Light-emitting diode (LED) light sources have recently been introduced to photoacoustic imaging (PAI). The LEDs enable a smaller footprint for PAI systems when compared to laser sources, thereby improving system portability and allowing for improved access. An LED-based PAI system has been employed to identify inflammatory arthritis in human hand joints. B-mode ultrasound (US), Doppler, and PAIs were obtained from 12 joints with clinically active arthritis, five joints with subclinically active arthritis, and 12 normal joints. The quantitative assessment of hyperemia in joints by PAI demonstrated statistically significant differences among the three conditions. The imaging results from the subclinically active arthritis joints also suggested that the LED-based PAI has a higher sensitivity to angiogenic microvascularity compared to US Doppler imaging. This initial clinical study on arthritis patients validates that PAI can be a potential imaging modality for the diagnosis of inflammatory arthritis.

Many studies have been carried out in order to detect and quantify the level of mental stress by means of different physiological signals. From the physiological point of view, stress promptly affects brain and cardiac function; therefore, stress can be assessed by analyzing the brain- and heart-related signals more efficiently. Signals produced by functional near-infrared spectroscopy (fNIRS) of the brain together with the heart rate (HR) are employed to assess the stress induced by the Montreal Imaging Stress Task. Two different versions of the HR are used in this study. The first one is the commonly used HR derived from the electrocardiogram (ECG) and is considered as the reference HR (RHR). The other is the HR computed from the fNIRS signal (EHR) by means of an effective combinational algorithm. fNIRS and ECG signals were simultaneously recorded from 10 volunteers, and EHR and RHR are derived from them, respectively. Our results showed a high degree of agreement [r  >  0.9, BAR (Bland Altman ratio) <5  %  ] between the two HR. A principal component analysis/support vector machine-based algorithm for stress classification is developed and applied to the three measurements of fNIRS, EHR, and RHR and a classification accuracy of 78.8%, 94.6%, and 62.2% were obtained for the three measurements, respectively. From these observations, it can be concluded that the EHR carries more useful information with regards to the mental stress than the RHR and fNIRS signals. Therefore, EHR can be used alone or in combination with the fNIRS signal for a more accurate and real-time stress detection and classification.

Selective retina therapy (SRT) targets the retinal pigment epithelium (RPE) with pulsed laser irradiation by inducing microbubble formation (MBF) at the intracellular melanin granula, which leads to selective cell disruption. The following wound healing process rejuvenates the chorio-retinal junction. Pulse energy thresholds for selective RPE effects vary intra- and interindividually. We present the evaluation of an algorithm that processes backscattered treatment light to detect MBF as an indicator of RPE cell damage since these RPE lesions are invisible during treatment. Eleven patients with central serous chorioretinopathy and four with diabetic macula edema were treated with a SRT system, which uses a wavelength of 527 nm, a repetition rate of 100 Hz, and a pulse duration of 1.7 μs. Fifteen laser pulses with stepwise increasing pulse energy were applied per treatment spot. Overall, 4626 pulses were used for algorithm parameter optimization and testing. Sensitivity and specificity were the metrics maximized through an automatic optimization process. Data were verified by fluorescein angiography. A sensitivity of 1 and a specificity of 0.93 were achieved. The method introduced in this paper can be used for guidance or automatization of microbubble-related treatments like SRT or selective laser trabeculoplasty.

Spatially resolved diffuse reflectance spectroscopy (srDRS) is a well-established technique for noninvasive, in vivo characterization of tissue optical properties toward diagnostic applications. srDRS has a potential for depth-resolved analysis of tissue, which is desired in various clinical situations. However, current fiber-based and photodiode-based systems have difficulties achieving this goal due to challenges in sampling the reflectance with a high enough resolution. We introduce a compact, low-cost architecture for srDRS based on the use of a multipixel imaging sensor and light-emitting diodes to achieve lensless diffuse reflectance imaging in contact with the tissue with high spatial resolution. For proof-of-concept, a prototype device, involving a commercially available complementary metal–oxide semiconductor coupled with a fiber-optic plate, was fabricated. Diffuse reflectance profiles were acquired at 645 nm at source-to-detector separations ranging from 480  μm to 4 mm with a resolution of 16.7  μm. Absorption coefficients (μ_a) and reduced scattering coefficients ( μs′) of homogeneous tissue-mimicking phantoms were measured with 4.2  ±  3.5  %   and 7.0  ±  4.6  %   error, respectively. The results obtained confirm the potential of our approach for quantitative characterization of tissue optical properties in contact imaging modality. This study is a first step toward the development of low-cost, wearable devices for skin condition diagnosis in vivo.

