We present methods for visualizing the dynamic response of biological samples to laser-induced heating. Our approach utilizes optical frequency-domain imaging to detect, spatially localize, and monitor unique dynamic signatures that arise within zones of active tissue denaturation. Since this information is precisely registered with high-resolution (~10 µm) cross sectional images, regions of thermally destroyed tissue can be mapped in relation to pre-existing morphology. Using porcine esophageal specimens ex vivo, we demonstrate that the extent and evolution of laser thermal damage can be assessed in real time.

Gold nanoparticles grown within the intracellular confines of living cells are introduced as potential surface-enhanced Raman scattering (SERS) substrates for confocal Raman spectrometry. Electron microscopy and a silver-enhanced reflectance laser scanning confocal microscopic approach were used to visualize the size, shape, and distribution of intracellularly grown gold nanoparticles (IGAuN) as small as 1 nm. Passive uptake as the conventional approach for delivering nanoparticles inside cells faces the insurmountable challenge of escaping the endosomal/lysosomal pathway. In contrast, IGAuN provides an unprecedented advantage of providing access to cytoplasm and nucleus.

We report on a simple correlation method for lifetime measurements using a random modulated excitation light source. We use an intensity correlation function of emission for lifetime analyses. In this method, no reference timing of the excitation is required. We apply the correlation method to measure phosphorescence decays and successfully demonstrate in the analysis of the phosphorescence decay from Pd(II) porphine in HeLa cells under aerobic and anaerobic conditions to understand the oxygen dynamics in individual cells. The method is applicable to faster decay time measurements down to a nanosecond range when the detection system is improved. Current fluorescence correlation setups can easily be modified for lifetime measurements, expanding the applicability in biological problems.

In the postgenomic era, imaging techniques are playing an important role in visualizing gene expression in vivo. This work represents the first demonstration of photoacoustic tomography (PAT) for reporter gene imaging. Rats inoculated with 9L/lacZ gliosarcoma tumor cells are imaged with PAT before and after injection of X-gal, a colorimetric assay for the lacZ-encoded enzyme β-galactosidase. Using far-red optical illumination, the genetically tagged tumors in rats are clearly visualized by PAT following the assay. The spatial resolution is quantified to be less than 400 μm, while 500-nM-level sensitivity is demonstrated. With the future development of new absorption-based reporter gene systems, it is anticipated that photoacoustic technology will provide a valuable tool for molecular imaging research.

An 800-nm 200-fs laser is used to produce DNA damage in rat kangaroo (PtK1) and human cystic fibrosis pancreatic adenoma carcinoma (CFPAC-1) cells. Immunofluorescence staining for DNA repair factors in irradiated cells displays localization of γH2AX, Nbs1, and Rad50 to the site of irradiation 3 to 30 min following laser exposure. It is concluded that the 200-fs near-infrared laser is an excellent source for the production and study of spatially defined regions of DNA damage.

The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a) excite and detect fluorescence at two distinct colors simultaneously, and b) perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein (GFP)-labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies

We develop a double-differential spectroscopic analysis method for broadband near-infrared (NIR, 650 to 1000 nm) absorption spectra. Application of this method to spectra of tumor-containing breast tissue reveals specific cancer biomarkers. In this method, patient-specific variations in molecular composition are removed by using the normal tissue as an internal control. The effects of concentration differences of the four major tissue absorbers (oxyhemoglobin, deoxyhemoglobin, water, and bulk lipid) between the tumor and normal tissue are accounted for to reveal small spectral components unique to cancer. From a pilot study of 15 cancer patients, we find these spectral components to be characterized by specific NIR absorption bands. Based on the spectral regions of absorption at about 760, 930, and 980 nm, we identify these biomarkers with changes in state or addition of lipid and/or water. To quantify spectral variation in the absorption bands, we construct the specific tumor component (STC) index. The STC index identifies regions of the breast with tumors.

The first studies of the propagation of ultrafast (<45 fs) pulses of intense infrared light through protein media reveal that supercontinuum (white light) generation is severely suppressed in the presence of the protein α-amylase, a potential stress marker in human saliva. The continuum suppression capacity is attributed to the electron scavenging property of the protein.

