Measuring distribution of dissolved oxygen in biological tissue is of prime interest for cancer diagnosis, prognosis, and therapy optimization. Tumor hypoxia indicates poor prognosis and resistance to radiotherapy. Despite its major clinical significance, no current imaging modality provides direct imaging of tissue oxygen. We present preliminary results demonstrating the potential of photoacoustic lifetime imaging (PALI) for noninvasive, 3-D imaging of tissue oxygen. The technique is based on photoacoustic probing of the excited state lifetime of methylene blue (MB) dye. MB is an FDA-approved water soluble dye with a peak absorption at 660 nm. A double pulse laser system (pump probe) is used to excite the dye and probe its transient absorption by detecting photoacoustic emission. The relaxation rate of MB depends linearly on oxygen concentration. Our measurements show high photoacoustic signal contrast at a probe wavelength of 810 nm, where the excited state absorption is more than four times higher than the ground state absorption. Imaging of a simple phantom is demonstrated. We conclude by discussing possible implementations of the technique in clinical settings and combining it with photodynamic therapy (PDT) for real-time therapy monitoring.

We developed a miniaturized high-resolution low-cost reflectance-mode fiber microscope (RFM) aimed at optical tissue biopsy applications using a polarized imaging configuration to suppress background noise from specular reflectance. The RFM is equipped with an air-cooled light-emitting-diode illumination module and a single 450-µm outer-diameter fiber bundle image guide compatible with a 20-gauge needle. The dual illumination and image acquisition nature of the fiber bundle simplifies the system and reduces the total cost. Imaging tests with a United States Air Force resolution target demonstrate a lateral resolution of ~3.5 µm. The performance of the imaging system is evaluated by examining samples of cells and excised human tissue. Still and video rate images are obtainable in reflectance mode using intrinsic contrast.

A fully integrated trimodality fluorescence, diffuse optical, and x-ray computed tomography (FT/DOT/XCT) system for small animal imaging is reported in this work. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration images using a multimodality approach. XCT offers anatomical information, while DOT provides the necessary background optical property map to improve FT image accuracy. The quantitative accuracy of this trimodality system is demonstrated in vivo. In particular, we show that a 2-mm-diam fluorescence inclusion located 8 mm deep in a nude mouse can only be localized when functional a priori information from DOT is available. However, the error in the recovered fluorophore concentration is nearly 87%. On the other hand, the fluorophore concentration can be accurately recovered within 2% error when both DOT functional and XCT structural a priori information are utilized together to guide and constrain the FT reconstruction algorithm.

We combine optical scatter imaging (OSI) with fluorescence imaging of mitochondria to investigate the spatial relationship between the optical scatter signal and the location and structure of mitochondria within endothelial cells undergoing apoptosis. The OSI data corroborate our previous results showing a decrease in the intensity ratio of wide-to-narrow angle scatter [optical scatter image ratio (OSIR)] during the first 60 min of apoptosis. In addition, we find here that this is followed by an increase in OSIR concurrent with mitochondrial fragmentation. We demonstrate that the dynamic change in light scattering is spatially associated with subcellular regions containing fluorescently labeled mitochondria, and remains absent from adjacent nonfluorescent regions dominated by other organelles. These results lend strong support to the hypothesis that mitochondria act as the source of the optical scatter changes measured at the onset of apoptosis.

A fiber confocal backscattering (FCBS) spectrometer is developed based on fiber confocal microscopy and light scattering theory. The FCBS spectrometer can provide imaging and spectral information simultaneously at the cellular scale. Normal stomach epithelial cell line GES-1 and cancerous cell line NCI-N87 are measured and their spectral results show that backscattering intensity from NCI-N87 cells is stronger than that from GES-1 cells in 500 to 800 nm, and the GES-1 cells scattering spectra show regular intensity changes, while the NCI-N87 cells do not. The experiments prove that the FCBS spectrometer is able to distinguish cancerous cells from normal stomach cells at the cellular level. The spectrometer could be further developed into a noninvasive optical technology for early cancer detection.

A combined ultrahigh resolution optical coherence tomography (UHROCT) and a electroretinography (ERG) system is presented for simultaneous imaging of the retinal structure and physiological response to light stimulation in the rodent eye. The 1060-nm UHROCT system provides ~3×5 µm (axial×lateral) resolution in the rat retina and time resolution of 22 µs. A custom-designed light stimulator integrated into the UHROCT imaging probe provides light stimuli with user-selected color, duration, and intensity. The performance of the combined system is demonstrated in vivo in healthy rats, and in a rat model of drug-induced outer retinal degeneration. Experimental results show correlation between the observed structural and physiological changes in the healthy and degenerated retina.

We report on the development of an integrated point-wise spectroscopy and autofluorescence (AF) endoscopic imaging technique for real-time in vivo tissue measurements at endoscopy. We implement a unique point spectrum optical design to realize real-time AF imaging and AF or diffuse reflectance (DR) spectroscopy measurements from a small area of tissue of interest on the AF image. We demonstrate that both the AF image and the point-wise AF/DR spectra can be simultaneously acquired from the oral cavity in vivo within 0.1 s, suggesting the potential of the integrated spectroscopy and endoscopic imaging technique developed to facilitate in vivo tissue diagnosis and characterization at endoscopy.

The upper eyelid is a biological tissue with complex structure, essential for the maintenance of an optically clear ocular surface due to its physical (blinking) effect. The Meibomian glands (MGs) are structures that lie beneath the surface of the inner eyelid and are partially responsible for the production of the superficial oily layer of the tear film. The MGs are only superficially visible under magnification when the eyelid is everted. We present for the first time in vivo 3-D images of healthy and inflamed human MGs. Tomograms were acquired from the tarsal plate of everted human eyelids with a 1060-nm ultrahigh-resolution optical coherence tomography (UHOCT) system, with ∼3μm×10μm (axial×lateral) resolution in biological tissue at the rate of 91,911 A-scans/s. Comparison with histology shows that the UHOCT images reveal a spatial distribution of structures that appear to correspond with the MGs’ acini and ducts (in healthy subjects), and accumulation of heterogeneous, highly scattering biological material and clear fluids in the visibly blocked glands. Noninvasive, volumetric high-resolution morphological imaging of the human tarsal area could have a significant impact in the clinical diagnosis of inflammatory and noninflammatory lid pathologies.

According to classical theory, visual acuity (VA) can be modeled using the intersection of the eye's modulation transfer function with a retinal threshold function. To date, there have been limited attempts to validate this methodology by comparing theory with actual measured data. We use the methodology to predict the visual acuity in the presence of defocus of a population of cataract patients implanted with diffractive multifocal intraocular lenses. For the prediction, we used a set of physiological eye models that include chromatic and higher order aberrations. We found that the simulations correlated strongly to the clinical outcomes (R²=0.93). While the simulated VA of the eye models was systematically 0.05 logMAR units lower (better acuity) than the clinical results, this difference was independent of defocus (p=0.98). These results show that when the simple and straightforward classical theory is applied using physiological eye models, accurate predictions of the VA, and through-focus VA of a population can be made. This method may be suited for predicting visual performance of new cataract and refractive treatments.