In breast surgery, a lack of knowledge about what is below the tissue surface may lead to positive tumor margins and iatrogenic damage. Diffuse reflectance spectroscopy (DRS) is a spectroscopic technique that can distinguish between healthy and tumor tissue making it a suitable technology for intraoperative guidance. However, because tumor surgeries are often performed with an electrosurgical knife, the effect of a coagulated tissue layer on DRS measurements must be taken into account. It is evaluated whether real-time DRS measurements obtained with a photonic electrosurgical knife could provide useful information of tissue properties also when tissue is coagulated and cut. The size of the coagulated area is determined and the effect of its presence on DR spectra is studied using ex vivo porcine adipose and muscle tissue. A coagulated tissue layer with a depth of 0.1 to 0.4 mm is observed after coagulating muscle with an electrosurgical knife. The results show that the effect of coagulating adipose tissue is negligible. Using the fat/water ratio’s calculated from the measured spectra of the photonic electrosurgical knife, it was possible to determine the distance from the instrument tip to a tissue transition during cutting. In conclusion, the photonic electrosurgical knife can determine tissue properties of coagulated and cut tissue and has, therefore, the potential to provide real-time feedback about the presence of breast tumor margins during cutting, helping surgeons to establish negative margins and improve patient outcome.

Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis and cirrhosis. Liver biopsy, considered the gold standard to diagnose NAFLD, shows significantly high rates of interobserver variability. Thus there is a need to develop tools that accurately categorize mild and advanced grades of steatosis in order to identify patients at higher risk of developing chronic liver disease. Diffuse reflectance spectroscopy (DRS) has proved to be useful in grading liver fibrosis and cirrhosis, without having been implemented for steatosis. We aim to categorize early and advanced stages of liver steatosis in a methionine–choline deficient (MCD) mouse model. C57bl/6 mice are fed either methionine–choline control or MCD diet during 2 or 8 weeks to induce mild and advanced steatosis. Liver samples are obtained and steatosis is evaluated by oil red O staining. Diffuse reflectance spectra are directly measured on ex vivo liver specimens, in a wavelength range of 400 to 800 nm. DRS is able to discriminate between early or advanced steatosis and healthy hepatic tissue with negligible error while showing high average sensitivity and specificity (0.94 and 0.95, respectively). Our results suggest that liver steatosis can be accurately evaluated by DRS, highlighting the importance of applied spectroscopic methods in assessing NAFLD.

We apply three optical coherence tomography (OCT) image analysis techniques to extract morphometric information from OCT images obtained on peripheral nerves of rat. The accuracy of each technique is evaluated against histological measurements accurate to +  /    −  1  μm. The three OCT techniques are: (1) average depth-resolved profile (ADRP), (2) autoregressive spectral estimation (AR-SE), and (3) correlation of the derivative spectral estimation (CoD-SE). We introduce a scanning window to the ADRP technique, which provides transverse resolution and improves epineurium thickness estimates—with the number of analyzed images showing agreement with histology increasing from 2  /  10 to 5  /  10 (Kruskal–Wallis test, α  =  0.05). A method of estimating epineurium thickness, using the AR-SE technique, showed agreement with histology in 6  /  10 analyzed images (Kruskal–Wallis test, α  =  0.05). Using a tissue sample in which histology identified two fascicles with an estimated difference in mean fiber diameter of 4  μm, the AR-SE and CoD-SE techniques both correctly identified the fascicle with larger fiber diameter distribution but incorrectly estimated the magnitude of this difference as 0.5  μm. The ability of the OCT signal analysis techniques to extract accurate morphometric details from peripheral nerves is promising but restricted in depth by scattering in adipose and neural tissues.

A high-speed polarization imaging instrument is demonstrated to be capable of measuring the collagen fiber alignment orientation and alignment strength during high-displacement rate dynamic loading at acquisition rates up to 10 kHz. The implementation of a high-speed rotating quarter wave plate and high-speed camera in the imaging system allows a minimum measurement acquisition time of 6 ms. Sliced tendon-to-bone insertion samples are loaded using a modified drop tower with an average maximum displacement rate of 1.25  m  /  s, and imaged using a high-speed polarization imaging instrument. The generated collagen fiber alignment angle and strength maps indicate the localized deformation and fiber realignment in tendon-to-bone samples during dynamic loading. The results demonstrate a viable experimental method to monitor collagen fiber realignment in biological tissue under high-displacement rate dynamic loading.