This PDF file contains the editorial “Photonics in the Auditory System” for JBO Vol. 12 Issue 02

The biophysical properties and organization of cell membranes regulate many membrane-based processes, including electromotility in outer hair cells (OHCs) of the cochlea. Studies of the membrane environment can be carried out by measuring the orientation of membrane-bound fluorophores using fluorescence polarization microscopy (FPM). Due to the cylindrical shape of OHCs, existing FPM theory developed for spherical cells is not applicable. We develop a new method for analyzing FPM data suitable for the quasi-cylindrical OHC. We present the theory for this model, as well as a study of the orientation of the fluorescent probe pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl) (di-8-ANEPPS) in the OHC membrane. Our results indicate that the absorption transition dipole moment of di-8-ANEPPS orients symmetrically about the membrane normal at 27 deg with respect to the plane of the membrane. The observed agreement between theoretical predictions and experimental measurements establishes the applicability of FPM to study OHC plasma membrane properties.

The transmembrane protein prestin is crucial to outer hair cell (OHC) electromotility and contributes to the sensitivity and frequency selectivity of mammalian hearing. The molecular mechanisms of electromotility remain unclear, but prestin is purported to function as both a voltage sensor and a molecular motor. Understanding the role of prestin requires characterizing its organization and behavior in the plasma membrane. Fluorescence recovery after photobleaching (FRAP) provides a powerful means to quantitatively study molecular diffusion. However, OHCs are inherently fragile ex vivo, and dynamic studies of prestin require model systems, such as human embryonic kidney (HEK) cells, expressing fluorescently labeled prestin. Utilizing this system, we provide the first direct, quantitative measurement of prestin lateral mobility. The results show remarkably different diffusion behavior for prestin-green fluorescent protein (GFP) as compared to a control protein, human somatostatin receptor 5 (SSTR5). Prestin-GFP FRAP experiments reveal immobile fractions approaching 50%, low effective diffusion coefficients, and recovery times slower than those of SSTR5. Secondary bleaching of a region reveals distinctly different diffusion parameters, which we propose reflect the transient confinement of prestin in the HEK cell. Although uncharacterized, intermolecular interactions between prestin and the membrane and/or cytoskeleton may be important for the unique properties of prestin in electromotile OHCs.

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.

We describe a novel confocal image acquisition system capable of measuring the sound-evoked motion of the organ of Corti. The hearing organ is imaged with a standard laser scanning confocal microscope during sound stimulation. The exact temporal relation between each image pixel and the sound stimulus is quantified. The motion of the structures under study is obtained by fitting a Fourier series to the time dimension of a continuous sequence of acquired images. Previous versions of this acquisition system used a simple search to find pixels with similar phase values. The Fourier series approach permits substantially faster image acquisition with reduced noise levels and improved motion estimation. The system is validated by imaging various vibrating samples attached to a feedback-controlled piezoelectric translator. When using a rigid sample attached to the translator, the system is capable of measuring motion with peak-to-peak amplitudes smaller than 50 nm with an error below 20% at frequencies between 50 and 600 Hz. Examples of image sequences from the inner ear are given, along with detailed performance characteristics of the method.

An optical coherence tomography (OCT) system is built to acquire in vivo both images and vibration measurements of the organ of Corti of the guinea pig. The organ of Corti is viewed through a ~300-μm-diam hole in the bony wall of the cochlea at the scala tympani of the first cochlear turn. In imaging mode, the image is acquired as reflectance R(x,z). In vibration mode, the basilar membrane (BM) or reticular lamina (RL) are selected by the investigator interactively from the R(x,z) image. Under software control, the system moves the scanning mirrors to bring the sensing volume of the measurement to the desired membrane location. In vivo images of the organ of Corti are presented, indicating reflectance signals from the BM, RL, tectorial membrane, and Reissner's membrane. The tunnel of Corti and the inner sulcus are also visible in the images. Vibrations of ±2 and ±22 nm are recorded in the BM in response to low and high sound levels at 14 kHz above a noise floor of 0.2 nm.

The cochlea is the mammalian organ of hearing. Its predominant vibratory element, the basilar membrane, is tonotopically tuned, based on the spatial variation of its mass and stiffness. The constituent collagen fibers of the basilar membrane affect its stiffness. Laser irradiation can induce collagen remodeling and deposition in various tissues. We tested whether similar effects could be induced within the basilar membrane. Trypan blue was perfused into the scala tympani of anesthetized mice to stain the basilar membrane. We then irradiated the cochleas with a 694-nm pulsed ruby laser at 15 or 180 J/cm². The mice were sacrificed 14 to 16 days later and collagen organization was studied. Polarization microscopy revealed that laser irradiation increased the birefringence within the basilar membrane in a dose-dependent manner. Electron microscopy demonstrated an increase in the density of collagen fibers and the deposition of new fibrils between collagen fibers after laser irradiation. As an assessment of hearing, auditory brainstem response (ABR) thresholds were found to increase moderately after 15 J/cm² and substantially after 180 J/cm². Our results demonstrate that collagen remodeling and new collagen deposition occurs within the basilar membrane after laser irradiation in a similar fashion to that found in other tissues.