Melanoma is a primary malignancy that is known to metastasize to the brain and often causes death. The ability to image the growth of brain melanoma in vivo can provide new insights into its evolution and response to therapies. In our study, we use a reflection mode photoacoustic microscopy (PAM) system to detect the growth of melanoma brain tumor in a small animal model. The melanoma tumor cells are implanted in the brain of a mouse at the beginning of the test. Then, PAM is used to scan the region of implantation in the mouse brain, and the growth of the melanoma is monitored until the death of the animal. It is demonstrated that PAM is capable of detecting and monitoring the brain melanoma growth noninvasively in vivo.

We propose a phasor representation for oscillations at a given frequency (or relatively narrow frequency band) of hemoglobin concentrations in tissue, as well as hemoglobin concentrations measured by near-infrared spectroscopy. This representation provides a straightforward visualization of the phase relationships between oscillations of oxy-, deoxy-, and total hemoglobin concentrations ([HbO], [Hb], and [HbT], respectively). We observe that measured oscillations of [Hb] and [HbO] with a phase difference that is neither 0 nor must result from the combination of different physiological processes that are out of phase (or time shifted) with respect to each other. Finally, we propose the use of cross-correlation phasors to map phase relationships and correlation levels among hemoglobin oscillations measured at spatially distinct locations at a given frequency. Such a representation may find a natural application in the study of functional connectivity networks in the brain.

This PDF file contains the editorial “Special Section Guest Editorial: Selected Topics in Biophotonics: Photodynamic Therapy and Optical Micromanipulation for Biophotonics ” for JBO Vol. 15 Issue 04

Photodynamic therapy (PDT) is reviewed using the treatment of skin tumors as an example of superficial lesions and prostate cancer as an example of deep-lying lesions requiring interstitial intervention. These two applications are among the most commonly studied in oncological PDT, and illustrate well the different challenges facing the two modalities of PDT-superficial and interstitial. They thus serve as good examples to illustrate the entire field of PDT in oncology. PDT is discussed based on the Lund University group's over 20 yr of experience in the field. In particular, the interplay between optical diagnostics and dosimetry and the delivery of the therapeutic light dose are highlighted. An interactive multiple-fiber interstitial procedure to deliver the required therapeutic dose based on the assessment of light fluence rate and sensitizer concentration and oxygen level throughout the tumor is presented.

The biomedical sciences have benefited immensely from photonics technologies in the last 50 years. This includes the application of minute forces that enable the trapping and manipulation of cells and single molecules. In terms of the area of biophotonics, optical manipulation has made a seminal contribution to our understanding of the dynamics of single molecules and the microrheology of cells. Here we present a review of optical manipulation, emphasizing its impact on the areas of single-molecule studies and single-cell biology, and indicating some of the key experiments in the fields.

A combined experimental and theoretical study is carried out to probe the rotational behavior of red blood cells (RBCs) in a single beam optical trap. We induce shape changes in RBCs by altering the properties of the suspension medium in which live cells float. We find that certain shape anisotropies result in the rotation of optically trapped cells. Indeed, even normal (healthy) RBCs can be made to rotate using linearly polarized trapping light by altering the osmotic stress the cells are subjected to. Hyperosmotic stress is found to induce shape anisotropies. We also probe the effect of the medium's viscosity on cell rotation. The observed rotations are modeled using a Langevin-type equation of motion that takes into account frictional forces that are generated as RBCs rotate in the medium. We observe good correlation between our measured data and calculated results.

The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

Recently, femtosecond laser pulses have been utilized for the targeted introduction of genetic matter into mammalian cells. This rapidly expanding and developing novel optical technique using a tightly focused laser light beam is called phototransfection. Extending previous studies [Stevenson et al., Opt. Express 14, 7125-7133 (2006)], we show that femtosecond lasers can be used to phototransfect a range of different cell lines, and specifically that this novel technology can also transfect mouse embryonic stem cell colonies with ~25% efficiency. Notably, we show the ability of differentiating these cells into the extraembryonic endoderm using phototransfection. Furthermore, we present two new findings aimed at optimizing the phototransfection method and improving applicability: first, the influence of the cell passage number on the transfection efficiency is explored and, second, the ability to enhance the transfection efficiency via whole culture treatments. Our results should encourage wider uptake of this methodology.

We prove the first application of near-infrared-absorbing gold nanorods (GNRs) for in vivo laser closure of a rabbit carotid artery. GNRs are first functionalized with a biopolymeric shell and then embedded in hyaluronan, which gives a stabilized and handy laser-activable formulation. Four rabbits undergo closure of a 3-mm longitudinal incision performed on the carotid artery by means of a 810-nm diode laser in conjunction with the topical application of the GNRs composite. An effective surgery is obtained by using a 40-W/cm2 laser power density. The histological and electron microscopy evaluation after a 30-day follow-up demonstrates complete healing of the treated arteries with full re-endothelization at the site of GNRs application. The absence of microgranuloma formation and/or dystrophic calcification is evidence that no host reaction to nanoparticles interspersed through the vascular tissue occurred. The observation of a reshaping and associated blue shift of the NIR absorption band of GNRs after laser treatment supports the occurrence of a self-terminating process, and thus of additional safety of the minimally invasive laser procedure. This study underlines the feasibility of using GNRs for in vivo laser soldering applications, which represents a step forward toward the introduction of nanotechnology-based therapies in minimally invasive clinical practices.

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.

Radiofrequency ablation (RFA) is the standard of care to cure many cardiac arrhythmias. Epicardial ablation for the treatment of ventricular tachycardia has limited success rates due in part to the presence of epicardial fat, which prevents proper rf energy delivery, inadequate contact of ablation catheter with tissue, and increased likelihood of complications with energy delivery in close proximity to coronary vessels. A method to directly visualize the epicardial surface during RFA could potentially provide feedback to reduce complications and titrate rf energy dose by detecting critical structures, assessing probe contact, and confirming energy delivery by visualizing lesion formation. Currently, there is no technology available for direct visualization of the heart surface during epicardial RFA therapy. We demonstrate that optical coherence tomography (OCT) imaging has the potential to fill this unmet need. Spectral domain OCT at 1310 nm is employed to image the epicardial surface of freshly excised swine hearts using a microscope integrated bench-top scanner and a forward imaging catheter probe. OCT image features are observed that clearly distinguish untreated myocardium, ablation lesions, epicardial fat, and coronary vessels, and assess tissue contact with catheter-based imaging. These results support the potential for real-time guidance of epicardial RFA therapy using OCT imaging.

The recently introduced new phase-dependent Doppler model for spectral domain optical coherence tomography (SD OCT) has shown that the simple linear relation between the Doppler phase shift and the axial velocity component of an obliquely moving sample is not valid. Additionally, for nearly transverse sample motion with high velocities the phase shift will approach a constant value. Consequently, for small Doppler angles the velocity measurement range of the phase-resolved Doppler analysis is limited in SD OCT. Since these undesirable small Doppler angles can not be prevented, for example, in the in vivo 3-D measurement, we introduce a novel method extending the limited velocity detection range taking the signal power decrease due to fringe washout in SD OCT into account. The signal damping of an obliquely moving sample is presented as a function of the axial and transverse displacement by a universally valid contour plot and does not correspond simply to the sum of the axial and transverse effect. A quantitative combination of the Doppler analysis and the signal-damping method is presented with a flow phantom model. The practicability of this new combined method is presented for the blood flow of the saphenous artery in the in vivo mouse model.

