Laser speckle contrast imaging (LSCI) offers a cost-effective means to image blood flow in vivo. However, it is not commonly used to image rodent retinas because of the challenges associated with imaging through the curved cornea and delivering light through the highly scattering lens. A solution to overcome these problems by using LSCI in conjunction with an endoscope to obtain high spatiotemporal blood flow images is described. Its utility is demonstrated by imaging blood flow changes in rat retinas using hyperoxic, hypercapnic, and visual (flicker) stimulations. Hypercapnia increases blood flow, hyperoxia decreases blood flow, and visual stimulation increases blood flow in the retina relative to basal conditions. The time-to-peak of the LSCI response to visual stimulation is also measured. This approach may prove useful to investigate dysregulation in blood flow-evoked responses in retinal diseases and to evaluate treatment strategies in rodents.

Using a 0.8-mm-diameter image guide fiber bundle consisting of 30,000 single-mode fibers and an external linear array transducer, we demonstrate a dual-mode photoacoustic system capable of ultrasound-guided microendoscope insertion and photoacoustic imaging. The array optical resolution photoacoustic microendoscopy (AOR-PAME) system is designed to visualize the placement of the distal end of an endoscopy probe several centimeters into tissue, transmit scanning focused laser pulses into tissues via the fiber bundle, and acquire the generated photoacoustic signals. A ytterbium-doped fiber laser is tightly focused and is scanned across the proximal tip of the image guide fiber bundle using a two-dimensional galvanometer scanning mirror system. The end of the fiber bundle is used in contact mode with the object. The capabilities of AOR-PAME are demonstrated by imaging carbon fiber networks embedded in tissue-mimicking phantoms and the ears of a 60-g rat. The lateral resolution and signal-to-noise ratio are measured as 9 μm and 40 dB, respectively.

Tissue characterization method in optical coherence tomography (OCT) for in situ histology of soft tissues is presented and demonstrated for mice skin. OCT allows direct noninvasive visualization of subsurface anatomy. It is currently used for in situ investigation of lesions in skin, vessels, retinal layers, oral, and bronchial cavitities. Although OCT images present high resolution information about tissue morphology, reporting requires a reader experienced in interpretation of the images, viz., identification of anatomical layers and structures constituting the organ based on OCT speckle appearance. Our approach characterizes tissues through transfer learning of tissue energy interaction statistical physics models of ballistic and near-ballistic photons. The clinical information yield with our approach is comparable to conventional invasive histology. On cross evaluation with a mice model experiment, the epidermis, papillary dermis, dermis, and adipose tissue constituting the mice skin are identified with an accuracy of 99%, 95%, 99%, and 98%, respectively. This high accuracy of characterizing heterogeneous tissues using OCT justifies the ability of our computational approach to perform in situ histology and can be extended to regular clinical practice for diagnosis of vascular, retinal, or oral pathologies.

We demonstrate a reflective, continuous-wave terahertz (THz) imaging system to acquire ex vivo images of fresh human colonic excisions. Reflection measurements of 5-mm-thick sections of colorectal tissues were obtained using a polarization-specific detection technique. Two-dimensional THz reflection images of both normal and cancerous colon tissues with a spatial resolution of 0.6 mm were acquired using an optically pumped far-infrared molecular gas laser. Good contrast has been observed between normal and tumorous tissues at 584 GHz frequency. The resulting THz reflection images compared with the tissue histology showed a correlation between cancerous region and increased reflection. We hypothesize that the imaging system and polarization techniques are capable of registering reflectance differences between cancerous and normal colon. However, further investigations are necessary to completely understand the source mechanism behind the contrast and confirm the hypothesis; if true, it likely represents the first continuous-wave THz reflection imaging technique to show sufficient contrast to identify colon tumor margins. Also, it may represent a significant step forward in clinical endoscopic application of THz technology to aid in in vivo colorectal cancer screening.

For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser.