Pulsed, mid-infrared lasers were recently investigated as a method to stimulate neural activity. There are significant benefits of optically stimulating nerves over electrically stimulating, in particular the application of more spatially confined neural stimulation. We report results from experiments in which the gerbil auditory system was stimulated by optical radiation, acoustic tones, or electric current. Immunohistochemical staining for the protein c-FOS revealed the spread of excitation. We demonstrate a spatially selective activation of neurons using a laser; only neurons in the direct optical path are stimulated. This pattern of c-FOS labeling is in contrast to that after electrical stimulation. Electrical stimulation leads to a large, more spatially extended population of labeled, activated neurons. In the auditory system, optical stimulation of nerves could have a significant impact on the performance of cochlear implants, which can be limited by the electric current spread.

The purpose of this study is to demonstrate the feasibility of broadband diffuse optical spectroscopy (DOS) for noninvasive optical monitoring of differentiating patterns of total tissue hemoglobin (THC), oxy- (OxyHb), and deoxyhemoglobin (DeOxyHb) concentrations during hypovolemic shock and subsequent fluid replacement with saline and whole blood. The goal of this DOS application is to determine the efficacy of resuscitation efforts at the tissue level rather than currently available indirect and invasive measurements of hemodynamic parameters. 16 New Zealand white rabbits are hemorrhaged 20% of their total blood volume. In resuscitated animals, shed blood volume is replaced with equal volume of crystalloid or whole blood (five animals each). Physiological variables (cardiac output, mean arterial pressure, systemic vascular resistance, hematocrit) are measured invasively, while (OxyHb) and (DeOxyHb) are measured during the interventions using broadband DOS. During the pure hypovolemic hemorrhages, the decrease in THC is mainly due to the decrease in (OxyHb), since the decrease in THC due to blood loss results in decreased tissue perfusion, with a resultant increased tissue extraction of oxygen. The hemorrhage with the whole blood resuscitation model shows significant changes in (OxyHb) during resuscitation phases due to the higher oxygen carrying capacity of whole blood, as opposed to the limited volume replacement effects and the decreased tissue oxygen content from the euvolemic anemia of the saline resuscitation. Broadband DOS noninvasive optical monitoring reveals distinct patterns of total tissue hemoglobin, oxy-, and deoxyhemoglobin during hemorrhage. Further studies are needed to confirm potential clinical utility and accuracy under more complex clinical conditions in animal models and patients.

The near-IR cytochrome c oxidase (CCO) signal has potential as a clinical marker of changes in mitochondrial oxygen utilization. We examine the CCO signal response to reduced oxygen delivery in the healthy human brain. We induced a reduction in arterial oxygen saturation from baseline levels to 80% in eight healthy adult humans, while minimizing changes in end tidal carbon dioxide tension. We measured changes in the cerebral concentrations of oxidized CCO (Δ[oxCCO]), oxyhemoglobin (Δ[HbO₂]), and deoxyhemoglobin (Δ[HHb]) using broadband near-IR spectroscopy (NIRS), and estimated changes in cerebral oxygen delivery (ecDO₂) using pulse oximetry and transcranial Doppler ultrasonography. Results are presented as median (interquartile range). At the nadir of hypoxemia ecDO₂ decreased by 9.2 (5.4 to 12.1)% (p<0.0001), Δ[oxCCO] decreased by 0.24 (0.06 to 0.28) micromoles/l (p<0.01), total hemoglobin concentration increased by 2.83 (2.27 to 4.46) micromoles/l (p<0.0001), and change in hemoglobin difference concentration (Δ[Hbdiff]=Δ[HbO₂]−Δ[HHb]) decreased by 12.72 (11.32 to 16.34) micromoles/l (p<0.0001). Change in ecDO₂ correlated with Δ[oxCCO] (r=0.78, p<0.001), but not with either change in total hemoglobin concentration or Δ[Hbdiff]. This is the first description of cerebral Δ[oxCCO] during hypoxemia in healthy adults. Studies are ongoing to investigate the clinical relevance of this signal in patients with traumatic brain injury.

