Suppression of the conjugate signal for broadband computed imaging via synthetic phase modulation.

We present synthetic-phase-modulated interferometric synthetic aperture microscopy (SPM-ISAM), a method to perform 3D object reconstructions from data acquired with confocal broadband interferometric microscopy (BIM) that reconstructs images virtually free of coherent and depth-dependent defocus artifacts. This is achieved by implementing a sinusoidal SPM method in combination with an ISAM reconstruction algorithm that uses relatively low-modulation frequencies compared with acquisition frequencies. A theoretical framework and numerical results are provided here.

Optical sectioning enhancement using higher-order moment signals in random speckle-structured illumination microscopy.

The optical sectioning capability of structured illumination using random speckle patterns is shown by simulations to improve when cumulants beyond the traditional second order are used as the image-forming signals. The improvement scales with the cumulant order, asymptotically approaching confocal performance. As actual experiments operate with finite-size sample estimators instead of true cumulants, purely statistical (nonoptical) effects can result in nonideal behavior. We analyze the finite ensemble effects along with the experimental effects of detector dynamic range through Monte Carlo simulations. Despite these real-world factors, we show when the third-order-derived signal can demonstrate improved sectioning at good signal-to-noise levels set by finite-sample effects.

Temperatures of the Ocular Surface, Lid, and Periorbital Regions of Sjögren's, Evaporative, and Aqueous-Deficient Dry Eyes Relative to Normals.

PURPOSE: To compare the temperatures of the ocular surface, eyelid, and periorbital skin in normal eyes with Sjögren's syndrome (SS) eyes, evaporative dry eyes (EDE), and aqueous deficient dry eyes (ADDE). METHODS: 10 eyes were analyzed in each age-matched group (normal, SS, EDE, and ADDE). A noninvasive infrared thermal camera captured two-dimensional images in three regions of interest (ROI) in each of three areas: the ocular surface, the upper eyelid, and the periorbital skin within a controlled environmental chamber. Mean temperatures in each ROI were calculated from the videos. Ocular surface time-segmented cooling rates were calculated over a 5-s blink interval. RESULTS: Relative to normal eyes, dry eyes had lower initial central OSTs (SS -0.71°C, EDE -0.55°C, ADDE -0.95°C, KW P<.0001) and lower central upper lid temperatures (SS -0.24°C, ADDE -0.51°C, and EDE -0.54°C, KW P<.0001). ADDE eyes had the lowest initial central OST (P<.0001), while EDE eyes had the lowest central lid temperature and lower periorbital temperatures (P<.0001). Over the 5-s interblink interval, the greatest rate of temperature loss occurred following eyelid opening, but varied by group (normals -0.52, SS -0.73, EDE -0.63, and ADDE -0.75°C/s). The ADDE group also had the most substantial heat loss over the 5-s interblink interval (-0.97°C). CONCLUSIONS: Differences in OST may be related to thermal differences in lids and periorbita along with an altered tear film. Thermography of the ocular surface, lids, and surrounding tissues may help to differentiate between different etiologies of dry eye.

Red Light Modulates Ultraviolet-Induced Gene Expression in the Epidermis of Hairless Mice.

OBJECTIVE: The purpose of this study was to investigate whether low-level light therapy (LLLT) was capable of modulating expression of ultraviolet (UV) light-responsive genes in vivo. MATERIALS AND METHODS: The effects of 670 nm light-emitting diode (LED) array irradiation were investigated in a hairless SHK-1 mouse epidermis model. Mice were given a single dose of UVA/UVB light, or three doses of red light (670 nm @ 8 mW/cm(2) x 312 sec, 2.5 J/cm(2) per session) spread over 24 h along with combinations of pre- and post-UV treatment with red light. Levels of 14 UV-responsive mRNAs were quantified 24 h after UV irradiation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: The transcription of mRNAs encoding for cluster of differentiation molecule 11b (CD11b) (p < 0.05) and interferon (IFN)-γ (p < 0.012) increased after irradiation with red light alone, whereas expression level of cyclooxygenase (COX)-2 (p < 0.02) was downregulated. Genes unresponsive to UV did not change their expression levels after exposure to red light either. Pretreatment with red light significantly modified response of Fos to UV exposure (p < 0.01). A synergy of UV and post-treatment with red light in reducing the transcription levels of CD11b (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.05) was observed. CONCLUSIONS: This is an initial observation that in mouse red light LLLT more often than not causes opposite gene expression changes or reduces those caused by moderate UVA-UVB irradiation.

Thermal analysis of dry eye subjects and the thermal impulse perturbation model of ocular surface.