This study aimed to investigate the effects of sustained hypoxic exposure on cerebral and muscle oxygenation and cardiorespiratory function at rest. Eleven healthy subjects inhaled a normobaric hypoxic (FiO ₂ =0.12 ) or normoxic (FiO ₂ =0.21 ) gas mixture for 4 h at rest, on two separated blinded sessions. Arterial oxygen saturation (SpO ₂ ), heart rate variability (HRV), end-tidal CO ₂ (EtCO ₂ ), and oxygenation of quadriceps muscle, prefrontal and motor cortices assessed by near-infrared spectroscopy (NIRS) were measured continuously during each session. Acute mountain sickness symptoms were evaluated at the end of each session. During a hypoxic session, SpO ₂ reduction (∼13% ) plateaued after 20 min, while deoxygenation pattern took 30 to 40 min at the cerebral sites to plateau (+5.3±1.6  μMol of deoxygenated-hemoglobin). Deoxygenation was more pronounced in the cerebral cortex compared to the muscle (+2.1±2.3  μMol of deoxygenated-hemoglobin), and NIRS-derived tissue perfusion index showed distinct profiles between the muscle (hypoperfusion) and the brain (hyperperfusion) with prolonged hypoxia. Changes in tissue oxygenation were not associated with cardiorespiratory responses (e.g., HRV, EtCO _{2 [}/sub] ) and altitude sickness symptom appearance during hypoxic sessions. These data demonstrate that sustained hypoxia elicits time delay in changes between arterial and tissue (especially cerebral) oxygenation, as well as a tissue-specific sensitivity.

Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK ₂ (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80 , and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis.

We evaluate the biological and physicochemical effects of a Fusarium oxysporum crude extract (CE) on the skin of healthy rats. The CE is topically applied and subsequently the skin is collected after 3, 6, 12, and 24 h. The samples are analyzed by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) and histomorphometric analysis. Terminal dUTP nick end labeling (TUNEL assay) is performed to detect both the cells in apoptosis and proliferation. There is a thickening of the epidermis after 6, 12, and 24 h and dermis after 12 and 24 h of CE application. A reduction of the dermis thickness is observed at 3 and 6 h. The treated skin shows higher labeling intensity by TUNEL at 3 h, while a higher intensity by proliferating cell nuclear antigen occurs at 3 and 12 h. FTIR-PAS data support the histology observations showing an increase in the absorption peaks in the dermis after the application of the CE. F. oxysporum CE permeated through the epidermis and the dermis, reaching the subcutaneous tissue, inducing cell apoptosis, and causing physicochemical changes in the organic molecules located in the dermis. This is the first known study associating histopathological and physical chemistry changes on healthy skin after the application of F. oxysporum CE.

A hyperspectral fluorescence lifetime imaging (FLIM) instrument is developed to study endogenous fluorophores in biological tissue as an optical biopsy tool. This instrument is able to spectrally, temporally, and spatially resolve fluorescence signal, thus providing multidimensional information to assist clinical tissue diagnosis. An acousto-optic tunable filter (AOTF) is used to realize rapid wavelength switch, and a photomultiplier tube and a high-speed digitizer are used to collect the time-resolved fluorescence decay at each wavelength in real time. The performance of this instrument has been characterized and validated on fluorescence tissue phantoms and fresh porcine skin specimens. This dual-arm AOTF design achieves high spectral throughput while allowing microsecond nonsequential, random wavelength switching, which is highly desirable for time-critical applications. In the results reported here, a motorized scanning stage is used to realize spatial scanning for two-dimensional images, while a rapid beam steering technique is feasible and being developed in an ongoing project.

Visibility of optical coherence tomography (OCT) images can be severely degraded by speckle noise. A computationally efficient despeckling approach that strongly reduces the speckle noise is reported. It is based on discrete wavelet transform (DWT), but eliminates the conventional process of threshold estimation. By decomposing an image into different levels, a set of sub-band images are generated, where speckle noise is additive. These sub-band images can be compounded to suppress the additive speckle noise, as DWT coefficients resulting from speckle noise tend to be approximately decorrelated. The final despeckled image is reconstructed by taking the inverse wavelet transform of the new compounded sub-band images. The performance of speckle reduction and edge preservation is controlled by a single parameter: the level of wavelet decomposition. The proposed technique is applied to intravascular OCT imaging of porcine carotid arterial wall and ophthalmic OCT images. Results demonstrate the effectiveness of this technique for speckle noise reduction and simultaneous edge preservation. The presented method is fast and easy to implement and to improve the quality of OCT images.