Fourier transform infrared microspectroscopy (FTIR-MSP) is potentially a powerful analytical method for identifying the spectral properties of biological activity in cells. The goal of the present research is the implementation of FTIR-MSP to study early spectral changes accompanying malignant transformation of cells. As a model system, cells in culture are infected by the murine sarcoma virus (MuSV), which induces malignant transformation. The spectral measurements are taken at various postinfection time intervals. To follow up systematically the progress of the spectral changes at early stages of cell transformation, it is essential first to determine and validate consistent and significant spectral parameters (biomarkers), which can evidently discriminate between normal and cancerous cells. Early stages of cell transformation are classified by an array of spectral biomarkers utilizing cluster analysis and discriminant classification function techniques. The classifications indicate that the first spectral changes are detectable much earlier than the first morphological signs of cell transformation. Our results point out that the first spectral signs of malignant transformation are observed on the first and third day of postinfection (PI) (for NIH/3T3 and MEF cell cultures, respectively), while the first visible morphological alterations are observed only on the third and seventh day, respectively. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent-free method for early detection of malignancy.

An IR-spectroscopy-based bedside device, coupled to a subcutaneously implanted microdialysis probe, is developed for quasicontinuous glucose monitoring with intermittent readouts at 10-min intervals, avoiding any sensor recalibration under long-term operation. The simultaneous estimation of the microdialysis recovery rate is possible using an acetate containing perfusate and determining its losses across the dialysis membrane. Measurements are carried out on four subjects, with experiments lasting either 8 or 28 h, respectively. Using the spectral interval data either from 1180 to 950 or 1560 to 1000 cm^-1, standard errors of prediction (SEPs) between 0.13 and 0.28 mM are achieved using multivariate calibration with partial least-squares (PLS) or classical least-squares (CLS) calibration models, respectively. The transfer of a PLS calibration model using the spectral and reference concentration data of the dialysates from the three 8-h-long experiments to a 28-h monitoring episode with another healthy subject is tested. Including microdialysis recovery for the determination of the interstitial glucose concentrations, an SEP of 0.24 mM is obtained versus whole blood glucose values. The option to determine other metabolites such as urea or lactate offers the possibility to develop a calibration- and reagent-free point-of-care analyzer.

Mid-IR semiconductor lasers of two wavelength bands, 5.4 and 9.6 μm, are applied to measure aqueous glucose concentration ranging from 0 to 500 mg/dL with Intralipid^® emulsion (0 to 8%) added as a fat simulator. The absorption coefficient µ_a is found linear with respect to glucose and Intralipid^® concentrations, and their specific absorption coefficients are obtained via linear regression. These coefficients are subsequently used to infer the concentrations and compare with known values. The objective is to evaluate the method accuracy. Glucose concentration is determined within ±21 mg/dL with 90% confidence and ±32 mg/dL with 99% confidence, using <1-mJ laser energy. It is limited by the apparatus mechanical error and not the photometric system noise. The expected uncertainties due to photometric noise are ±6 and ±9 mg/dL with 90 and 99% confidence, respectively. The uncertainty is fully accounted for by the system intrinsic errors, allowing rigorous inference of the confidence level. Intralipid^® is found to have no measurable effect on glucose determination. Further analysis suggests that a few mid-IR wavelengths may be sufficient, and that the laser technique offers advantages with regard to accuracy, speed, and sample volume, which can be small, ~0.4×10^-7 mL for applications such as microfluidic or microbioarray monitoring.

Atherosclerotic and normal rabbit aorta samples show a marked difference in chemical composition governed by the water, lipid, and protein content. The strongly overlapping infrared absorption features of the different constituents, and the complexity of the tissue matrix, render tissue classification by direct evaluation of molecular spectroscopic characteristics obtained from IR reflectance or attenuated total reflectance (ATR) measurements virtually impossible. We apply multivariate analysis and classification techniques based on partial least squares regression (PLS) and linear discriminant analysis to IR spectroscopic data obtained by IR-ATR measurements and reflectance IR microscopy with high predictive accuracy during blind testing. Training data are collected from atherosclerotic and normal rabbit aorta samples. These results demonstrate the potential of IR spectroscopy combined with multivariate classification strategies for the in-vitro identification of normal and atherosclerotic aorta tissue. The prospect for future in-vivo measurement concepts is also discussed.