Optical coherence tomography (OCT) can provide new insight into disease progression and therapy by enabling nondestructive, serial imaging of in vivo cancer models. In previous studies, we have shown the utility of endoscopic OCT for identifying adenomas in the azoxymethane-treated mouse model of colorectal cancer and tracking disease progression over time. Because of improved imaging speed made possible through Fourier domain imaging, three-dimensional imaging of the entire mouse colon is possible. Increased amounts of data can facilitate more accurate classification of tissue but require more time on the part of the researcher to sift through and identify relevant data. We present quantitative software for automatically identifying potentially diseased areas that can be used to create a two-dimensional "disease map" from a three-dimensional Fourier domain OCT data set. In addition to sensing inherent changes in tissue that occur during disease development, the algorithm is sensitive to exogeneous highly scattering gold nanoshells that can be targeted to disease biomarkers. The results of the algorithm were compared to histological diagnosis. The algorithm was then used to assess the ability of gold nanoshells targeted to epidermal growth factor receptor in vivo to enable functional OCT imaging.

An experimental design for noninvasive assessment of neural retinal tissue function with enhanced sensitivity is presented. By matching the response detection to a defined flicker frequency stimulus similar to heterodyne detection, the response signal will be shifted out of the low-frequency noise and the specificity of response detection will be strongly enhanced. Optimal measurement parameters are discussed, such as the function and timing of the response function to a single flash stimulus. The results indicate responses on the order of 200 ms that have been probed with our frequency-encoded approach using 5 Hz flickering. Preliminary results indicate the feasibility of our measurement concept to assess small changes in reflectivity with enhanced sensitivity. A functional tomogram for response localization and quantification is introduced.

The characterization of tissue morphology in murine models of pathogenesis has traditionally been carried out by excision of affected tissues with subsequent immunohistological examination. Excision-based histology provides a limited two-dimensional presentation of tissue morphology at the cost of halting disease progression at a single time point and sacrifice of the animal. We investigate the use of noninvasive reflectance mode confocal scanning laser microscopy (rCSLM) as an alternative tool to biopsy in documenting epidermal hyperplasia in murine models exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An automated technique utilizing average axial rCSLM reflectance profiles is used to extract epidermal thickness values from rCSLM data cubes. In comparisons to epidermal thicknesses determined from hematoxylin and eosin (H&E) stained tissue sections, we find no significant correlation to rCSLM-derived thickness values. This results from method-specific artifacts: physical alterations of tissue during H&E preparation in standard histology and specimen-induced abberations in rCSLM imaging. Despite their disagreement, both histology and rCSLM methods reliably measure statistically significant thickness changes in response to TPA exposure. Our results demonstrate that in vivo rCSLM imaging provides epithelial biologists an accurate noninvasive means to monitor cutaneous pathogenesis.

The chest wall underneath breast tissue distorts light reflection measurements, especially measurements obtained from distant source-detector pairs. For patients with a chest wall located at a shallower depth, the chest-wall effect needs to be considered in the image reconstruction procedure. Following our previous studies, this work systemically evaluates the performance of a two-layer model-based reconstruction using the finite element method, and compares it with the performance of the semi-infinite model. The results obtained from simulations and phantom experiments show that the two-layer model improves the light quantification of the targets. The improvements are attributed to improved background estimation and more accurate weight matrix calculation using a two-layer model compared to the semi-infinite model. Fitted two-layer background optical properties obtained from a group of ten patients with chest walls located less than 2 cm deep are more representative of breast tissue and chest-wall optical properties.

The structure of the antimalarial drug halofantrine is analyzed by means of density functional theory (DFT) calculations, IR, and Raman spectroscopy. Strong, selective enhancements of the Raman bands of halofantrine at 1621 and 1590 cm−1 are discovered by means of UV resonance Raman spectroscopy with excitation wavelength exc=244 nm. These signal enhancements can be exploited for a localization of small concentrations of halofantrine in a biological environment. The Raman spectrum of halofantrine is calculated by means of DFT calculations [B3LYP/6-311+G(d,p)]. The calculation is very useful for a thorough mode assignment of the Raman bands of halofantrine. The strong bands at 1621 and 1590 cm−1 in the UV Raman spectrum are assigned to combined CC stretching vibrations in the phenanthrene ring of halofantrine. These bands are considered as putative marker bands for interactions with the biological target molecules. The calculation of the electron density demonstrates a strong distribution across the phenanthrene ring of halofantrine, besides the electron withdrawing effect of the Cl and CF3 substituents. This strong and even electron density distribution supports the hypothesis of stacking as a possible mode of action of halofantrine. Complementary IR spectroscopy is performed for an investigation of vibrations of polar functional groups of the halofantrine molecule.

All-polymer chips with integrated optical waveguides and microfluidic channels were built as a platform for fluorescence excitation of biological samples. Their functionality has been shown for four cases: (i) fluorescence of labeled phospholipids inside a microfluidic channel, (ii) fluorescence of stained cells inside a microfluidic channel, (iii) fluorescence of labeled phospholipids on top of a polymer waveguide, and (iv) fluorescence of stained cells on top of a polymer waveguide.

Bacterial colonies play an important role in the isolation and identification of bacterial species, and plating on a petri dish is still regarded as the gold standard for confirming the cause of an outbreak situation. A bacterial colony consists of millions of densely packed individual bacteria along with matrices such as extracellular materials. When a laser is directed through a colony, complicated structures encode their characteristic signatures, which results in unique forward scattering patterns. We investigate the connection between the morphological parameters of a bacterial colony and corresponding forward scattering patterns to understand bacterial growth morphology. A colony elevation is modeled with a Gaussian profile, which is defined with two critical parameters: center thickness and diameter. Then, applying the scalar diffraction theory, we compute an amplitude modulation via light attenuation from multiple layers of bacteria while a phase modulation is computed from the colony profile. Computational results indicate that center thickness plays a critical role in the total number of diffraction rings while the magnitude of the slope of a colony determines the maximum diffraction angle. Experimental validation is performed by capturing the scattering patterns, monitoring colony diameters via phase contrast microscope, and acquiring the colony profiles via confocal displacement meter.

Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 µM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 µm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

The optical properties of antiphotobleaching and the advantage of long-term fluorescence observation of quantum dots are fully adopted to study the effects of iron on the endocytosis of transferrin. Quantum dots are labeled for transferrin and endocytosis of transferrin in HeLa cells is observed under the normal state, iron overloading, and an iron-deficient state. In these three states, the fluorescence undergoes a gradual process of first dark, then light, and finally dark, indicating the endocytosis of transferrin. The fluorescence intensity analysis shows that a platform emerges when fluorescence changes to a certain degree in the three states. Experienced a same period of time after platform, the fluorescence strength of cells in the normal state is 1.2 times the first value, and the iron-deficiency state is 1.4 times, but the iron overloading state was 0.85 times. We also find that the average fluorescence intensity in cells detected by the spectrophotometer in the iron-deficiency state is almost 7 times than that in a high iron state. All this proves that iron overloading would slow the process, but iron deficiency would accelerate endocytosis. We advance a direct observational method that may contribute to further study of the relationship of iron and transferrin.