In order to diagnose cancer, a sample must be removed, prepared, and examined under a microscope, which is expensive, invasive, and time consuming. Fiber optic fluorescence endomicroscopy, where an image guide is used to obtain high-resolution images of tissue in vivo, has shown promise as an alternative to conventional biopsies. However, the resolution of standard endomicroscopy is limited by the fiber bundle sampling frequency and out-of-focus light. A system is presented which incorporates a plastic, achromatic objective to increase the sampling and which provides optical sectioning via structured illumination to reject background light. An image is relayed from the sample by a fiber bundle with the custom 2.1-mm outer diameter objective lens integrated to the distal tip. The objective is corrected for the excitation and the emission wavelengths of proflavine (452 and 515 nm). It magnifies the object onto the fiber bundle to improve the system’s lateral resolution by increasing the sampling. The plastic lenses were fabricated via single-point diamond turning and assembled using a zero alignment technique. Ex vivo images of normal and neoplastic murine mammary tissues stained with proflavine are captured. The system achieves higher contrast and resolves smaller features than standard fluorescence endomicroscopy.

A cross-correlation-based method is proposed to quantitatively measure transverse flow velocity using optical resolution photoacoustic (PA) microscopy enhanced with a digital micromirror device (DMD). The DMD is used to alternately deliver two spatially separated laser beams to the target. Through cross-correlation between the slow-time PA profiles measured from the two beams, the speed and direction of transverse flow are simultaneously derived from the magnitude and sign of the time shift, respectively. Transverse flows in the range of 0.50 to 6.84  mm/s are accurately measured using an aqueous suspension of 10-μm-diameter microspheres, and the root-mean-squared measurement accuracy is quantified to be 0.22  mm/s . The flow measurements are independent of the particle size for flows in the velocity range of 0.55 to 6.49  mm/s , which was demonstrated experimentally using three different sizes of microspheres (diameters: 3, 6, and 10 μm). The measured flow velocity follows an expected parabolic distribution along the depth direction perpendicular to the flow. Both maximum and minimum measurable velocities are investigated for varied distances between the two beams and varied total time for one measurement. This technique shows an accuracy of 0.35  mm/s at 0.3-mm depth in scattering chicken breast, making it promising for measuring flow in biological tissue.

The results of quantum dot (QD) probe preparation for multiplexed single-cell array staining and analysis are reported. By controlling the reaction temperature, time, and ratio of Cd to Se, multicolor CdSe QDs emitting fluorescence ranging from purple to red in a safer, simpler, and more convenient way than traditional methods is obtained. To detect cells using these oil-soluble QDs, they are first coated with water-soluble thioglycolic aid (TGA) so that biocompatible multiwavelength bioprobes can be obtained. QDs’ surface is somewhat damaged when binding TGA to QDs is found, which results in a reduction of QDs’ emission wavelength and a slight blue shift of QDs’ emission wavelength after water-soluble modification with TGA. Comparison of the emission spectrum showed that it is negligible, and the fluorescent properties of QDs capped by TGA are still satisfactory. Living cells are then stained with multiplexed probes by conjugating TGA-QDs with antibodies specific to these cell antigens. Changes in fluorescence intensity can indicate change in the relative quantity of antigens expressed in the same cell caused by external stimulus, offering effective methods to multiplexed optical analysis of single cells.

A photoacoustic contrast agent that is based on bis-carboxylic acid derivative of indocyanine green (ICG) covalently conjugated to single-wall carbon nanotubes (ICG/SWCNT) is presented. Covalently attaching ICG to the functionalized SWCNT provides a more robust system that delivers much more ICG to the tumor site. The detection sensitivity of the new contrast agent in a mouse tumor model is demonstrated in vivo by our custom-built photoacoustic imaging system. The summation of the photoacoustic tomography (PAT) beam envelope, referred to as the “PAT summation,” is used to demonstrate the postinjection light absorption of tumor areas in ICG- and ICG/SWCNT-injected mice. It is shown that ICG is able to provide 33% enhancement at approximately 20 min peak response time with reference to the preinjection PAT level, while ICG/SWCNT provides 128% enhancement at 80 min and even higher enhancement of 196% at the end point of experiments (120 min on average). Additionally, the ICG/SWCNT enhancement was mainly observed at the tumor periphery, which was confirmed by fluorescence images of the tumor samples. This feature is highly valuable in guiding surgeons to assess tumor boundaries and dimensions in vivo and to achieve clean tumor margins to improve surgical resection of tumors.