In this study, we explore the usage of ocular surface temperature (OST) decay patterns to distinguished between dry eye patients with aqueous deficient dry eye (ADDE) and meibomian gland dysfunction (MGD). The OST profiles of 20 dry eye subjects were measured by a long-wave infrared thermal camera in a standardized environment (24 °C, and relative humidity (RH) 40%). The subjects were instructed to blink every 5 s after 20 âˆ¼ 25 min acclimation. Exponential decay curves were fit to the average temperature within a region of the central cornea. We find the MGD subjects have both a higher initial temperature (p < 0.022) and a higher asymptotic temperature (p < 0.007) than the ADDE subjects. We hypothesize the temperature difference among the subpopulations is due to tear volume and heat transfer mechanisms. To study the validity of our claim, we develop a mathematical model, referred to as the thermal impulse perturbation (TIP) model. We conclude that long-wave-infrared thermal imaging is a plausible tool in assisting with the classification of dry eye patient.

Optical segmentation of unprocessed breast tissue for margin assessment.

Visual and tactual examination of unprocessed breast specimens is the standard for intraoperative surgical margin assessment in the United States. However, this procedure does not provide surgeons or pathologists with microscopic views of the tissue, which makes it difficult to accurately assess margin status or the extent of the disease, especially in non-palpable cases. We use a combination of spectral and polarization macroscopic imaging to optically segment the adipose and collagen tissues thus highlighting regions suspected of containing epithelium in order to facilitate optical microscopy techniques. A small study on five lumpectomy and mastectomy samples showed a sensitivity of 70% ± 20% and specificity of 50% ± 10% for adipose segmentation and a sensitivity of 50% ± 20% and specificity of 50% ± 20% for collagen segmentation. This sensitivity and specificity are sufficient for providing morphological information to the pathologist in order to guide microscopic examination of regions likely to be of clinical significance.

Design and validation of two optical beacons for guidewire localization in breast-conserving surgery.

Stereotactically placed guidewires are used for indicating the location of a nonpalpable carcinoma in breast-conserving surgery. Pathologists use the end of the embedded guidewire to guide sectioning during intraoperative margin assessment, but they do not currently have a tool to indicate the location of the guidewire end for informed sectioning. We present analysis and experimental testing of two optical methods for localizing the end of an embedded fiber-optic guidewire: the first uses irradiance emitted from the fiber to indicate the location of the guidewire end, while the second system uses the fiber optic to create a photoacoustic pulse for localization. Both systems locate the end of the guidewire within ±5 mm, which ensures that the lesion of interest is bisected during sectioning. The accuracy of the irradiance-based beacon is influenced by standard margin paints, so the photoacoustic beacon proved more useful under current tissue-handling protocols.

A coherent model for turbid imaging with confocal microscopy.

We present an engineering model of coherent imaging within a turbid volume, such as human tissues, with a confocal microscope. The model is built to analyze the statistical effect of aberrations and multiply scattered light on the resulting image. Numerical modeling of theory is compared with experimental results. We describe the construction of a stable phantom that represents the statistical effect of object turbidity on the image recorded. The model and phantom can serve as basis for system optimization in turbid imaging.

Effect of airflow exposure on the tear meniscus.

Purpose. To compare the effect of airflow exposure on the tear meniscus and blink frequency in normal and evaporative dry eye subjects. Methods. In 9 normal subjects and 9 short tear breakup time (SBUT) dry eye subjects, lower tear meniscus height (TMH) and area (TMA) and blink frequency were measured with anterior segment optical coherence tomography (OCT) before and after 5 minutes of airflow exposure (1.5 ± 0.5 m/s). Results. In SBUT dry eyes, both TMH and TMA decreased significantly (P = 0.027, P = 0.027) with a significant increase of blink frequency after airflow exposure, while significant increase in TMA was found in normal eyes. Conclusion. Measurement of the tear meniscus with anterior segment OCT seems to be useful as a noninvasive and objective method for evaluating the effect of airflow on tear film.

Integrated multimodal metrology for objective and noninvasive tear evaluation.

The clinical tests used to assess tear film and diagnose dry eye are invasive and produce results that are different from natural tear characteristics. There is a need to objectively and noninvasively assess tear parameters under controlled environmental circumstances to refine dry eye diagnosis and therapy. We have developed multimodal tear imaging systems integrated in a chamber in which individual environmental factors can be precisely varied to investigate their impacts on tear parameters. With the custom-built high-resolution wavefront sensor combined with placido disc, it is possible to objectively detect two-dimensional tear breakups in real time and evaluate its impact on visual quality. Micrometer ultra-high resolution optical coherence tomography (OCT) enables us to quantify thickness and volume of the tear over the cornea and tear menisci. The ocular surface imaging ellipsometer uses polarized illumination from which both the lipid refractive index and thickness can be measured at a very high resolution. Using an enhanced thermal camera, we measure the ocular surface temperature noninvasively, which makes it possible to study spatial and temporal changes in tear evaporation. The multimodal deployment of these four components in the controlled chamber will assist in better differentiating the various clinical dry eye entities and will lead to the development of specific dry eye treatments.

