This editorial by the journal’s Editor in Chief, Brian Pogue, explains the need for a new type of paper.

The enamel scattering coefficient decreases markedly with increasing wavelength from the visible to the near-infrared (NIR). However, beyond 1300 nm, the scattering coefficient is difficult to measure, and it is not known whether light scattering continues to decrease significantly at longer wavelengths. It is hypothesized that water absorption is a major contributor to the contrast between sound and demineralized enamel beyond 1300 nm since deeply penetrating photons in sound enamel are likely absorbed by water. Reflectance images of demineralization on tooth surfaces were acquired at wavelengths near 1450, 1860, 1880, and 1950 nm. The magnitude of water absorption is similar at 1450 and 1880 nm but varies markedly between 1860, 1880, and 1950 nm. Multispectral comparisons of lesion contrast provide insight into the mechanism responsible for higher contrast at longer NIR wavelengths. The highest contrast was at 1950 nm; however, the markedly higher contrast at 1880 compared to 1450 nm and similar contrast between 1860 and 1880 nm suggests that the enamel scattering coefficient continues to decrease beyond 1300 nm, and that reduced light scattering in sound enamel is most responsible for the higher lesion contrast at longer NIR wavelengths. This has important implications for the choice of wavelengths for caries detection and diagnostic devices, including the performance of optical coherence tomography beyond 1300 nm.

Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.

For fluorescence molecular tomography, higher spatial resolution can be achieved using minimally scattered early photons. Conventional reconstruction methods of early photon tomography (EPT) are based on the integral of temporal point spread function (TPSF), which may lead to poor image quality due to systematic noise and time mismatch between the TPSF data and forward model. The derivative of the rising portion of TPSF is proposed to be used in EPT to increase the performance of reconstruction because the derivative is less sensitive to noise and time mismatch than the integral. A method based on projected Tikhonov regularization with the reconstructed result of steepest descent algorithm as a priori information is developed. Using the derivative of TPSF, the method can achieve high spatial resolution in phantom experiments and is capable of separating targets with an edge–edge distance of 1.5 mm.

Optical technologies may be applied to multiple facets of spinal surgery from diagnostics to intraoperative image guidance to therapeutics. In diagnostics, the current standard remains cross-sectional static imaging. Optical surface scanning tools may have an important role; however, significant work is required to clearly correlate surface metrics to radiographic and clinically relevant spinal anatomy and alignment. In the realm of intraoperative image guidance, optical tracking is widely developed as the current standard of instrument tracking, however remains compromised by line-of-sight issues and more globally cumbersome registration workflows. Surface scanning registration tools are being refined to address concerns over workflow and learning curves, and allow real-time update of tissue deformation; however, the line-of-sight issues plaguing instrument tracking remain to be addressed. In therapeutics, optical applications exist in both visualization, in the form of endoscopes, and ablation, in the form of lasers. Further work is required to extend the feasibility of laser ablation to multiple tissues, including disc, bone, and tumor, in a safe and time-efficient manner. Finally, we postulate some of the short- and long-term opportunities for future growth of optical techniques in the context of spinal surgery. Particular emphasis is placed on intraoperative image guidance, the area of the authors’ primary expertise.

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell–extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell–ECM interaction during angiogenesis, wound healing, and metastasis.

F18-fluorodeoxyglucose (FDG)–positron emission tomography (PET) along with computed tomography (CT) is a standard method for assessing brown adipose tissue (BAT) activity. We tested the usefulness of near-infrared time-resolved spectroscopy (NIRTRS) as a simple and noninvasive method for evaluating BAT density (BAT-d) by examining the effects of some factors known to influence BAT activity. The total hemoglobin concentration as a parameter of BAT-d was evaluated using NIRTRS in the supraclavicular region in 413 Japanese individuals. The associations were analyzed between BAT-d and sex, age, the percentages of body fat (%BF), visceral fat (VF), and the seasonal ambient temperature (AmT) fluctuations. Age was associated with decreased BAT-d (P  <  0.05). There was no sex difference in the BAT-d, except for those in their twenties. Multivariate analyses revealed that %BF and VF were correlated with BAT-d, and the lower AmT (around 4°C or 5°C) for 4 and 6 weeks prior to the measurement day was associated with an increase in the BAT-d. Our NIRTRS results were analogous to those reported with FDG18-PET  /  CT, indicating the usefulness of NIRTRS. BAT-d might increase during the 4 and 6 weeks after the AmT decreases to lower than 4°C or 5°C.