We present a comparative study of scattering from healthy red blood cells RBCs and diseased RBCs with deformed shapes. Scattering problems involving three-dimensional RBCs are formulated accurately with the electric and magnetic current combined-field integral equation and solved efficiently by the multilevel fast multipole algorithm. We compare scattering cross section values obtained for different RBC shapes and different orientations. In this way, we determine strict guidelines to distinguish deformed RBCs from healthy RBCs and to diagnose various diseases using scattering cross section values. The results may be useful for designing new and improved flow cytometry procedures.

Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 µg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.

The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithm is shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations is minimized.

Human perception of 2.0-µm infrared laser irradiation has become significant in such disparate fields as law enforcement, neuroscience, and pain research. Several recent studies have found damage thresholds for single-pulse and continuous wave irradiations at this wavelength. However, the only publication using multiple-pulse irradiations was investigating the cornea rather than skin. Literature has claimed that the 2.0-µm light characteristic thermal diffusion time was as long as 300-ms. Irradiating the skin with 2.0-µm lasers to produce sensation should follow published recommendations to use pulses on the order of 10 to 100 ms, which approach the theoretical thermal diffusion time. Therefore, investigation of the heating of skin for a variety of laser pulse combinations was undertaken. Temperatures of ex vivo pig skin were measured at the surface and at three depths from pulse sequences of six different duty factors. Differences were found in temperature rise per unit exposure that did not follow a linear relation to duty factor. The differences can be explained by significant heat conduction during the pulses. Therefore, the common heat modeling assumption of thermal confinement during a pulse may need to be experimentally verified if the pulse approaches the theoretical thermal confinement time.

The use of microneedles as a method of circumventing the barrier properties of the stratum corneum is receiving much attention. Although skin disruption technologies and subsequent transdermal diffusion rates are being extensively studied, no accurate data on depth and closure kinetics of microneedle-induced skin pores are available, primarily due to the cumbersome techniques currently required for skin analysis. We report on the first use of optical coherence tomography technology to image microneedle penetration in real time and in vivo. We show that optical coherence tomography (OCT) can be used to painlessly measure stratum corneum and epidermis thickness, as well as microneedle penetration depth after microneedle insertion. Since OCT is a real-time, in-vivo, nondestructive technique, we also analyze skin healing characteristics and present quantitative data on micropore closure rate. Two locations (the volar forearm and dorsal aspect of the fingertip) have been assessed as suitable candidates for microneedle administration. The results illustrate the applicability of OCT analysis as a tool for microneedle-related skin characterization.

Optical topography (OT) based on near-infrared spectroscopy is a noninvasive technique for mapping the relative concentration changes in oxygenated and deoxygenated hemoglobin (oxy- and deoxy-Hb, respectively) in the human cerebral cortex. In our previous study, we developed a small and light wearable optical topography (WOT) system that covers the entire forehead for monitoring prefrontal activation. In the present study, we examine whether the WOT system is applicable to OT measurement while walking, which has been difficult with conventional OT systems. We conduct OT measurements while subjects perform an attention-demanding (AD) task of balancing a ping-pong ball on a small card while walking. The measured time course and power spectra of the relative concentration changes in oxy- and deoxy-Hb show that the step-related changes in the oxy- and deoxy-Hb signals are negligible compared to the task-related changes. Statistical assessment of the task-related changes in the oxy-Hb signals show that the dorsolateral prefrontal cortex and rostral prefrontal area are significantly activated during the AD task. These results suggest that our functional imaging technique with the WOT system is applicable to OT measurement while walking, and will be a powerful tool for evaluating brain activation in a natural environment.

Many endoscopic, intravascular, and transvaginal applications require light to be delivered through optical fibers in a reflection mode. For photoacoustic imaging in reflection geometry, the front-face reflectivity of the ultrasound transducer face imposes a boundary condition that affects the light fluence and its distribution inside a turbid medium. Understanding and characterizing this boundary condition is critical for maximizing tissue illumination and therefore the signal-to-noise ratio of the photoacoustic signal. We systematically analyze the light fluence under three typical commercial transducer faces having reflection coefficients of 1.4, 18, and 28%, and compare the results to a transducer face with 60% coefficient at the laser wavelength of 750 nm. Monte Carlo simulations and experimental results show that light fluence and distribution obtained inside a turbid medium with the use of the 60% reflection coefficient transducer face has a significant improvement over the others, especially at shallower depths.

We report a high-speed, dispersion-encoded, full-range (DEFR) swept-source optical coherence tomography system for in vivo ocular imaging and biometry of small animals. The fast DEFR algorithm removes the depth ambiguity, gives access to objects located at the zero delay position, and doubles the sampling depth to 2×5.0 mm (at −101 to −71 dB sensitivity) in a single scan using 2048 samples/depth scan 0.43 nm line width of a light source operating at 1056 nm with 70 nm tuning range. The acquisition speed (frames of 512 depth scans in 18.3 ms) permits precise on-line monitoring during positioning and provides cross-sectional views of the mouse eye. Preliminary studies demonstrate high-throughput, reproducible assessment of multiple biometric features (e.g., day-to-day reproducibility of axial length measurement ±5.3 µm) that is insensitive to eye motion sufficient for long-term monitoring.

A depth compensation algorithm (DCA) can effectively improve the depth localization of diffuse optical tomography (DOT) by compensating the exponentially decreased sensitivity in the deep tissue. In this study, DCA is investigated based on computer simulations, tissue phantom experiments, and human brain imaging. The simulations show that DCA can largely improve the spatial resolution of DOT in addition to the depth localization, and DCA is also effective for multispectral DOT with a wide range of optical properties in the background tissue. The laboratory phantom experiment demonstrates that DCA can effectively differentiate two embedded objects at different depths in the medium. DCA is further validated by human brain imaging using a finger-tapping task. To our knowledge, this is the first demonstration to show that DCA is capable of accurately localizing cortical activations in the human brain in three dimensions.

Since the birth of "Dolly" as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

Multispectral images of skin contain information on the spatial distribution of biological chromophores, such as blood and melanin. From this, parameters such as blood volume and blood oxygenation can be retrieved using reconstruction algorithms. Most such approaches use some form of pixelwise or volumetric reconstruction code. We explore the use of principal component analysis (PCA) of multispectral images to access blood volume and blood oxygenation in near real time. We present data from healthy volunteers under arterial occlusion of the forearm, experiencing ischemia and reactive hyperemia. Using a two-layered analytical skin model, we show reconstruction results of blood volume and oxygenation and compare it to the results obtained from our new spectral analysis based on PCA. We demonstrate that PCA applied to multispectral images gives near equivalent results for skin chromophore mapping and quantification with the advantage of being three orders of magnitude faster than the reconstruction algorithm.

Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be time-consuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging (MPT-FLIM) we assess ischemic necrosis of ex vivo skin by NAD(P)H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media (Dulbecco Modified Eagle Medium) at −20, 4, and 37 °C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD(P)H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime (Πm) from ~1000 to 2000 ps. Additionally, significant distortions in NAD(P)H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4 °C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.

Using the liquid crystal spatial light modulator (LC-SLM) as the wavefront corrector, an open-loop adaptive optics (AO) system for fundus imaging in vivo is constructed. Compared with the LC-SLM closed-loop AO system, the light energy efficiency is increased by a factor of 2, which is helpful for the safety of fundus illumination in vivo. In our experiment, the subjective accommodation method is used to precorrect the defocus aberration, and three subjects with different myopia 0, −3, and −5 D are tested. Although the residual wavefront error after correction cannot to detected, the fundus images adequately demonstrate that the imaging system reaches the resolution of a single photoreceptor cell through the open-loop correction. Without dilating and cyclopleging the eye, the continuous imaging for 8 s is recorded for one of the subjects.