There is a need for cost effective, quantitative tissue spectroscopy and imaging systems in clinical diagnostics and pre-clinical biomedical research. A platform that utilizes a commercially available light-emitting diode (LED) based projector, cameras, and scaled Monte Carlo model for calculating tissue optical properties is presented. These components are put together to perform spatial frequency domain imaging (SFDI), a model-based reflectance technique that measures and maps absorption coefficients (μ _a ) and reduced scattering coefficients (μ ′ _s ) in thick tissue such as skin or brain. We validate the performance of the flexible LED and modulation element (FLaME) system at 460, 530, and 632 nm across a range of physiologically relevant μ _a values (0.07 to 1.5  mm ⁻¹ ) in tissue-simulating intralipid phantoms, showing an overall accuracy within 11% of spectrophotometer values for μ _a and 3% for μ ′ _s . Comparison of oxy- and total hemoglobin fits between the FLaME system and a spectrophotometer (450 to 1000 nm) is differed by 3%. Finally, we acquire optical property maps of a mouse brain in vivo with and without an overlying saline well. These results demonstrate the potential of FLaME to perform tissue optical property mapping in visible spectral regions and highlight how the optical clearing effect of saline is correlated to a decrease in μ ′ _s of the skull.

Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

We develop a coherent anti-Stokes Raman scattering (CARS) microscopy system equipped with a tunable picosecond laser for high-speed wavelength scanning. An acousto-optic tunable filter (AOTF) is integrated in the laser cavity to enable wavelength scanning by varying the radio frequency waves applied to the AOTF crystal. An end mirror attached on a piezoelectric actuator and a pair of parallel plates driven by galvanometer motors are also introduced into the cavity to compensate for changes in the cavity length during wavelength scanning to allow synchronization with another picosecond laser. We demonstrate fast spectral imaging of 3T3-L1 adipocytes every 5  cm ⁻¹ in the Raman spectral region around 2850  cm ⁻¹ with an image acquisition time of 120 ms. We also demonstrate fast switching of Raman shifts between 2100 and 2850  cm ⁻¹ , corresponding to CD ₂ symmetric stretching and CH ₂ symmetric stretching vibrations, respectively. The fast-switching CARS images reveal different locations of recrystallized deuterated and nondeuterated stearic acid.

We propose and test various strategies for the creation of artery phantoms mimicking different kinds of diseased arteries when imaged by intravascular optical coherence tomography (IVOCT). We first review the method for making healthy artery phantoms. We then describe the procedure to fabricate diseased artery phantoms with intima thickening, lipid pool, thin-capped fibroatheroma, calcification, and restenosis (homogeneous and layered) after stent apposition. For each case, a phantom is fabricated, an IVOCT image is obtained, and the image is compared to that of a real artery.

The mouse ischemic hindlimb model is used widely for studying collateral artery growth (i.e., arteriogenesis) in response to increased shear stress. Nonetheless, precise measurements of regional shear stress changes along individual collateral arteries are lacking. Our goal is to develop and verify trans-illumination laser speckle flowmetry (LSF) for this purpose. Studies of defibrinated bovine blood flow through tubes embedded in tissue-mimicking phantoms indicate that trans-illumination LSF better maintains sensitivity with an increasing tissue depth when compared to epi-illumination, with an ∼50% reduction in the exponential decay of the speckle velocity signal. Applying trans-illuminated LSF to the gracilis muscle collateral artery network in vivo yields both improved sensitivity and reduced noise when compared to epi-illumination. Trans-illuminated LSF images reveal regional differences in collateral artery blood velocity after femoral artery ligation and are used to measure an ∼2 -fold increase in the shear stress at the entrance regions to the muscle. We believe these represent the first direct measurements of regional shear stress changes in individual mouse collateral arteries. The ability to capture deeper vascular signals using a trans-illumination configuration for LSF may expand the current applications for LSF, which could have bearing on determining how shear stress magnitude and direction regulate arteriogenesis.

Diffuse optical tomography (DOT) is a noninvasive, nonionizing imaging modality that uses near-infrared light to visualize optically relevant chromophores. A recently developed dynamic DOT imaging system enables the study of hemodynamic effects in the breast during a breath-hold. Dynamic DOT imaging was performed in a total of 21 subjects (age 54±10  years ) including 3 healthy subjects and 18 subjects with benign (n=8 ) and malignant (n=14 ) masses. Three-dimensional time-series images of the percentage change in oxygenated and deoxygenated hemoglobin concentrations ([HbO 2 ] and [Hb]) from baseline are obtained over the course of a breath-hold. At a time point of 15 s following the end of the breath-hold, [Hb] in healthy breasts has returned to near-baseline values (1.6%±0.5% ), while tumor-bearing breasts have increased levels of [Hb] (6.8%±3.6% , p<0.01 ). Further, healthy subjects have a higher correlation between the breasts over the course of the breath-hold as compared with the subjects with breast cancer (healthy: 0.96±0.02 ; benign: 0.89±0.02 ; malignant: 0.78±0.23 , p<0.05 ). Therefore this study shows that dynamic features extracted from DOT measurements can differentiate healthy and diseased breast tissues. These features provide a physiologic method for identifying breast cancer without the need for ionizing radiation.