A new method is described for obtaining a 3-D reconstruction of a bioluminescent light source distribution inside a living animal subject, from multispectral images of the surface light emission acquired on charge-coupled device (CCD) camera. The method uses the 3-D surface topography of the animal, which is obtained from a structured light illumination technique. The forward model of photon transport is based on the diffusion approximation in homogeneous tissue with a local planar boundary approximation for each mesh element, allowing rapid calculation of the forward Green's function kernel. Absorption and scattering properties of tissue are measured a priori as input to the algorithm. By using multispectral images, 3-D reconstructions of luminescent sources can be derived from images acquired from only a single view. As a demonstration, the reconstruction technique is applied to determine the location and brightness of a source embedded in a homogeneous phantom subject in the shape of a mouse. The technique is then evaluated with real mouse models in which calibrated sources are implanted at known locations within living tissue. Finally, reconstructions are demonstrated in a PC3M-luc (prostate tumor line) metastatic tumor model in nude mice.

Breast calcifications can be found in both benign and malignant lesions, and the composition of these calcifications can indicate the possible disease state. As current practices such as mammography and histopathology examine the morphology of the specimen, they cannot reliably distinguish between the two types of calcification, which frequently are the only mammographic features that indicate the presence of a cancerous lesion. Raman spectroscopy is an optical technique capable of obtaining biochemical information of a sample in situ. We demonstrate for the first time the noninvasive recovery of Raman spectra of calcified materials buried within a chicken breast tissue slab 16 mm thick, achieved using transmission Raman spectroscopy. The spectra of both calcium hydroxyapatite (HAP) and calcium oxalate monohydrate (COM) are obtained and chemically identified. The experimental geometry and gross insensitivity of the Raman signal to the depth of the calcified lesion makes the concept potentially well suited for probing human female breasts, in conjunction with existing mammography or ultrasound, to provide complementary data in the early diagnosis of breast cancer.

Raman spectroscopy is used to study low-wave-number (≤20 cm^-1) acoustic vibrations of the M13 phage. A well-defined Raman line is observed at around 8.5 cm^-1. The experimental results are compared with theoretical calculations based on an elastic continuum model and appropriate Raman selection rules derived from a bond polarizability model. The observed Raman mode is shown to belong to one of the Raman-active axial modes of the M13 phage protein coat. It is expected that the detection and characterization of this low-frequency vibrational mode can be used for applications in biomedical nanotechnology such as for monitoring the process of virus functionalization and self-assembly.

The nonlinear optical interference of two successively generated coherent anti-Stokes Raman scattering (CARS) signals from two different samples placed in series is demonstrated for the imaging performance, in which a collinear phase matching geometry is used. The relative phase of two CARS signals is controlled by a phase-shifting unit made of dispersive glass materials of which the thickness can be precisely varied. The clear interference fringes are observed as the thickness of the phase-shifting unit changes. The interference effect is then utilized to achieve a better quality CARS image of a biological tissue taken from a mouse skin. Placing the tissue in the second sample position and performing raster scans of the laser beams on it, we can acquire a CARS image of higher contrast compared to the normal image obtained without interferometric implementation.

Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes—laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-μm histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI_max), which includes collagen and elastin, increases with animal weight (R²=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R²=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI_max at R²=0.60).

The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenz[α]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells has short (0.29±0.03 ns) and long lifetime components (2.03±0.06 ns), attributed to free and protein-bound NADH, respectively. Low-grade precancers (mild to moderate dysplasia) and high-grade precancers (severe dysplasia and carcinoma in situ) are discriminated from normal tissues by their decreased protein-bound NADH lifetime (p<0.05). Inhibition of cellular glycolysis and oxidative phosphorylation in cell monolayers produces an increase and decrease, respectively, in the protein-bound NADH lifetime (p<0.05). Results indicate that the decrease in protein-bound NADH lifetime with dysplasia is due to a shift from oxidative phosphorylation to glycolysis, consistent with the predictions of neoplastic metabolism. We demonstrate that multiphoton FLIM is a powerful tool for the noninvasive characterization and detection of epithelial precancers in vivo.