By modifying a clinical ultrasound array system, we develop a novel handheld photoacoustic probe for image-guided needle biopsy. The integration of optical fiber bundles for pulsed laser light delivery enables photoacoustic image-guided insertion of a needle into rat axillary lymph nodes with accumulated indocyanine green (ICG). Strong photoacoustic contrast of the needle is achieved. After subcutaneous injection of the dye in the left forepaw, sentinel lymph nodes are easily detected, in vivo and in real time, beneath 2-cm-thick chicken breast overlaying the axillary region. ICG uptake in axillary lymph nodes is confirmed with fluorescence imaging both in vivo and ex vivo. These results demonstrate the clinical potential of this handheld photoacoustic system for facile identification and needle biopsy of sentinel lymph nodes for cancer staging and metastasis detection in humans.

Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.

Multiharmonic optical microscopy has been widely applied in biomedical research due to its unique capability to perform noninvasive studies of biomaterials. In this study, virtual biopsy based on back-propagating multiple optical harmonics, combining second and third harmonics, is applied in unfixed rat tympanic membrane. We show that third harmonic generation can provide morphologic information on the epithelial layers of rat tympanic membrane as well as radial collagen fibers in middle fibrous layers, and that second harmonic generation can provide information on both radial and circular collagen fibers in middle fibrous layers. Through third harmonic generation, the capillary and red blood cells in the middle fibrous layer are also noted. Additionally, the 3-D relationship to adjacent bony structures and spatial variations in thickness and curvature are obtained. Our study demonstrates the feasibility of using a noninvasive optical imaging system for comprehensive evaluation of the tympanic membrane.

Noncontact optical imaging of curved objects can result in strong artifacts due to the object's shape, leading to curvature biased intensity distributions. This artifact can mask variations due to the object's optical properties, and makes reconstruction of optical/physiological properties difficult. In this work we demonstrate a curvature correction method that removes this artifact and recovers the underlying data, without the necessity of measuring the object's shape. This method is applicable to many optical imaging modalities that suffer from shape-based intensity biases. By separating the spatially varying data (e.g., physiological changes) from the background signal (dc component), we show that the curvature can be extracted by either averaging or fitting the rows and columns of the images. Numerical simulations show that our method is equivalent to directly removing the curvature, when the object's shape is known, and accurately recovers the underlying data. Experiments on phantoms validate the numerical results and show that for a given image with 16.5% error due to curvature, the method reduces that error to 1.2%. Finally, diffuse multispectral images are acquired on forearms in vivo. We demonstrate the enhancement in image quality on intensity images, and consequently on reconstruction results of blood volume and oxygenation distributions.

Cavernous nerves course along the surface of the prostate gland and are responsible for erectile function. These nerves are at risk of injury during surgical removal of a cancerous prostate gland. In this work, a combination of segmentation, denoising, and edge detection algorithms are applied to time-domain optical coherence tomography (OCT) images of rat prostate to improve identification of cavernous nerves. First, OCT images of the prostate are segmented to differentiate the cavernous nerves from the prostate gland. Then, a locally adaptive denoising algorithm using a dual-tree complex wavelet transform is applied to reduce speckle noise. Finally, edge detection is used to provide deeper imaging of the prostate gland. Combined application of these three algorithms results in improved signal-to-noise ratio, imaging depth, and automatic identification of the cavernous nerves, which may be of direct benefit for use in laparoscopic and robotic nerve-sparing prostate cancer surgery.

We demonstrate quantitative analysis and error correction of optical coherence tomography (OCT) retinal images by using a custom-built, computer-aided grading methodology. A total of 60 Stratus OCT (Carl Zeiss Meditec, Dublin, California) B-scans collected from ten normal healthy eyes are analyzed by two independent graders. The average retinal thickness per macular region is compared with the automated Stratus OCT results. Intergrader and intragrader reproducibility is calculated by Bland-Altman plots of the mean difference between both gradings and by Pearson correlation coefficients. In addition, the correlation between Stratus OCT and our methodology-derived thickness is also presented. The mean thickness difference between Stratus OCT and our methodology is 6.53 µm and 26.71 µm when using the inner segment/outer segment (IS/OS) junction and outer segment/retinal pigment epithelium (OS/RPE) junction as the outer retinal border, respectively. Overall, the median of the thickness differences as a percentage of the mean thickness is less than 1% and 2% for the intragrader and intergrader reproducibility test, respectively. The measurement accuracy range of the OCT retinal image analysis (OCTRIMA) algorithm is between 0.27 and 1.47 µm and 0.6 and 1.76 µm for the intragrader and intergrader reproducibility tests, respectively. Pearson correlation coefficients demonstrate R2>0.98 for all Early Treatment Diabetic Retinopathy Study (ETDRS) regions. Our methodology facilitates a more robust and localized quantification of the retinal structure in normal healthy controls and patients with clinically significant intraretinal features.

While photoacoustic imaging is capable of producing high-resolution biomedical images with optical absorption contrast, optical property quantification has thus far remained challenging. One reason for this is that laser-induced photoacoustic signal amplitudes are proportional to not only the local optical absorption coefficient, but also the local laser fluence in the tissue. Unfortunately, local laser fluence is often unknown, but could possibly be estimated if local bulk tissue optical properties were known. One method to estimate tissue optical properties is a technique known as oblique incidence diffuse reflectance (OIR). We report on an integrated OIR and photoacoustic imaging system and demonstrate, using phantom experiments, improved ability to quantitatively estimate optical properties of a turbid medium.

Methods of wide-field fluorescence microscopy for measuring membrane dynamics of living cells are described, including spectral imaging as well as anisotropy imaging of the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). Plasma membranes are selected by illumination with an evanescent electromagnetic field and distinguished from intracellular membranes assessed by whole-cell illumination. While fluorescence spectra of laurdan appeared red-shifted with decreasing membrane stiffness, fluorescence anisotropy and rotational correlation times were reduced with increasing membrane fluidity. Membrane stiffness was found to increase with decreasing temperature and increasing amounts of cholesterol and was always higher for the plasma membrane than for intracellular membranes. These effects may have some clinical relevance in the research of drug resistance or cell aging.

Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

We investigate optical coherence tomography (OCT) as a method for imaging bone. The OCT images are compared directly to those of the standard methods of bone histology and microcomputed tomography (µCT) on a single, fixed human femoral trabecular bone sample. An advantage of OCT over bone histology is its noninvasive nature. OCT also images the lamellar structure of trabeculae at slightly higher contrast than normal bone histology. While µCT visualizes the trabecular framework of the whole sample, OCT can image additionally cells with a penetration depth limited approximately to 1 mm. The most significant advantage of OCT, however, is the absence of toxic effects (no ionizing radiation), i.e., continuous images may be made and individual cell tracking may be performed. The penetration depth of OCT, however, limits its use to small animal models and small bone organ cultures.