The prevention and treatment of spinal cord injury are focused upon the maintenance of spinal cord blood flow, yet no technology exists to monitor spinal cord ischemia. We recently demonstrated continuous monitoring of spinal cord ischemia with diffuse correlation and optical spectroscopies using an optical probe. Prior to clinical translation of this technology, it is critically important to demonstrate the safety profile of spinal cord exposure to the required light. To our knowledge, this is the first report of in situ safety testing of such a monitor. We expose the spinal cord to laser light utilizing a custom fiber-optic epidural probe in a survival surgery model (11 adult Dorset sheep). We compare the tissue illumination from our instrument with the American National Standards Institute maximum permissible exposures. We experimentally evaluate neurological and pathological outcomes of the irradiated sheep associated with prolonged exposure to the laser source and evaluate heating in ex vivo spinal cord samples. Spinal cord tissue was exposed to light levels at ∼18  ×   the maximum permissible exposure for the eye and ∼  (  1  /  3  )    ×   for the skin. Multidisciplinary testing revealed no functional neurological sequelae, histopathologic evidence of laser-related injury to the spinal cord, or significant temperature changes in ex vivo samples. Low tissue irradiance and the lack of neurological, pathological, and temperature changes upon prolonged exposure to the laser source offer evidence that spinal cord tissues can be monitored safely with near-infrared optical probes placed within the epidural space.

A GPU-based Monte Carlo software (MCtet) was developed to calculate the light propagation in arbitrarily shaped objects, like a human tooth, represented by a tetrahedral mesh. A unique feature of MCtet is a concept to realize different kinds of light-sources illuminating the complex-shaped surface of an object, for which no preprocessing step is needed. With this concept, it is also possible to consider photons leaving a turbid media and reentering again in case of a concave object. The correct implementation was shown by comparison with five other Monte Carlo software packages. A hundredfold acceleration compared with central processing units-based programs was found. MCtet can simulate anisotropic light propagation, e.g., by accounting for scattering at cylindrical structures. The important influence of the anisotropic light propagation, caused, e.g., by the tubules in human dentin, is shown for the transmission spectrum through a tooth. It was found that the sensitivity to a change in the oxygen saturation inside the pulp for transmission spectra is much larger if the tubules are considered. Another “light guiding” effect based on a combination of a low scattering and a high refractive index in enamel is described.

Mitochondrial oxidative flashes (mitoflashes) are oxidative burst events in mitochondria. It is crosslinked with numerous mitochondrial molecular processes and related with pivotal cell functions such as apoptosis and respiration. In previous research, mitoflashes were found as spontaneous occasional events. It would be observed more frequently if cells were treated with proapoptotic chemicals. We show that multiple mitoflashes can be initiated by a single femtosecond-laser stimulation that was tightly focused on a diffraction-limited spot in the mitochondrial tubular structure. The mitoflash events triggered by different photostimulations are quantified and analyzed. The width and amplitude of mitoflashes are found very sensitive to stimulation parameters including laser power, exposure duration, and total incident laser energy. This study provides a quantitative investigation on the photostimulated mitoflashes. It may thus demonstrate such optical method to be a promising technique in future mitochondrial research.

Examination of the retinal circulation in patients with retinal diseases is a clinical routine for ophthalmologists. In the present work, an automatic method is proposed for measuring the global retinal circulation in fluorescein angiography (FA). First, the perfusion region in FA images is segmented using a multiscale line detector. Then, the time evolution of the perfusion area is modeled using damped least-squares regression. Based on the perfusion area profile, some circulation parameters are defined to describe quantitatively the global retinal circulation. The effectiveness of the proposed method is tested using our own and public datasets, with reasonable results and satisfactory accuracy compared with manual measurement. The proposed method has good computing efficiency and thus has potential to be used in clinical practice for evaluation of global retinal circulation.

Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals’ neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.

We demonstrate in vivo endoscopic optical coherence tomography (OCT) imaging in the forward direction using a flexible fiber bundle (FB). In comparison to current conventional forward-looking probe schemes, our approach simplifies the endoscope design by avoiding the integration of any beam steering components in the distal probe end due to two-dimensional scanning of a focused light beam over the proximal FB surface. We describe the challenges that arise when OCT imaging with an FB is performed, such as multimoding or cross coupling. The performance of different FBs varying in parameters, such as numerical aperture, core size, core structure, and flexibility, was consequently compared, and image quality degrading artifacts were described in detail. Based on our findings, we propose an optimal FB design for endoscopic OCT imaging.