The acousto-optic deflector (AOD) is highly preferred in laser scanning microscopy for its fast scanning ability and random-addressing capability. However, its application in two-photon microscopy is frustrated by the dispersion of the AOD, which results in beam distortion and pulse lengthening. We report the analysis of simultaneous compensation for the angular dispersion and temporal dispersion of the AOD by merely introducing a single dispersive element such as a prism or a grating. Besides serving as a scanner, the AOD is also a part of the compressor pair by integrating the dispersive nature of the AO interaction. This compensation principle is effective for both one-dimensional (1-D) AOD and two-dimensional (2-D) AOD scanning. Switching from a 1-D to a 2-D system requires proper optical alignment with the compensation element, but does not involve any new components. Analytical expressions are given to illustrate the working principle and to help with understanding the design of the system. Fluorescence images of beads and cells are shown to demonstrate the performance of two-photon microscopy when applying this compensated 2-D AOD as scanner.

Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2A^pro), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Förster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2A^pro released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.

Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660/710 nm] or IRDye® 800CW (ex/em: 785/830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye® 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.

Optical coherence tomography (OCT) is an imaging modality that enables assessment of tissue structural characteristics. Studies have indicated that OCT is a useful method to assess both blood vessel morphology and the response of a vessel to a deployed stent. We evaluated the ability of OCT to visualize the cellular lining of a tissue-engineered blood vessel mimic (BVM) and the response of this lining to a bare metal stent. We develop a side-firing endoscope that obtains intraluminal, longitudinal scans within the sterile bioreactor environment, enabling time-serial assessment. Seventeen BVMs are imaged with the endoscopic OCT system. The BVMs are then evaluated via fluorescence microscopy and/or standard histologic techniques. We determine that (1) the OCT endoscope can be repeatedly inserted without visible damage to the BVM cellular lining, (2) OCT provides a precise measure of cellular lining thickness with good correlation to measurements obtained from histological sections, and (3) OCT is capable of monitoring the accumulation of cellular material in response to a metallic stent. Our studies indicate that OCT is a useful technique for monitoring the BVM cellular lining, and that OCT may facilitate the use of BVMs for early stage device assessment.

We report the assessment of cerebral blood flow (CBF) changes with a wide-field laser Doppler imager based on a CCD camera detection scheme, in vivo, in mice. The setup enables the acquisition of data in minimally invasive conditions. In contrast with conventional laser Doppler velocimeters and imagers, the Doppler signature of moving scatterers is measured in the frequency domain, by detuning a heterodyne optical detection. The quadratic mean of the measured frequency shift is used as an indicator of CBF. We observe a significant variability of this indicator in an experiment designed to induce blood flow changes.

In optical Doppler measurements, the path length of the light is unknown. To facilitate quantitative measurements, we develop a phase-modulated Mach-Zehnder interferometer with separate fibers for illumination and detection. With this setup, path-length-resolved dynamic light scattering measurements of multiple scattered light in static and dynamic turbid media are performed. Optical path length distributions spanning a range from 0 to 11 mm are measured from the area under the phase modulation peak around the modulation frequency in the power spectrum. A Doppler-broadened phase modulation interference peak is observed that shows an increase in the average Doppler shift with optical path length, independent of absorption. Validation of the estimated path length distributions is done by measuring their deformation for increasing absorption and comparing these observations with predictions based on Lambert-Beer's law.

(Partial Abstract) Confocal microscopy can provide real-time, 2-D and 3-D images of the cellular morphology and tissue architecture features that pathologists use to detect precancerous lesions without the need for tissue removal, sectioning, and staining. The utility of 3-D confocal image stacks of epithelial tissue for detecting dysplasia has not yet been explored. We aim to extract morphometry and tissue architecture information from 2-D confocal reflectance images and 3-D image stacks from fresh, unstained cervical biopsies and compare their potential for detecting dysplasia. Nine biopsies are obtained from eight patients; confocal images are acquired pre- and postacetic acid at multiple epithelial depths in 1.5 μm-intervals. Postacetic acid images are processed to segment cell nuclei; after segmentation, 2-D images taken at 50 μm below the tissue surface, and the entire 3-D image stacks are processed to extract morphological and architectural features. Data are analyzed to determine which features gave the best separation between normal and high-grade cervical precancer. Most significant differences are obtained from parameters extracted from the 3-D image stacks. However, in all cases where the 2-D features were multiplicatively scaled by the depth of acquisition divided by the epithelial thickness or scaled by the scattering coefficient, the significance level is equal to or greater than the comparable feature extracted from the 3-D image stacks. A linear discriminant function previously developed to separate 19 samples of normal tissue and high-grade cervical precancer based on the nuclear-to-cytoplasm (N/C) ratio and epithelial scattering coefficient is prospectively applied to the nine biopsies examined to determine the accuracy with which it could separate normal tissue from cervical intra epithelial neoplasia (CIN) 2/3. For the entire data set of 28 biopsies, a sensitivity and specificity of 100% is produced using this discriminant function;