Live microscopy techniques (i.e., differential interference contrast, confocal microscopy, etc.) have enabled the understanding of the mechanisms involved in cells and tissue formation. In long-term studies, special care must be taken in order to avoid sample damage, restricting the applicability of the different microscopy techniques. We demonstrate the potential of using third-harmonic generation (THG) microscopy for morphogenesis/embryogenesis studies in living Caenorhabditis elegans (C. elegans). Moreover, we show that the THG signal is obtained in all the embryo development stages, showing different tissue/structure information. For this research, we employ a 1550-nm femtosecond fiber laser and demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Additionally, this has the important advantage that the THG signal is emitted at visible wavelengths (516 nm). Therefore, standard collection optics and detectors operating near maximum efficiency enable an optimal signal reconstruction. All this, to the best of our knowledge, demonstrates for the first time the noninvasiveness and strong potential of this particular wavelength to be used for high-resolution four-dimensional imaging of embryogenesis using unstained C. elegans in vivo samples.

The extraction of 3-D morphological information about thick objects is explored in this work. We extract this information from 3-D differential interference contrast (DIC) images by applying a texture detection method. Texture extraction methods have been successfully used in different applications to study biological samples. A 3-D texture image is obtained by applying a local entropy-based texture extraction method. The use of this method to detect regions of blastocyst mouse embryos that are used in assisted reproduction techniques such as in vitro fertilization is presented as an example. Results demonstrate the potential of using texture detection methods to improve morphological analysis of thick samples, which is relevant to many biomedical and biological studies. Fluorescence and optical quadrature microscope phase images are used for validation.

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

The advantages and limitations of using a beating heart model in the development of intravascular optical coherence tomography are discussed. The model fills the gap between bench experiments, performed on phantoms and excised arteries, and whole animal in-vivo preparations. The beating heart model is stable for many hours, allowing for extended measurement times and multiple imaging sessions under in-vivo conditions without the complications of maintaining whole-animal preparation. The perfusate supplying the heart with nutrients can be switched between light scattering blood to a nonscattering perfusate to allow the optical system to be optimized without the need of an efficient blood displacement strategy. Direct access to the coronary vessels means that there is no need for x-ray fluoroscopic guidance of the catheter to the heart, as is the case in whole animal preparation. The model proves to be a valuable asset in the development of our intravascular optical coherence tomography technology.

Osteoarthritis (OA) is a common musculoskeletal disorder often diagnosed during arthroscopy. In OA, visual color changes of the articular cartilage surface are typically observed. We demonstrate in vitro the potential of visible light spectral imaging (420 to 720 nm) to quantificate these color changes. Intact bovine articular cartilage samples (n=26) are degraded both enzymatically using the collagenase and mechanically using the emery paper (P60 grit, 269 µm particle size). Spectral images are analyzed by using standard CIELAB color coordinates and the principal component analysis (PCA). After collagenase digestion, changes in the CIELAB coordinates and projection of the spectra to PCA eigenvector are statistically significant (p<0.05). After mechanical degradation, the grinding tracks could not be visualized in the RGB presentation, i.e., in the visual appearance of the sample to the naked eye under the D65 illumination. However, after projecting to the chosen eigenvector, the grinding tracks are revealed. The tracks are also seen by using only one wavelength, i.e., 469 nm, however, the contrast in the projection image is 1.6 to 2.5 times higher. Our results support the idea that the spectral imaging can be used for evaluation of the integrity of the cartilage surface.

Novel methods and technologies that could extend and complement the capabilities of the prevailing fluorescence microscope in following the cell cycle under different perturbations are highly desirable in the area of biological and biomedical imaging. We report a newly designed instrument for long-term light scattering live cell imaging based on integrating a homebuilt environmental cell incubation minichamber and an angled dark-field illumination system into a conventional inverted light microscope. Peptide-conjugated gold nanoparticles that are selectively delivered to either the cytoplasmic or nuclear region of the cell are used as light scattering contrast agents. The new system enables us to carry out continuous and intermittence-free dark-field live cell imaging over several tens of hours. A variety of applications of this imaging system are demonstrated, such as monitoring the nuclear uptake of peptide-conjugated gold nanoparticles, tracking the full cycle of cancer cells from birth to division, following the chromosome dynamics during cell mitosis, and observing the intracellular distribution of gold nanoparticles after cell division. We also discuss the overall effect of nuclear targeting gold nanoparticles on the cell viability of parent and daughter cells.

Karyotyping is an important process to classify chromosomes into standard classes and the results are routinely used by the clinicians to diagnose cancers and genetic diseases. However, visual karyotyping using microscopic images is time-consuming and tedious, which reduces the diagnostic efficiency and accuracy. Although many efforts have been made to develop computerized schemes for automated karyotyping, no schemes can get be performed without substantial human intervention. Instead of developing a method to classify all chromosome classes, we develop an automatic scheme to detect abnormal metaphase cells by identifying a specific class of chromosomes (class 22) and prescreen for suspicious chronic myeloid leukemia (CML). The scheme includes three steps: (1) iteratively segment randomly distributed individual chromosomes, (2) process segmented chromosomes and compute image features to identify the candidates, and (3) apply an adaptive matching template to identify chromosomes of class 22. An image data set of 451 metaphase cells extracted from bone marrow specimens of 30 positive and 30 negative cases for CML is selected to test the scheme's performance. The overall case-based classification accuracy is 93.3% (100% sensitivity and 86.7% specificity). The results demonstrate the feasibility of applying an automated scheme to detect or prescreen the suspicious cancer cases.

Digital in-line holographic microscopy (DIHM) is a modern approach capable of achieving micron-range lateral and depth resolutions in three-dimensional imaging. DIHM in combination with numerical imaging reconstruction uses an extremely simplified setup while retaining the advantages provided by holography with enhanced capabilities derived from algorithmic digital processing. We introduce superresolved DIHM incoming from time and angular multiplexing of the sample spatial frequency information and yielding in the generation of a synthetic aperture (SA). The SA expands the cutoff frequency of the imaging system, allowing submicron resolutions in both transversal and axial directions. The proposed approach can be applied when imaging essentially transparent (low-concentration dilutions) and static (slow dynamics) samples. Validation of the method for both a synthetic object (U.S. Air Force resolution test) to quantify the resolution improvement and a biological specimen (sperm cells biosample) are reported showing the generation of high synthetic numerical aperture values working without lenses.

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

We present a parametric optical coherence tomography (OCT) technique to improve contrast between malignant and healthy non-neoplastic tissue. The technique incorporates a fully automated method to extract tissue attenuation characteristics. Results are represented visually as a parametric en face image, where the parameter used for contrast is indicative of the relative optical attenuation coefficient of the tissue. We present the first parametric OCT images of human lymph nodes containing malignant cells, and demonstrate improved tissue contrast over en face OCT images.

A novel method for rapidly detecting metastatic breast cancer within excised sentinel lymph node(s) of the axilla is presented. Elastic scattering spectroscopy (ESS) is a point-contact technique that collects broadband optical spectra sensitive to absorption and scattering within the tissue. A statistical discrimination algorithm was generated from a training set of nearly 3000 clinical spectra and used to test clinical spectra collected from an independent set of nodes. Freshly excised nodes were bivalved and mounted under a fiber-optic plate. Stepper motors raster-scanned a fiber-optic probe over the plate to interrogate the node's cut surface, creating a 20×20 grid of spectra. These spectra were analyzed to create a map of cancer risk across the node surface. Rules were developed to convert these maps to a prediction for the presence of cancer in the node. Using these analyses, a leave-one-out cross-validation to optimize discrimination parameters on 128 scanned nodes gave a sensitivity of 69% for detection of clinically relevant metastases (71% for macrometastases) and a specificity of 96%, comparable to literature results for touch imprint cytology, a standard technique for intraoperative diagnosis. ESS has the advantage of not requiring a pathologist to review the tissue sample.

