow the clinical use of multiphoton endoscopy for diagnosing cancer via "optical biopsy". The purpose of this study was to establish MPM optical diagnostic features for liver cancer and evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis. Firstly, we performed a pilot study to establish the MPM diagnostic features by investigating 60 surgical specimens, and found that high-resolution MPM images clearly demonstrated apparent differences between benign and malignant liver lesions in terms of their tissue architecture and cell morphology. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were identified by MPM images, which were comparable to hematoxylin-eosin staining images. Secondly, we performed a blinded study to evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis by investigating another 164 specimens, and found that the sensitivity, specificity, and accuracy of MPM diagnosis was 96.32%, 96.43%, and 96.34%, respectively. In conclusion, it is feasible to use MPM to diagnose liver cancer and differentiate benign and malignant liver lesions. This preclinical study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive "optical biopsy" for liver lesions in the near future.

Intravascular optical coherence tomography (IV-OCT) is a catheter-based high-resolution imaging technique able to visualize the inner wall of the coronary arteries and implanted devices in vivo with an axial resolution below 20 μm. IV-OCT is being used in several clinical trials aiming to quantify the vessel response to stent implantation over time. However, stent analysis is currently performed manually and corresponding images taken at different time points are matched through a very labor-intensive and subjective procedure. We present an automated method for the spatial registration of IV-OCT datasets. Stent struts are segmented through consecutive images and three-dimensional models of the stents are created for both datasets to be registered. The two models are initially roughly registered through an automatic initialization procedure and an iterative closest point algorithm is subsequently applied for a more precise registration. To correct for nonuniform rotational distortions (NURDs) and other potential acquisition artifacts, the registration is consecutively refined on a local level. The algorithm was first validated by using an in vitro experimental setup based on a polyvinyl-alcohol gel tubular phantom. Subsequently, an in vivo validation was obtained by exploiting stable vessel landmarks. The mean registration error in vitro was quantified to be 0.14 mm in the longitudinal axis and 7.3-deg mean rotation error. In vivo validation resulted in 0.23 mm in the longitudinal axis and 10.1-deg rotation error. These results indicate that the proposed methodology can be used for automatic registration of in vivo IV-OCT datasets. Such a tool will be indispensable for larger studies on vessel healing pathophysiology and reaction to stent implantation. As such, it will be valuable in testing the performance of new generations of intracoronary devices and new therapeutic drugs.

We determined the effect of aggregation and coating thickness of gold on the luminescence of nanoparticles engulfed by macrophages and in gelatin phantoms. Thin gold-coated iron oxide nanoclusters (nanoroses) have been developed to target macrophages to provide contrast enhancement for near-infrared optical imaging applications. We compare the brightness of nanoroses luminescent emissions in response to 635 nm laser excitation to other nanoparticles including nanoshells, nanorods, and Cy5 conjugated iron oxide nanoparticles. Luminescent properties of all these nanoparticles were investigated in monomeric and aggregated form in gelatin phantoms and primary macrophage cell cultures using confocal microscopy. Aggregation of the gold nanoparticles increased luminescence emission and correlated with increased surface mass of gold per nanoparticle (nanoshells 37±14.30×10−3 brightness with 1.23×10−4 wt of gold (g)/nanoparticle versus original nanorose 1.45±0.37×10−3 with 2.10×10−16 wt of gold/nanoparticle, p<0.05). Nanoshells showed greater luminescent intensity than original nanoroses or Cy5 conjugated iron oxide nanoparticles when compared as nanoparticles per macrophage (38±10 versus 11±2.8 versus 17±6.5, p<0.05, respectively, ANOVA), but showed relatively poor macrophage uptake (1025±128 versus 7549±236 versus 96,000  nanoparticles/cell, p<0.05, student t-test nanoshells versus nanoroses). Enhancement of gold fluorescent emissions by nanoparticles can be achieved by reducing the thickness of the gold coating, by clustering the gold on the surface of the nanoparticles (nanoshells), and by clustering the gold nanoparticles themselves.

Collagen is the most prominent protein in the human body, making up 30% of the total protein content. Quantitative studies have shown structural differences between collagen fibers of the normal and diseased tissues, due to the remodeling of the extracellular matrix during the pathological process. The dominant orientation, which is an important characteristic of collagen fibers, has not been taken into consideration for quantitative collagen analysis. Based on the conventional gray level co-occurrence matrix (GLCM) method, the authors proposed the orientation-dependent GLCM (OD-GLCM) method by estimating the dominant orientation of collagen fibers. The authors validated the utility of the OD-GLCM method on second harmonic generation (SHG) microscopic images of tendons from rats with different ages. Compared with conventional GLCM method, the authors' method has not only improved the discrimination between different tissues but also provided additional texture information of the orderliness of collagen fibers and the fiber size. The OD-GLCM method was further applied to the differentiation of the preliminary SHG images of normal and cancerous human pancreatic tissues. The combination of SHG microscopy and the OD-GLCM method might be helpful for the evaluation of diseases marked with abnormal collagen morphology.