In this study, 532-nm picosecond and 800-nm femtosecond lasers are used in combination with fluorescently labeled tubulin to further elucidate microtubule depolymerization and the effect lasers may have on the resulting depolymerization. Depolymerization rates of targeted single microtubules are dependent on location with respect to the nucleus. Microtubules located near the nucleus exhibit a significantly faster depolymerization rate when compared to microtubule depolymerization rates near the periphery of the cell. Microtubules cut with the femtosecond laser depolymerize at a slower rate than unirradiated controls (p=0.002), whereas those cut with the picosecond laser depolymerize at the same rate as unirradiated controls (p=0.704). Our results demonstrate the ability of both the picosecond and femtosecond lasers to cut individual microtubules. The differences between the two ablation results are discussed.

The retinal injury threshold dose for laser exposure varies as a function of the irradiated area on the retina. Zuclich reported thresholds for laser-induced retinal injury from 532 nm, nanosecond-duration laser exposures that varied as the square of the diameter of the irradiated area on the retina. We report data for 0.1-s-duration retinal exposures to 514-nm, argon laser irradiation. Thresholds for macular injury at 24 h are 1.05, 1.40, 1.77, 3.58, 8.60, and 18.6 mJ for retinal exposures at irradiance diameters of 20, 69, 136, 281, 562, and 1081 µm, respectively. These thresholds vary as the diameter of the irradiated retinal area. The relationship between the retinal injury threshold and retinal irradiance diameter is a function of the exposure duration. The 0.1-s-duration data of this experiment and the nanosecond-duration data of Zuclich show that the ED50 (50% effective dose) for exposure to a highly collimated beam does not decrease relative to the value obtained for a retinal irradiance diameter of 100 μm. These results can form the basis to improve current laser safety guidelines in the nanosecond-duration regime. These results are relevant for ophthalmic devices incorporating both wavefront correction and retinal exposure to a collimated laser.

An optical fiber sensor for measuring the pH in interstitial fluid is described. Microdialysis is the approach followed for extracting the sample from the subcutaneous adipose tissue. The interstitial fluid drawn flows through a microfluidic circuit formed by a microdialysis catheter in series with a pH glass capillary. The pH indicator (phenol red) is covalently immobilized on the internal wall of the glass capillary. An optoelectronic unit that makes use of LEDs and photodetectors is connected to the sensing capillary by means of optical fibers. Optical fibers are used to connect the interrogating unit to the sensing capillary. A resolution of 0.03 pH units and an accuracy of 0.07 pH units are obtained. Preliminary in vivo tests are carried out in pigs with altered respiratory function.

Application of a fiber optic biosensor (FOB) to the real-time investigation of the interaction kinetics between FITC-conjugated monoclonal sheep anti-human C-reactive protein (CRP) antibody and CRP isoforms on the surface of optical fiber is described. Recently, both the native pentameric CRP (pCRP), an acute phase protein belonging to pentraxin family, and an isoform of pCRP, modified CRP (mCRP), have been suggested to have proinflammation effects on vascular cells in acute myocardial infarction (AMI). In current studies, we generate mCRP from pCRP, and use several methods including fluorescence spectral properties, circular dichroism, analytical ultracentrifuge, and Western blotting to demonstrate their differences in physical and chemical properties as well as the purity of pCRP and mCRP. In addition, we design and implement an FOB to study the real-time qualitative and quantitative biomolecular recognition of CRP isoforms. Specifically, the association and dissociation rate constants of the reaction between FITC-conjugated monoclonal sheep anti-human CRP antibody and the pCRP and mCRP are determined. The feasibility of our current approach to measure the association and dissociation rate constants of the reaction between tested CRP isoforms was successfully demonstrated.