Histopathological image analysis of stained tissue slides is routinely used in tumor detection and classification. However, diagnosis requires a highly trained pathologist and can thus be time-consuming, labor-intensive, and potentially risk bias. Here, we demonstrate a potential complementary approach for diagnosis. We show that multiphoton microscopy images from unstained, reproductive tissues can be robustly classified using deep learning techniques. We fine-train four pretrained convolutional neural networks using over 200 murine tissue images based on combined second-harmonic generation and two-photon excitation fluorescence contrast, to classify the tissues either as healthy or associated with high-grade serous carcinoma with over 95% sensitivity and 97% specificity. Our approach shows promise for applications involving automated disease diagnosis. It could also be readily applied to other tissues, diseases, and related classification problems.

Tomographic phase microscopy (TPM) is a unique imaging modality to measure the three-dimensional refractive index distribution of transparent and semitransparent samples. However, the requirement of the dense sampling in a large range of incident angles restricts its temporal resolution and prevents its application in dynamic scenes. Here, we propose a graphics processing unit-based implementation of a deep convolutional neural network to improve the performance of phase tomography, especially with much fewer incident angles. As a loss function for the regularized TPM, the ℓ₁-norm sparsity constraint is introduced for both data-fidelity term and gradient-domain regularizer in the multislice beam propagation model. We compare our method with several state-of-the-art algorithms and obtain at least 14 dB improvement in signal-to-noise ratio. Experimental results on HeLa cells are also shown with different levels of data reduction.

Paired-agent kinetic modeling protocols provide one means of estimating cancer cell-surface receptors with in vivo molecular imaging. The protocols employ the coadministration of a control imaging agent with one or more targeted imaging agent to account for the nonspecific uptake and retention of the targeted agent. These methods require the targeted and control agent data be converted to equivalent units of concentration, typically requiring specialized equipment and calibration, and/or complex algorithms that raise the barrier to adoption. This work evaluates a kinetic model capable of correcting for targeted and control agent signal differences. This approach was compared with an existing simplified paired-agent model (SPAM), and modified SPAM that accounts for signal differences by early time point normalization of targeted and control signals (SPAM_PN). The scaling factor model (SPAM_SF) outperformed both SPAM and SPAM_PN in terms of accuracy and precision when the scale differences between targeted and imaging agent signals (α) were not equal to 1, and it matched the performance of SPAM for α  =  1. This model could have wide-reaching implications for quantitative cancer receptor imaging using any imaging modalities, or combinations of imaging modalities, capable of concurrent detection of at least two distinct imaging agents (e.g., SPECT, optical, and PET/MR).

Remodeling of the extracellular matrix in human ovarian cancer can be manifested in increased collagen concentration, changes in alignment within fibrils/fibers and/or up-regulation of different collagen isoforms. We used pixel-based second harmonic generation (SHG) polarization microscopy analyses to probe these molecular changes in human ovarian tissues [normal stroma, benign tumors, and high-grade serous (HGS) tumors] by: (i) determination of the α-helical pitch angle via the single-axis molecular model, (ii) collagen alignment within fibrils via SHG anisotropy, and (iii) chirality via SHG circular dichroism (SHG-CD). Pixel approaches are required due to the complex structure of the matrix that lacks a high degree of fiber alignment. The largest differences in the helical pitch angle were between normal stroma and benign tumors, consistent with gene expression showing the Col III isoform is up-regulated in the latter. The data were not consistent with up-regulation of Col III in HGS tumors as previous reports have suggested. The different tissues also displayed differing SHG anisotropies and SHG-CD responses, consistent with either Col III incorporation or randomization of Col I alignment within benign and malignant tumors. Additionally, the high-grade tumors displayed higher collagen concentration, where this desmoplasia is consistent with the higher fiber density in these tissues. These results collectively indicate that the fibril assemblies are distinct in all tissues, where these differences likely result from the synthesis of collagen rather than remodeling of existing collagen. Importantly, these analyses are label-free and interrogate subresolution collagen structure on intact tissues, without the need for conventional structural biology tools.

Here, we present dual-dimensional microscopy that captures both two-dimensional (2-D) and light-field images of an in-vivo sample simultaneously, synthesizes an upsampled light-field image in real time, and visualizes it with a computational light-field display system in real time. Compared with conventional light-field microscopy, the additional 2-D image greatly enhances the lateral resolution at the native object plane up to the diffraction limit and compensates for the image degradation at the native object plane. The whole process from capturing to displaying is done in real time with the parallel computation algorithm, which enables the observation of the sample’s three-dimensional (3-D) movement and direct interaction with the in-vivo sample. We demonstrate a real-time 3-D interactive experiment with Caenorhabditis elegans.