A multifunctional line scanning ophthalmoscope (mLSO) was designed, constructed, and tested on human subjects. The mLSO could sequentially acquire wide-field, confocal, near-infrared reflectance, fluorescein angiography (FA), and indocyanine green angiography (ICGA) retinal images. The system also included a retinal tracker (RT) and a photodynamic therapy laser treatment port. The mLSO was tested in a pilot clinical study on human subjects with and without retinal disease. The instrument exhibited robust retinal tracking and high-contrast line scanning imaging. The FA and ICGA angiograms showed a similar appearance of hyper- and hypo-pigmented disease features and a nearly equivalent resolution of fine capillaries compared to a commercial flood-illumination fundus imager. An mLSO-based platform will enable researchers and clinicians to image human and animal eyes with a variety of modalities and deliver therapeutic beams from a single automated interface. This approach has the potential to improve patient comfort and reduce imaging session times, allowing clinicians to better diagnose, plan, and conduct patient procedures with improved outcomes.

Multiphoton microscopy has emerged in the past decade as a promising tool for noninvasive skin imaging. Our aim was to evaluate the potential of multiphoton microscopy to detect topical corticosteroids side effects within the epidermis and to provide new insights into their dynamics. Healthy volunteers were topically treated with clobetasol propionate on a small region of their forearms under overnight occlusion for three weeks. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.

Studies examining acute oxygenation and perfusion changes in irradiated skin are limited. Hyperspectral imaging (HSI), a method of wide-field, diffuse reflectance spectroscopy, provides noninvasive, quantified measurements of cutaneous oxygenation and perfusion. This study examines whether HSI can assess acute changes in oxygenation and perfusion following irradiation. Skin on both flanks of nude mice (n = 20) was exposed to 50 Gy of beta radiation from a strontium-90 source. Hyperspectral images were obtained before irradiation and on selected days for three weeks. Skin reaction assessment was performed concurrently with HSI. Desquamative injury formed in all irradiated areas. Skin reactions were first seen on day 7, with peak formation on day 14, and resolution beginning by day 21. HSI demonstrated increased tissue oxygenation on day 1 before cutaneous changes were observed (p<0.001). Further increases over baseline were seen on day 14, but returned to baseline levels by day 21. For perfusion, similar increases were seen on days 1 and 14. Unlike tissue oxygenation, perfusion was decreased below baseline on day 21 (p<0.002). HSI allows for complete visualization and quantification of tissue oxygenation and perfusion changes in irradiated skin, and may also allow prediction of acute skin reactions based on early changes seen after irradiation.

Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was recognized as regions of motion by creating differential phase-variance (DPV) tomograms: multiple B-scans of individual slices through the retina were collected and the variance of the phase differences was calculated. DPV captured the small vessels and the meshwork of capillaries associated with the inner retina in en-face images over 4  mm². The swept-source laser at 1060 nm offered the needed phase sensitivity to perform DPV and generated en-face images that capture motion in the inner choroidal layer exceeding the capabilities of previous spectrometer-based instruments. In comparison with the power Doppler phase-shift method, DPV provided better visualization of the foveal avascular zone in en-face images.

Brain tumors cause significant morbidity and mortality even when benign. Completeness of resection of brain tumors improves quality of life and survival; however, that is often difficult to accomplish. The goal of this study was to evaluate the feasibility of using multimodal confocal imaging for intraoperative detection of brain neoplasms. We have imaged different types of benign and malignant, primary and metastatic brain tumors. We correlated optical images with histopathology and evaluated the possibility of interpreting confocal images in a manner similar to pathology. Surgical specimens were briefly stained in 0.05  mg/ml aqueous solution of methylene blue (MB) and imaged using a multimodal confocal microscope. Reflectance and fluorescence signals of MB were excited at 642 nm. Fluorescence emission of MB was registered between 670 and 710 nm. After imaging, tissues were processed for hematoxylin and eosin (H&E) histopathology. The results of comparison demonstrate good correlation between fluorescence images and histopathology. Reflectance images provide information about morphology and vascularity of the specimens, complementary to that provided by fluorescence images. Multimodal confocal imaging has the potential to aid in the intraoperative detection of microscopic deposits of brain neoplasms. The application of this technique may improve completeness of resection and increase patient survival.

Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

In pulsed ultrasound modulated optical tomography (USMOT), an ultrasound (US) pulse performs as a scanning probe within the sample as it propagates, modulating the scattered light spatially distributed along its propagation axis. Detecting and processing the modulated signal can provide a 1-dimensional image along the US axis. A simple model is developed wherein the detected signal is modelled as a convolution of the US pulse and the properties (ultrasonic/optical) of the medium along the US axis. Based upon this model, a maximum likelihood (ML) method for image reconstruction is established. For the first time to our knowledge, the ML technique for an USMOT signal is investigated both theoretically and experimentally. The ML method inverts the data to retrieve the spatially varying properties of the sample along the US axis, and a signal proportional to the optical properties can be acquired. Simulated results show that the ML method can serve as a useful reconstruction tool for a pulsed USMOT signal even when the signal-to-noise ratio (SNR) is close to unity. Experimental data using 5 cm thick tissue phantoms (scattering coefficient μs = 6.5  cm−1, anisotropy factor g = 0.93) demonstrate that the axial resolution is 160 μm and the lateral resolution is 600 μm using a 10 MHz transducer.