Toxoplasmosis is an important zoonosis in public health because domestic cats are the main agents responsible for the transmission of this disease in Brazil. We investigate a method for diagnosing toxoplasmosis based on Raman spectroscopy. Dispersive near-infrared Raman spectra are used to quantify anti-Toxoplasma gondii (IgG) antibodies in blood sera from domestic cats. An 830-nm laser is used for sample excitation, and a dispersive spectrometer is used to detect the Raman scattering. A serological test is performed in all serum samples by the enzyme-linked immunosorbent assay (ELISA) for validation. Raman spectra are taken from 59 blood serum samples and a quantification model is implemented based on partial least squares (PLS) to quantify the sample's serology by Raman spectra compared to the results provided by the ELISA test. Based on the serological values provided by the Raman/PLS model, diagnostic parameters such as sensitivity, specificity, accuracy, positive prediction values, and negative prediction values are calculated to discriminate negative from positive samples, obtaining 100, 80, 90, 83.3, and 100%, respectively. Raman spectroscopy, associated with the PLS, is promising as a serological assay for toxoplasmosis, enabling fast and sensitive diagnosis.

Functional connectivity has become one of the important approaches to understanding the functional organization of the human brain. Recently, functional near-infrared spectroscopy (fNIRS) was demonstrated as a feasible method to study resting-state functional connectivity (RSFC) in the sensory and motor systems. However, whether such fNIRS-based RSFC can be revealed in high-level and complex functional systems remains unknown. In the present study, the feasibility of such an approach is tested on the language system, of which the neural substrates have been well documented in the literature. After determination of a seed channel by a language localizer task, the correlation strength between the low frequency fluctuations of the fNIRS signal at the seed channel and those at all other channels is used to evaluate the language system RSFC. Our results show a significant RSFC between the left inferior frontal cortex and superior temporal cortex, components both associated with dominant language regions. Moreover, the RSFC map demonstrates left lateralization of the language system. In conclusion, the present study successfully utilized fNIRS-based RSFC to study a complex and high-level neural system, and provides further evidence for the validity of the fNIRS-based RSFC approach.

Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1-dioctadecyl-3,3,3,3-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

An in vivo study was conducted to verify the ability of laser fluorescence (LF) to assess the activity status of occlusal caries in primary teeth, using different air-drying times. Occlusal sites (707) were examined using LF (DIAGNOdent) after air-drying for 3 s and 15 s, and the difference between readings (DIF15 s-3 s) was calculated. For concurrent validation of LF, visual criteria-Nyvad (NY) and Lesion Activity Assessment associated with the International Caries Detection and Assessment System (LAA-ICDAS)-were the reference standards for lesion activity. Histological exam using a pH-indicator dye (0.1% methyl red) was performed in 46 exfoliated/extracted teeth for criterion validation. LF readings and DIF15 s-3 s were compared using Kruskall-Wallis and Mann-Whitney tests. Receiver operating characteristic analyses were performed and validity parameters calculated, considering the caries activity assessment. Using NY, active lesions (3 s: 30.0±29.3; 15 s: 34.2±30.6) presented higher LF readings than inactive lesions (3 s: 17.0±16.3; 15 s: 19.2±17.3; p<0.05), different from LAA-ICDAS. Active cavitated caries resulted in higher LF readings (3 s: 50.3±3.5; 15 s: 54.7±30.2) than inactive cavitated caries (3 s: 19.9±16.3; 15 s: 22.8±16.8). Therefore, LF can distinguish cavitated active and inactive lesions classified by NY, but not by LAA-ICDAS; however, this difference might be related to the visual system rather than to LF. The air-drying time could be an alternative to improve the caries activity assessment; however, longer air-drying time is suggested to be tested subsequently.

In this study, we exploit the sensitivity offered by surface-enhanced Raman scattering (SERS) for the direct detection of thrombin using the thrombin-binding aptamer (TBA) as molecular receptor. The technique utilizes immobilized silver nanoparticles that are functionalized with thiolated thrombin-specific binding aptamer, a 15-mer (5'-GGTTGGTGTGGTTGG-3') quadruplex forming oligonucleotide. In addition to the Raman vibrational bands corresponding to the aptamer and blocking agent, new peaks (mainly at 1140, 1540, and 1635 cm−¹) that are characteristic of the protein are observed upon binding of thrombin. These spectral changes are not observed when the aptamer-nanoparticle assembly is exposed to a nonbinding protein such as bovine serum albumin (BSA). This methodology could be further used for the development of label-free biosensors for direct detection of proteins and other molecules of interest for which aptamers are available.

There is an urgent clinical need to monitor the intravenous delivery and bioavailability of circulating nanoparticles used in cancer therapy. This work presents the use of photoplethysmography for the noninvasive real-time estimation of vascular gold nanoshell concentration in a murine subject. We develop a pulse photometer capable of accurately measuring the photoplethysmogram in mice and determining the ratio of pulsatile changes in optical extinction between 805 and 940 nm, commonly referred to as R. These wavelengths are selected to correspond to the extinction properties of gold nanoshells. Six 30-s measurements (5 min, 2, 4, 6, 8, 10 h) are taken under light anesthesia to observe the change in R as the nanoparticles clear from the circulation. Our model describes the linear fit (R2=0.85) between R and the concentration of nanoparticles measured via ex vivo spectrophotometric and instrumental neutron activation analysis. This demonstrates the utility of this technique in support of clinical nanoparticle therapies.

The fluorescence spectra of human cancerous and normal prostate tissues obtained by the selective excitation wavelength of 340 nm were measured. The contributions of principle biochemical components to tissue fluorescence spectra were investigated using the method of multivariate curve resolution with alternating least squares. The results show that there is a reduced contribution from the emission of collagen and increased contribution from nicotinamide adenine dinucleotide (NADH) in cancerous tissues as compared with normal tissue. This difference is attributed to the changes of relative contents of NADH and collagen during cancer development. This research may present a potential native biomarker for prostate cancer detection.

Myocardial infarction leads to structural remodeling of the myocardium, in particular to the loss of cardiomyocytes due to necrosis and an increase in collagen with scar formation. Stem cell regenerative treatments have been shown to alter this remodeling process, resulting in improved cardiac function. As healthy myocardial tissue is highly fibrous and anisotropic, it exhibits optical linear birefringence due to the different refractive indices parallel and perpendicular to the fibers. Accordingly, changes in myocardial structure associated with infarction and treatment-induced remodeling will alter the anisotropy exhibited by the tissue. Polarization-based linear birefringence is measured on the myocardium of adult rat hearts after myocardial infarction and compared with hearts that had received mesenchymal stem cell treatment. Both point measurement and imaging data show a decrease in birefringence in the region of infarction, with a partial rebound back toward the healthy values following regenerative treatment with stem cells. These results demonstrate the ability of optical polarimetry to characterize the micro-organizational state of the myocardium via its measured anisotropy, and the potential of this approach for monitoring regenerative treatments of myocardial infarction.