Atherosclerosis is a leading cause of mortality in industrialized countries. In addition to “traditional” systemic risk factors for atherosclerosis, the geometry and motion of coronary arteries may contribute to individual susceptibility to the development and progression of disease in these vessels. To be able to test this, we have developed a high-speed (∼40 frames per second) microscope-based stereo-imaging system to quantify the motion of epicardial coronary arteries of mice. Using near-infrared nontargeted quantum dots as an imaging contrast agent, we synchronously acquired paired images of a surgically exposed murine heart, from which the three-dimensional geometry of the coronary arteries was reconstructed. The reconstructed geometry was tracked frame by frame through the cardiac cycle to quantify the in vivo motion of the vessel, from which displacements, curvature, and torsion parameters were derived. Illustrative results for a C57BL/6J mouse are presented.

Conflict processing is crucial for humans and has been investigated using hemodynamic and electrophysiological measures. However, because most previous research has studied hemodynamic and electrophysiological measures separately, the relationship between these two measures in conflict processing is poorly understood. In our study, we measure near-infrared spectroscopy (NIRS) and event-related potential (ERP) signals simultaneously in a Chinese color-word matching Stroop task and examine the relationship between the conflict-related hemodynamic signal in the prefrontal cortex (PFC) and electrophysiological signal. The results show significant Stroop effects for behavioral, NIRS (oxy-hemoglobin: HbO ₂ ), and ERP [N450, late positive complex (LPC)] data. The significant N450 Stroop effect occurs before the behavioral response to incongruent stimuli, while the evident LPC Stroop effect occurs after it, suggesting that only N450 is associated with conflict processing. Additionally, N450 Stroop effects during the early and later phases are negatively correlated with HbO ₂ Stroop effects in the left PFC and in the bilateral PFC, respectively. These results suggest that N450 reflects conflict detection and resolution, the left PFC may be involved in conflict detection, and the bilateral PFC is engaged in conflict resolution. Overall, the analysis of the correlation between hemodynamic and electrophysiological signals is useful for studying human brain function.

We introduce immunofluorescence and automated image processing protocols for serial tumor sections to objectively and efficiently quantify tumor microvasculature following antivascular therapy. To determine the trade-off between tumor subsampling and throughput versus microvessel quantification accuracy, we provide a mathematical model that accounts for tumor-specific vascular heterogeneity. This mathematical model can be applied broadly to define tumor volume samplings needed to reach statistical significance, depending on the biomarker in question and the number of subjects. Here, we demonstrate these concepts for tumor microvessel density and total microvascularity (TMV) quantification in whole pancreatic ductal adenocarcinoma tumors ex vivo. The results suggest that TMV is a more sensitive biomarker for detecting reductions in tumor vasculature following antivascular treatment. TMV imaging is a broadly accessible technique that offers robust assessment of antivascular therapies, and it offers promise as a tool for developing high-throughput assays to quantify treatment-induced microvascular alterations for therapeutic screening and development.

Transdermal drug delivery, transporting the drug molecules through epidermis to dermis, has been extensively investigated but not studied in dynamic detail. The objective of this study is to monitor the dynamical changes of drug permeation and that of polarization of skin tissue with oleic acid treatment. We utilize two-photon fluorescence microscopy (TPM) to investigate the dynamics of transdermal drug delivery in skin excised from the abdominal region of euthenized nude mice with sulforhodamine B (SRB) modeling as a drug and Laurdan serving as a polarity indicator. The treatment of oleic acid increases the permeation rate of SRB, quickly reaching to the steady state of permeation. Increases in polarity within the skin tissue (in both intercellular and intracellular region of stratum corneum) are observed in SRB permeation enhanced by oleic acid treatment. TPM has successfully demonstrated the ability to study spatial distribution of transport dynamics in oleic acid-enhanced transdermal delivery.

Photoacoustic (PA) image reconstruction of data acquired with conventional linear arrays suffers from incompleteness due to limited bandwidth and limited view. This problem is compounded by the dominance of boundary signals suppressing other weaker PA sources. In this study, we propose a method that uses a naturally coregistered ultrasound image to enhance the PA image reconstruction, with the dual aim to reconstruct the sources that suffer from incompleteness and reveal weak sources near a strong boundary. In this method, an ultrasound image provides the input for a PA wave field simulation. The simulated PA field can be combined with the measured fields to complete the partial reconstruction or to suppress the dominant boundary signals. Experimental validation of the method was performed with two different phantoms and in vivo data from the lower arm of a healthy volunteer.