Digital pathology via whole-slide imaging (WSI) systems has recently been approved for the primary diagnostic use in the US. Acquiring whole-slide images with spectral information at each pixel permits the use of multiplexed antibody labeling and allow for the measurement of cellularly resolved chemical information. Here, we report the development of a high-throughput terapixel hyperspectral WSI system using prism-based slit-array dispersion. We demonstrate a slit-array detection scheme for absorption-based measurements and a slit-array projection scheme for fluorescence-based measurements. The spectral resolution and spectral range in the reported schemes can be adjusted by changing the orientation of the slit-array mask. We use our system to acquire 74 5-megapixel brightfield images at different wavelengths in ∼1  s, corresponding to a throughput of 0.375  gigapixels  /  s. A terapixel whole-slide spatial–spectral data cube can be obtained in ∼45  min. The reported system is compatible with existing WSI systems and can be developed as an add-on module for whole-slide spectral imaging. It may find broad applications in high-throughput chemical imaging with multiple antibody labeling. The use of slit array for structured illumination may also provide insights for developing high-throughput hyperspectral confocal imaging systems.

Tumor hypoxia is a critical indicator of poor clinical outcome in patients with cancers of the breast, cervix, and oral cavity. The ability to noninvasively and reliably monitor tumor oxygenation both prior to and during therapy can aid in identifying poor treatment response earlier than is currently possible and lead to effective changes in treatment regimen. Diffuse reflectance spectroscopy (DRS) has been used in several studies to measure tissue scattering, total hemoglobin content (THb), and vascular oxygenation (sO2) in tissue. In this study, we validate in vivo DRS-based measurements of vascular oxygenation using immunohistochemical staining of tumor hypoxia using pimonidazole, an established hypoxia marker. Using tumor xenografts grown from two different head and neck cell lines—UM-SCC-22B and UM-SCC-47—we demonstrate statistically significant negative correlations between tumor hypoxic fraction (HF) and THb (r  =    −  0.45; p  =  0.04) and sO2 (r  =    −  0.50; p  =  0.02). In addition, we also found a statistically significant positive correlation between HF and mean reduced scattering coefficient (r  =  0.60; p  =  0.005). Our results demonstrate that DRS-based measures of sO2 can provide reliable indirect measurements of tumor hypoxia that can be of significant utility in preclinical and clinical studies.

With better surgical outcomes, quicker recovery times, decreased postoperative pain, and reduced scarring at the surgical site, the application of minimally invasive surgery (MIS) has gained a lot of prominence in the last 30 years. This change in surgical practice has taken away the ability of a surgeon to palpate for the presence of a blood vessel as would occur in an open procedure. They instead must rely on a laparoscopic video camera feed that unfortunately cannot detect the presence of a blood vessel hidden beneath tissue. In certain scenarios, a surgeon can accidentally cut a blood vessel, which can lead to severe, even fatal, complications. Here, we show that by adding a near-infrared LED and a photodiode onto the opposing jaws of laparoscopic graspers, blood vessels buried under tissue can be detected. We show the results of Monte Carlo simulations to support our theory that the blood vessels ranging from 3 to 6 mm buried under up to 1 cm of tissue can be detected and quantified. This technology could be added to already existing laparoscopic tools that have limited surface areas on the jaws to assist surgeons during MIS procedures.

The aim was to determine the precision of a noninvasive near-infrared spectroscopy (NIRS)-based tissue oximeter (OxyPrem v1.3). Using a linear mixed-effects model, we quantified the variability for cerebral tissue oxygenation (StO2) measurements in 35 preterm neonates to be 2.64%, a value that meets the often-articulated clinicians’ demand for a precise tissue oxygenation measurement. We showed that the variability of StO2 values measured was dominated by spontaneous systemic hemodynamic fluctuations during the measurement, meaning that precision of the instrument was actually even better. Based on simultaneous and continuous measurements of peripheral arterial oxygenation and cerebral StO2 with a second sensor, we were able to determine and quantify the physiological instability precisely. We presented different methods and analyses aiming at reducing this systematic physiological error of in vivo precision assessments. Using these methods, we estimated the precision of the OxyPrem tissue oximeter to be   ≤  1.85  %  . With our study, we deliver relevant information to establish highly precise cerebral oxygenation measurements with NIRS-based oximetry, facilitating the further development toward a substantially improved diagnosis and treatment of patients with respect to brain oxygenation.

We present a fiber-optic dual-confocal laser caliper method for noncontact high-precision sensing and measuring thickness and refractive index of intraocular lens (IOL) implants. The principle of the method is based on sensing and measuring the confocal intensity response of the laser beam reflection from the opposite object surfaces, which provides the advanced feature of having no limitations on the object shape, thickness, and transparency. Using single-mode optical fibers and a 658-nm laser source, the thickness measurement accuracy was assessed to be as high as 5  μm. In addition, refractive index of a transparent object with thickness smaller than the working distance of the focusing lenses can be measured. The thickness and refractive index of a planoconvex IOL were measured with a high accuracy.