An experimental technique to reveal the effects of dental polymer contraction is established to choose the most appropriate polymerization technique. Tooth deformation following a dental filling polymerization is analyzed using double-exposure holographic interferometry. A caries-free, extracted human molar is mounted in dental gypsum and different cavity preparations and fillings are made on the same tooth. Dental composite fillings are polymerized by an LED light source especially designed for this purpose. Holographic interferograms are made for occlusal (class I), occlusomesial (class II), and mesioocclusodistal (class II MOD) cavities and fillings. Maximum intercuspal deformation ranges from 2 μm for the class I cavity to 14 μm for the MOD class cavity. A finite element method (FEM) is used to calculate von Mises stress on a simplified tooth model, based on experimental results. The stress varies between 50 and 100 MPa, depending on the cavity type.

3-D pulsed digital holography is a noninvasive optical method used to measure the depth position of breast tumor tissue immersed in a semisolid gel model. A master gel without inhomogeneities is set to resonate at an 810 Hz frequency; then, an identically prepared gel with an inhomogeneity is interrogated with the same resonant frequency in the original setup. Comparatively, and using only an out-of-plane sensitive setup, gel surface displacement can be measured, evidencing an internal inhomogeneity. However, the depth position cannot be measured accurately, since the out-of-plane component has the contribution of in-plane surface displacements. With the information gathered, three sensitivity vectors can be obtained to separate contributions from x, y, and z vibration displacement components, individual displacement maps for the three orthogonal axes can be built, and the inhomogeneity's depth position can be accurately measured. Then, the displacement normal to the gel surface is used to find the depth profile and its cross section. Results from the optical data obtained are compared and correlated to the inhomogeneity's physically measured position. Depth position is found with an error smaller than 1%. The inhomogeneity and its position within the gel can be accurately found, making the method a promising noninvasive alternative to study mammary tumors.

A coherent light beam is used to interrogate the focal region within a tissue-mimicking phantom insonified by an ultrasound transducer. The ultrasound-tagged photons exiting from the object carry with them information on local optical path length fluctuations caused by refractive index variations and medium vibration. Through estimation of the force distribution in the focal region of the ultrasound transducer, and solving the forward elastography problem for amplitude of vibration of tissue particles, we observe that the amplitude is directed along the axis of the transducer. It is shown that the focal region interrogated by photons launched along the transducer axis carries phase fluctuations owing to both refractive index variations and particle vibration, whereas the photons launched perpendicular to the transducer axis carry phase fluctuations arising mainly from the refractive index variations, with only smaller contribution from vibration of particles. Monte-Carlo simulations and experiments done on tissue-mimicking phantoms prove that as the storage modulus of the phantom is increased, the detected modulation depth in autocorrelation is reduced, significantly for axial photons and only marginally for the transverse-directed photons. It is observed that the depth of modulation is reduced to a significantly lower and constant value as the storage modulus of the medium is increased. This constant value is found to be the same for both axial and transverse optical interrogation. This proves that the residual modulation depth is owing to refractive index fluctuations alone, which can be subtracted from the overall measured modulation depth, paving the way for a possible quantitative reconstruction of storage modulus. Moreover, since the transverse-directed photons are not significantly affected by storage modulus variations, for a quantitatively accurate read-out of absorption coefficient variation, the interrogating light should be perpendicular to the focusing ultrasound transducer axis.

Glaucoma represents the second leading cause of blindness worldwide. While both age and intraocular pressure (IOP) are well-recognized risk factors for this disease, the underlying pathologic process involves the accelerated death of retinal ganglion cells (RGCs) that is associated with progressive loss of vision. The loss of RGCs has been postulated to occur primarily by injury to axons in the optic nerve head (ONH) due to its anatomic features and the mechanical vulnerability of the lamina cribrosa, the specialized ONH zone comprised of collagen beams that define the channels or pores through which axon bundles exit the eye. Recent advances in multiphoton microscopy using femtosecond lasers that generate second harmonic (SH) signals from collagen allows for direct optical imaging of the lamina cribrosa. We assess the application of SH generated microscopy (SHG) to the study of the ONH, and test the general hypothesis that increasing intraocular pressure in the same eye results in the movement of ONH collagen beams leading to distortion of the lamina cribrosa channels and compression of the axon bundles.

Fluorescence microscopy has become the method of choice in the majority of life-science applications. We describe development and use of mirror slides to significantly enhance the fluorescence signal using standard air microscope objectives. This technique offers sufficient gain to achieve high-sensitivity imaging, together with wide field of observation and large depth of focus, two major breakthroughs for routine analysis and high-throughput screening applications on cells and tissue samples.