A picogram-sensitive optical microfluidic biosensor using an integrated polycarbazole photodiode is developed. The photodetector is mainly composed of the blend heterojunction of poly [N-9 ′ -heptadecanyl-2,7-carbazole-alt-5,5-(4 ′ ,7 ′ -di-2-thienyl-2 ′ ,1 ′ ,3 ′ -benzothiadiazole)] (PCDTBT) and [6,6]-phenyl C ₇₁ -butyric acid methyl ester (PC ₇₀ BM ) and the poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) as the hole transport layer. Analyte detection is accomplished via a chemiluminescent immunoassay performed in a poly(dimethylsiloxane)-gold-glass hybrid microchip, on which antibodies were immobilized and chemiluminescent horseradish peroxidase-luminol-peroxide reactions were generated. Enhanced sensor response to the chemiluminescent light is achieved by optimizing the thickness of PCDTBT:  PC ₇₀ BM and PEDOT:PSS. Using the optimized polycarbazole photodiode for detecting the human thyroid-stimulating hormone as the model target, the integrated biosensor demonstrates an excellent linearity in the range of 0.03 to 10  ng/ml with an analytical sensitivity of 68  pg/ml . The sensor response shows high specificity and reproducibility. Hormone detection in clinical samples is further demonstrated and compared with a commercial enzyme-linked immunosorbent assay. The integrated device reported here has potential to detect other hormonal compounds or protein targets. Moreover, the presented concept enables the development of miniaturized, low-cost but highly sensitive optical microfluidic biosensors based on integrated polymer photodetectors with high potential for point-of-care diagnostics.

Optical characterization of biological tissue in field carcinogenesis offers a method with which to study the mechanisms behind early cancer development and the potential to perform clinical diagnosis. Previously, low-coherence enhanced backscattering spectroscopy (LEBS) has demonstrated the ability to discriminate between normal and diseased organs based on measurements of histologically normal-appearing tissue in the field of colorectal (CRC) and pancreatic (PC) cancers. Here, we implement the more comprehensive enhanced backscattering (EBS) spectroscopy to better understand the structural and optical changes which lead to the previous findings. EBS provides high-resolution measurement of the spatial reflectance profile P(r _s ) between 30 microns and 2.7 mm, where information about nanoscale mass density fluctuations in the mucosa can be quantified. A demonstration of the length-scales at which P(r _s ) is optimally altered in CRC and PC field carcinogenesis is given and subsequently these changes are related to the tissue’s structural composition. Three main conclusions are made. First, the most significant changes in P(r _s ) occur at short length-scales corresponding to the superficial mucosal layer. Second, these changes are predominantly attributable to a reduction in the presence of subdiffractional structures. Third, similar trends are seen for both cancer types, suggesting a common progression of structural alterations in each.

The fundamental limitations of photoacoustic microscopy for detecting optically absorbing molecules are investigated both theoretically and experimentally. We experimentally demonstrate noise-equivalent detection sensitivities of 160,000 methylene blue molecules (270 zeptomol or 2.7×10 ⁻¹⁹mol ) and 86,000 oxygenated hemoglobin molecules (140 zeptomol) using narrowband continuous-wave photoacoustics. The ultimate sensitivity of photoacoustics is fundamentally limited by thermal noise, which can present in the acoustic detection system as well as in the medium itself. Under the optimized conditions described herein and using commercially available detectors, photoacoustic microscopy can detect as few as 100s of oxygenated hemoglobin molecules. Realizable improvements to the detector may enable single molecule detection of select molecules.

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease, with a prevalence of 0.5 to 1% in the general population. Imaging can possibly aid in early diagnosis, crucial to effective personalized therapeutic strategies and treatment follow-up. The intravenous administration of indocyanine green (ICG) has been considered for identifying synovial hyperperfusion as an RA physiological biomarker. However, while the distribution of ICG in the human hand is a time-dependent process, the particular biodistribution dynamic patterns established following intravenous administration have not yet been studied. For this reason, the dynamic relationships of ICG distribution in the human hand in RA patients using a method based on principal component analysis are analyzed. In vivo analyses were corroborated by simulations of clinical scenarios using a finite element method. Observations of spatiotemporal characteristics are contrasted to fluorescence intensity images and magnetic resonance images of the hand joints, employed as the anatomical and diagnostic reference. Processing results for 450 joints from 5 healthy volunteers and 10 patients show that image features obtained from the spatiotemporal analysis offer good congruence with synovitis and reveal better detection performance compared to observations of raw fluorescence intensity images.