Multichannel microscopy is frequently used to study intermolecular interactions and spatial relationships between biomolecules and organelles or vesicles in cells. Based on multichannel images, quantitative colocalization analysis can provide valuable information about cellular internalization, vesicular transport, and the intracellular kinetics and location of biomolecules. However, such analyses should be performed carefully, because quantitative colocalization parameters have different interpretations and can be highly affected by image quality. We use quantitative three-dimensional colocalization analysis of deconvolved and chromatic-registered confocal images to study the dissociation of double-labeled pDNA-chitosan polyplexes in HeLa cells and their colocalization with early endosomes. Two chitosans that form polyplexes with highly different transfection efficacies are compared. Pearson's correlation coefficient, Manders' colocalization coefficients, and the intensity correlation quotient are estimated to determine the intracellular localization of polyplexes, free pDNA, and free chitosans. Differences are observed in the amount of uptake, and in the intracellular pathways and rates of dissociation for the two chitosans. The results support previous findings that polyplexes formed by self-branched, glycosylated chitosan oligomers are more favorable for cellular uptake and intracellular trafficking to the nucleus compared with polyplexes formed by linear chitosans.

This clinical study was to demonstrate the applicability of diffuse reflectance (DR) intensity ratio R620/R575 in the quantification and discrimination of periodontitis and gingivitis from healthy gingiva. DR spectral measurements were carried out with white-light illumination from 70 healthy sites in 30 healthy volunteers, and 63 gingivitis- and 58 periodontitis-infected sites in 60 patients. Clinical parameters such as probing pocket depth, clinical attachment level, and gingival index were recorded in patient population. Diagnostic accuracies for discrimination of gingivitis and periodontitis from healthy gingiva were determined by comparison of spectral signatures with clinical parameters. Divergence of average DR spectral intensity ratio between control and test groups was studied using analysis of variance. The mean DR spectrum on normalization at 620 nm showed marked differences between healthy tissue, gingivitis, and periodontitis. Hemoglobin concentration and apparent SO₂ (oxygen saturation) were also calculated for healthy, gingivitis, and periodontitis sites. DR spectral intensities at 545 and 575 nm showed a decreasing trend with progression of disease. Among the various DR intensity ratios studied, the R620/R575 ratio provided a sensitivity of 90% and specificity of 94% for discrimination of healthy tissues from gingivitis and a sensitivity of 91% and specificity of 100% for discrimination of gingivitis from periodontitis.

Ultrashort pulsed lasers in bone ablation show promise for many orthopedic applications. To minimize collateral tissue damage and control the ablation process, the ablation threshold fluence must be well characterized. Using an amplified femtosecond laser (170 fs, 800 nm, 1 kHz), the ablation threshold on unaltered porcine cortical bone was measured using the D² method at multiple incident pulse numbers ranging from 25 to 1000 pulses per spot. The lowered threshold at greater pulse numbers indicated an incubation effect. Using a power law model, the incubation coefficient of unaltered porcine cortical bone was found to be 0.89 ± 0.03. Through extrapolation, the single-pulse ablation threshold was found to be 3.29 ± 0.14  J/cm².

The thulium fiber laser (TFL) is currently being studied as an alternative to the conventional holmium:YAG (Ho:YAG) laser for lithotripsy. The diode-pumped TFL may be electronically modulated to operate with variable parameters (e.g., pulse rate, pulse duration, and duty cycle) for studying the influence of pulse train mode on stone ablation rates. The TFL under study was operated at 1908 nm, 35-mJ pulse energy, and 500-μs pulse duration, in a train of 5 micro-pulses, with macro-pulse rates of 10 Hz, compared with conventional TFL operation at 10 to 50 Hz. TFL energy was delivered through 100-μm-core fibers in contact with human uric acid (UA) and calcium oxalate monohydrate (COM) stones. Mass removal rates, optical coherence tomography, and light microscopy were used to analyze the ablation craters. Stone retropulsion and fiber tip degradation studies also were conducted for these laser parameters. TFL operation in micro-pulse train (MPT) mode resulted in a factor of two increase in the ablation rate of 414±94  μg/s and 122±24  μg/s for the UA and COM stones, respectively, compared to 182±69  μg/s and 60±14  μg/s with standard pulse trains delivered at 50 Hz (P<0.05). Stone retropulsion remained minimal (<2  mm after 1200 pulses) for both pulse modes. Fiber burnback was significant for both pulse modes and was higher for COM stones than UA stones. TFL operation in MPT mode results in increased stone ablation rates which, with further optimization, may approach levels comparable to Ho:YAG laser lithotripsy in the clinic.

A book review of a recently released title.