The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry.

The high transparency of dental enamel in the near-infrared (NIR) at 1310 nm can be exploited for imaging dental caries without the use of ionizing radiation. The objective of this study is to determine whether the lesion contrast derived from NIR imaging in both transmission and reflectance can be used to estimate lesion severity. Two NIR imaging detector technologies are investigated: a new Ge-enhanced complementary metal-oxide-semiconductor (CMOS)-based NIR imaging camera, and an InGaAs focal plane array (FPA). Natural occlusal caries lesions are imaged with both cameras at 1310 nm, and the image contrast between sound and carious regions is calculated. After NIR imaging, teeth are sectioned and examined using polarized light microscopy (PLM) and transverse microradiography (TMR) to determine lesion severity. Lesions are then classified into four categories according to lesion severity. Lesion contrast increases significantly with lesion severity for both cameras (p<0.05). The Ge-enhanced CMOS camera equipped with the larger array and smaller pixels yields higher contrast values compared with the smaller InGaAs FPA (p<0.01). Results demonstrate that NIR lesion contrast can be used to estimate lesion severity.

We present an approach to adaptively adjust the spectral window sizes for optical spectra feature extraction. Previous studies extracted features from spectral windows of a fixed width. In our algorithm, piecewise linear regression is used to adaptively adjust the window sizes to find the maximum window size with reasonable linear fit with the spectrum. This adaptive windowing technique ensures the signal linearity in defined windows; hence, the adaptive windowing technique retains more diagnostic information while using fewer windows. This method was tested on a data set of diffuse reflectance spectra of oral mucosa lesions. Eight features were extracted from each window. We performed classifications using linear discriminant analysis with cross-validation. Using windowing techniques results in better classification performance than not using windowing. The area under the receiver-operating-characteristics curve for windowing techniques was greater than a nonwindowing technique for both normal versus mild dysplasia (MD) plus severe high-grade dysplasia or carcinama (SD) (MD+SD) and benign versus MD+SD. Although adaptive and fixed-size windowing perform similarly, adaptive windowing utilizes significantly fewer windows than fixed-size windows (number of windows per spectrum: 8 versus 16). Because adaptive windows retain most diagnostic information while reducing the number of windows needed for feature extraction, our results suggest that it isolates unique diagnostic features in optical spectra.

Development of a noninvasive vasectomy technique may eliminate male fear of complications and result in a more popular procedure. This study explores application of an optical clearing agent (OCA) to scrotal skin to reduce laser power necessary for successful noninvasive laser vasectomy and eliminate scrotal skin burns. A mixture of dimethyl sulfoxide and glycerol was noninvasively delivered into scrotal skin using a pneumatic jet device. Near-infrared laser radiation was delivered in conjunction with cryogen spray cooling to the skin surface in a canine model, ex vivo and in vivo. Burst pressure (BP) measurements were conducted to quantify strength of vas closure. A 30-min application of OCA improved skin transparency by 26±3%, reducing average power necessary for successful noninvasive laser vasectomy from 9.2 W without OCA (BP=291±31 mmHg) to 7.0 W with OCA (BP=292±19 mmHg). Control studies without OCA at 7.0 W failed to coagulate the vas with burst pressures (82±28 mmHg) significantly below typical ejaculation pressures (136±29 mmHg). Application of an OCA reduced the laser power necessary for successful noninvasive thermal coagulation of the vas by ~25%. This technique may result in use of a less expensive laser and eliminate the formation of scrotal skin burns during the procedure.

To overcome the inconsecutive drawback of shadow and schlieren photography, the complete dynamics of cavitation bubble oscillation or ablation products induced by a single holmium laser pulse [2.12μm , 300μs (FWHM)] transmitted in different core diameter (200, 400, and 600μm ) fibers is recorded by means of high-speed photography. Consecutive images from high-speed cameras can stand for the true and complete process of laser-water or laser-tissue interaction. Both laser pulse energy and fiber diameter determine cavitation bubble size, which further determines acoustic transient amplitudes. Based on the pictures taken by high-speed camera and scanned by an optical coherent microscopy (OCM) system, it is easily seen that the liquid layer at the distal end of the fiber plays an important role during the process of laser-tissue interaction, which can increase ablation efficiency, decrease heat side effects, and reduce cost.

Modern operating microscopes offer high power illumination to ensure optimal visualization, but can also cause thermal damage. The aim of our study is to quantify the thermal effects in vivo and discuss conditions for safe use. In a pilot study on volunteers, we measured the temperature at the skin surface during microscope illumination, including the influence of anaesthesia and the effects of staining, draping, or moistening of the skin. Irradiation within the limit given by safety regulations (200 mW/cm²) results in skin surface temperature of 43 °C. Higher intensities (forearm 335 mW/cm², back 250 mW/cm²) are tolerated, resulting in reversible hyperaemia. At a very high illumination intensity (750 mW/cm²), pain occurs within 30 s at temperatures of 46 °C±1 °C (hand and forearm), and 43 °C±2 °C (back), respectively. Anaesthesia has no distinct effect on the temperature, whereas staining and drapes result in much higher temperatures (<100 °C). Moistening at practicable flow rates can reduce temperature efficiently when combined with a light absorbing and water absorbent drape. In conclusion, surgeons must be aware that surgical microscope illumination without protective means can cause skin temperatures to rise much above pain threshold, which in our study serves as a (conservative) benchmark for potential damage.

The possibility of in vivo investigation of the pharmacokinetics of photosensitizers by means of fluorescence transillumination imaging is demonstrated. An animal is scanned in the transilluminative configuration by a single source and detector pair. Transillumination is chosen as an alternative approach to reflection imaging. In comparison with the traditional back-reflection technique, transillumination is preferable for photosensitizer detection due to its higher sensitivity to deep-seated fluorophores. The experiments are performed on transplantable mouse cervical carcinomas using three drugs: photosens, alasens, and fotoditazin. For quantitative evaluation of the photosensitizer concentration in tumor tissue the fluorescence signal is calibrated using tissue phantoms. We show that the kinetics of photosensitizer tumor uptake obtained by transillumination imaging in vivo agree with data of standard ex vivo methods. The described approach enables rapid and cost-effective study of newly developed photosensitizers in small animals.

The influence of emission parameters in low-level-light therapy on cellular responses is not yet fully understood. This study assessed the impact of various light delivery modes on collagen production in human primary fibroblast cultured in monolayers after three treatments with red light-emitting diode illumination (630 nm, 8 J/cm2). Human type I collagen was measured in cell culture supernatants with procollagen type I C-peptide enzyme immunoassay. Results demonstrated that, 72 h post-baseline, specific microsecond pulsing patterns had a more favorable impact on the ability of fibroblasts to produce collagen de novo than comparative conditions of continuous wave, pulsed 50% duty cycle, and millisecond pulsing domains. The cascade of events leading to collagen production by red illumination may be explained by the photodissociation of nitric oxide from cytochrome c oxidase. Short and intermittent light delivery might enhance this cellular event.

This PDF file contains the errata for “JBO Vol. 15 Issue 04 Paper JBO ERR-JUL2010-1” for JBO Vol. 15 Issue 04