Expanding our previously proposed “time segment analysis” for a two-layered turbid medium, this study attempted to selectively determine the absorption coefficient (μ_a ) of the bottom layer in a four-layered human head model with time-domain near-infrared measurements. The difference curve in the temporal profiles of the light attenuation between an object and a reference medium, which are obtained from Monte Carlo simulations, is divided into segments along the time axis, and a slope for each segment is calculated to obtain the depth-dependent μ _a (μ ^seg _a) . The reduced scattering coefficient (μ _s ′ ) of the reference is determined by curve fitting with the temporal point spread function derived from the analytical solution of the diffusion equation to the time-resolved reflectance of the object. The deviation of μ ^seg _a from the actual μ_a is expressed by a function of the ratio of μ ^seg _a in an earlier time segment to that in a later segment for mediums with different optical properties and thicknesses of the upper layers. Using this function, it is possible to determine the μ_a of the bottom layer in a four-layered epoxy resin-based phantom. These results suggest that the method reported here has potential for determining the μ_a of the cerebral tissue in humans.

Fourier transform infrared (FTIR) microspectroscopy has been used to estimate the spatial proteoglycan (PG) and collagen contents in articular cartilage (AC). However, it is not clear whether the results of FTIR analyses are consistent between different species. Our aim was to clarify how three different FTIR PG parameters in use, i.e., the integrated absorbance in the carbohydrate region, the carbohydrate/amide I ratio, and the second derivative peak at 1062  cm ⁻¹ , can indicate the densitometrically assessed (reference method) spatial PG content in a sample set consisting of osteoarthritic human and bovine AC samples. The results show that all the parameters can accurately reflect the PG content, when the species are analyzed separately. When all samples are pooled, the correlation with the reference method is high (r=0.760 , n=104 ) for the second derivative peak at 1062  cm ⁻¹ and is significantly lower (p<0.05 ) for the carbohydrate region (r=0.587 , n=104 ) and for the carbohydrate/amide I ratio (r=0.579 , n=104 ). Therefore, the analysis of the carbohydrate region may provide inconsistent results, if the cartilage samples from different species are in use. Based on the present study, second derivative analysis yields more consistent results for human and bovine cartilages.

Various leg exercises have been recommended to prevent deep vein thrombosis (DVT), a condition where a blood clot forms in the deep veins, especially during long-haul flights. Accessing the benefit of each of these exercises in avoiding the DVT, which can be fatal, is important in the context of suggesting the correct and the most beneficial exercises. Present work aims at demonstrating the fiber Bragg grating (FBG)–based sensing methodology for measuring surface strains generated on the skin of the calf muscle to evaluate the suggested airline exercises to avoid DVT. As the dataset in the experiment involves multiple subjects performing these exercises, an inertial measurement unit has been used to validate the repetitiveness of each of the exercises. The surface strain on the calf muscle obtained using the FBG sensor, which is a measure of the calf muscle deformation, has been compared against the variation of blood velocity in the femoral vein of the thigh measured using a commercial electronic-phased array color Doppler ultrasound system. Apart from analyzing the effectiveness of suggested exercises, a new exercise which is more effective in terms of strain generated to avoid DVT is proposed and evaluated.

A thin photothermal (PT) endoscope (∼80  μm ) for the noninvasive/minimally invasive hybrid-optical diagnosis of biological specimens is demonstrated. The technique has the unique advantage that the pump laser delivery fiber itself acts as the thermal wave sensor, which is a Bragg grating. It detects only the conductive component of the PT signal, thus enabling an emissivity independent measurement. The device is slidable through a syringe needle and PT analysis of exposed organs with limited accessibility for conventional PT techniques, and constricted regions can be examined noninvasively. For regions buried in thick tissues, a minimally invasive injection mode may be considered. Temperature measurement sensitivity is about 0.03°C. The amplitude and phase channels are sensitive up to about 3 and 10 kHz, respectively. The endoscope has been used for the simultaneous estimation of flow velocity, absorption coefficient, and diffusivity for a phantom-blood flow. The endoscopically estimated values are in agreement with true flow velocities over a range of 1 to 1000  cm ⁻¹ . The endoscope has been used for the optical biopsy of goat bone marrow.