Colonoscopy is routinely performed for colorectal cancer screening but lacks the capability to accurately characterize precursor lesions and early cancers. High-resolution microendoscopy (HRME) is a low-cost imaging tool to visualize colorectal polyps with subcellular resolution. We present a computer-aided algorithm to evaluate HRME images of colorectal polyps and classify neoplastic from benign lesions. Using histopathology as the gold standard, clinically relevant features based on luminal morphology and texture are quantified to build the classification algorithm. We demonstrate that adenomatous polyps can be identified with a sensitivity and specificity of 100% and 80% using a two-feature linear discriminant model in a pilot test set. The classification algorithm presented here offers an objective framework to detect adenomatous lesions in the colon with high accuracy and can potentially improve real-time assessment of colorectal polyps.

Resolutions higher than the optical diffraction limit are often desired in the context of cellular imaging and the study of disease progression at the cellular level. However, three-dimensional super-resolution imaging without reliance on exogenous contrast agents has so far not been achieved. We present nanoscale photoacoustic tomography (nPAT), an imaging modality based on the photoacoustic effect. nPAT can achieve a dramatic improvement in the axial resolution of the photoacoustic imaging. We derive the theoretical resolution and sensitivity of nPAT and demonstrate that nPAT can achieve a maximum axial resolution of 9.2 nm. We also demonstrate that nPAT can theoretically detect smaller numbers of molecules (∼273) than conventional photoacoustic microscopy due to its ability to detect acoustic signals very close to the photoacoustic source. We simulate nPAT imaging of malaria-infected red blood cells (RBCs) using digital phantoms generated from real biological samples, showing nPAT imaging of the RBC at different stages of infection. These simulations show the potential of nPAT to nondestructively image RBCs at the nanometer resolutions for in vivo samples without the use of exogenous contrast agents. Simulations of nPAT-enabled functional imaging show that nPAT can yield insight into malarial metabolism and biocrystallization processes. We believe that the experimental realization of nPAT has important applications in biomedicine.

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.

Phenotype analysis of yeast cell requires high-throughput imaging and automatic analysis of abundant image data. At first, each cell needs to be segmented and labeled in the bright-field images. However, the ambiguous boundary of bright-field yeast cell images leads to the failure of traditional segmentation algorithms. We propose a segmentation algorithm based on the morphological characteristics of yeast cells. Seed points are first identified along the cell contour and then connected by an edge tracing approach. In this way, “ill-detected” noise points are removed so that edges of yeast cells can be successfully extracted in bright-field images with sparsely distributed cells. In densely packed images, yeast cells with normal morphology can also be correctly segmented and labeled.

Medical knowledge of the skeleton including its structures has improved constantly over the past decades. Advanced imaging methods, mechanical testing and optical techniques have revealed insights into bone architecture and composition. Most of these advancements were possible due to the ex vivo investigation of biological tissues. Investigations of fresh tissue are generally preferred over preserved or fixed samples. However, chemical fixation is sometimes inevitable due to histological procedures or logistical reasons. The aim of this study was to investigate whether short-term chemical fixation with formaldehyde affects bone quality parameters obtained from Raman spectroscopy and if these effects last for intermediate sample storage of several hours. As formaldehyde induces cross-links to the organic components in bone tissue, we hypothesized that collagen-related parameters are particularly affected. Femurs of eight 17-week-old C57BL/6 mice were extracted and divided into two groups (N  =  8  /  group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12 h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4 h, to assess whether intermediate storage time influenced the obtained results. Based on resultant spectra, mineral-to-matrix ratio, carbonate-to-phosphate ratio, carbonate-to-amide I ratio, mineral crystallinity and collagen maturity were determined. Carbonate-to-phosphate ratio was the only parameter showing a significant difference between the first and the subsequent measurements. For both groups, ratios showed a decrease in carbonate substitution compared to the first measurement (percentage decrease: 3.1% in fixed, 4.7% in unfixed). Collagen maturity of samples, which were short-term fixed with formaldehyde, was significantly lower than of fresh, unfixed samples (percentage difference: 3.8%). Our study shows that Raman spectroscopy is able to detect changes in collagen structure initiated by formaldehyde and that changes in short-term fixed samples are minimally influencing bone material properties measured with Raman spectroscopy.

Continuous noninvasive measurement of intraocular pressure (IOP) is an important tool in the evaluation process for glaucoma. We present a methodology enabling high-precision, noncontact, reproducible, and continuous monitoring of IOP based on the value of the damping factor of transitional oscillations obtained at the surface of the eye after terminating its stimulation by a sound wave. The proposed configuration includes projection of a laser beam and usage of a fast camera for analyzing the temporal–spatial variations of the speckle patterns backscattered from the iris or the sclera following the above-mentioned sound waves external stimulation. The methodology was tested on an artificial eye and a carp fish eye under varying pressure as well as on human eyes.