Ocular microtremor (OMT) is an involuntary fixational eye movement. We identify the implications of biospeckle for a noninvasive, laser speckle correlation technique to measure OMT. Biospeckle from the in-vivo eye is characterized and, using the resulting characteristics, a mathematical model to describe the biospeckle from the eye is designed and tested. Through in-vivo measurements, biospeckle is shown to disrupt the temporal stability of the speckle images over time. However, provided each speckle image is cross correlated with the previous image within a sufficiently short time, the stability of speckle images captured from the eye is shown to be adequate to measure OMT-like displacements.

Infrared neural stimulation (INS) is becoming an important complementary tool to electrical stimulation. Since the mechanism of INS is photothermal, describing the laser-induced heat distribution is fundamental to determining the relationship between stimulation pulses and neural responses. This work developed both a framework describing the time evolution of the heat distribution induced by optical fluence and a new method to extract thermal criteria (e.g., temperature change and rate of change) for neural activation. To solve the general problem of describing the temperature distribution, a Green’s function solution to the heat diffusion equation was determined and convolved with the optical fluence. This provided a solution in the form of a single integral over time, from which closed-form solutions can be determined for special cases. This work also yielded an expression for thermal relaxation time, which provides a rigorous description of thermal confinement for INS. The developed framework was then applied to experimental data from the cochlea to extract the minimum temperature increase and rate of that increase to stimulate the cochlear spiral ganglion. This result, and similar analyses applied to other neural systems, can then shed light on the fundamental mechanism for INS and aid the development of optical neuroprostheses.

The effects of laser polarization on the efficacy of near-infrared low-level laser therapy for spinal cord injury (SCI) are presented. Rat spinal cords were injured with a weight-drop device, and the lesion sites were directly irradiated with a linearly polarized 808-nm diode laser positioned either perpendicular or parallel to the spine immediately after the injury and daily for five consecutive days. Functional recovery was assessed daily by an open-field test. Regardless of the polarization direction, functional scores of SCI rats that were treated with the 808-nm laser irradiation were significantly higher than those of SCI alone group (Group 1) from day 5 after injury. The locomotive function of SCI rats irradiated parallel to the spinal column (Group 3) was significantly improved from day 10 after injury, compared to SCI rats treated with the linear polarization perpendicular to the spinal column (Group 2). There were no significant differences in ATP contents in the injured tissue among the three groups. We speculate that the higher efficacy with parallel irradiation is attributable to the deeper light penetration into tissue with anisotropic scattering.

The aim is to investigate femtosecond laser ablation as an alternative method for enamel etching used before bonding orthodontic brackets. A focused laser beam is scanned over enamel within the area of bonding in a saw tooth pattern with a varying number of lines. After patterning, ceramic brackets are bonded and bonding quality of the proposed technique is measured by a universal testing machine. The results are compared to the conventional acid etching method. Results show that bonding strength is a function of laser average power and the density of the ablated lines. Intrapulpal temperature changes are also recorded and observed minimal effects are observed. Enamel surface of the samples is investigated microscopically and no signs of damage or cracking are observed. In conclusion, femtosecond laser exposure on enamel surface yields controllable patterns that provide efficient bonding strength with less removal of dental tissue than conventional acid-etching technique.

Common methods to characterize treatment efficacy based on morphological imaging may misrepresent outcomes and exclude effective therapies. Using a three-dimensional model of ovarian cancer, two functional treatment response metrics are used to evaluate photodynamic therapy (PDT) efficacy: total volume, calculated from viable and nonviable cells, and live volume, calculated from viable cells. The utility of these volume-based metrics is corroborated using independent reporters of photodynamic activity: viability, a common fluorescence-based ratiometric analysis, and photosensitizer photobleaching, which is characterized by a loss of fluorescence due in part to the production of reactive species during PDT. Live volume correlated with both photobleaching and viability, suggesting that it was a better reporter of PDT efficacy than total volume, which did not correlate with either metric. Based on these findings, live volume and viability are used to probe the susceptibilities of tumor populations to a range of PDT dose parameters administered using 0.25, 1, and 10 μM benzoporphyrin derivative (BPD). PDT with 0.25 μM BPD produces the most significant reduction in live volume and viability and mediates a substantial shift toward small nodules. Increasingly sophisticated bioengineered models may complement current treatment planning approaches and provide unique opportunities to critically evaluate key parameters including metrics of therapeutic response.