Noninvasive, objective, multimodal tear dynamics evaluation of 5 over-the-counter tear drops in a randomized controlled trial.

PURPOSE: To assess the ability of a noninvasive, objective, multimodal system to compare the efficacy and optical quality of 4 different groups of 5 over-the-counter tear drops. METHODS: Wavefront sensing and optical coherence tomography (OCT) were used to objectively assess visual quality and tear volume, respectively, after the over-the-counter tear drops [2 polyethylene glycol (PEG)-400, 1 carboxymethyl cellulose (CMC), 1 polyvinyl alcohol/PEG-400 (PVA), and 1 glycerine/polysorbate-80] were administered to 23 patients with aqueous-deficient dry eye in a randomized, controlled, double-masked trial. Measurements at each of the 5 visits were taken from both eyes at baseline and 5, 20, and 40 minutes after drop instillation. RESULTS: At 5 minutes after drop instillation, the 2 PEG drops showed significant worsening of visual quality (-128.87%, P = 0.001) compared with CMC, PVA, and glycerine drops. Tear volume was elevated significantly with PEG and CMC drops at 5 minutes (+33.2%, P = 0.002; +29.9%, P < 0.001) compared with the other groups. Subjects with a Schirmer score of less than 5 mm at 5 minutes showed improvement in visual quality with PEG drops, whereas those with a fluorescein staining score of 1 to 2 showed improvement with both PEG and CMC drops. A trend toward decreasing visual quality with increasing tear meniscus was noted. CONCLUSIONS: Noninvasive multimodal imaging has the potential to evaluate tear dynamics and the efficacy of artificial tears objectively. Certain artificial tear components and the temporary increase in tear volume after instillation of artificial tears may contribute to transient reduction of visual quality.

Micrometer axial resolution OCT for corneal imaging.

An optical coherence tomography (OCT) for high axial resolution corneal imaging is presented. The system uses 375 nm bandwidth (625 to 1000 nm) from a broadband supercontinuum light source. The system was developed in free space to minimize image quality degradation due to dispersion. A custom-designed spectrometer based on a Czerny Turner configuration was implemented to achieve an imaging depth of 1 mm. Experimentally measured axial resolution was 1.1 µm in corneal tissue and had a good agreement with the theoretically calculated resolution from the envelope of the spectral interference fringes. In vivo imaging was carried out and thin corneal layers such as the tear film and the Bowman's layer were quantified in normal, keratoconus, and contact lens wearing eyes, indicating the system's suitability for several ophthalmic applications.

Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin.

Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level.

Simultaneous measurement of tear film dynamics using wavefront sensor and optical coherence tomography.

PURPOSE. To investigate tear film dynamics using simultaneous measurements of ocular aberrations and lower tear meniscus. METHODS. Simultaneous measurements of wavefront aberration and lower tear meniscus were performed for 11 normal eyes and 7 eyes with short tear film break-up time (SBUT) dry eye, with a tear film break-up time shorter than 5 seconds, using a wavefront sensor and an anterior segment optical coherence tomography (OCT). During the measurement, the subjects were instructed to blink every 6 seconds for a total of 30 seconds. From the measured aberration, root mean square (RMS) wavefront error and volume modulation transfer function (vMTF) induced by changes in tear film dynamics were calculated for a 5-mm pupil. Lower tear meniscus height (TMH) and area (TMA) were estimated from the cross-sectional OCT images of lower tear meniscus. RESULTS. There was a positive correlation between RMS and tear meniscus dimensions and a negative correlation between vMTF and tear meniscus in both groups. There were moderate negative correlations between the postblink initial RMS change and baseline TMH (R = -0.61) and TMA (R = -0.54) in SBUT dry eyes that were stronger than in normal eyes (R = -0.37, R = -0.38). CONCLUSIONS. Tear meniscus dimensions increase with RMS over time, and tear quantity before blink has a significant role in maintaining initial optical integrity, especially in SBUT dry eye. Simultaneous measurement of optical quality and tear meniscus has the potential to improve understanding of tear stability in normal eyes and dry eyes.

Confocal microscopy of unfixed breast needle core biopsies: a comparison to fixed and stained sections.

BACKGROUND: Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation. METHODS: Needle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology. RESULTS: The pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue. CONCLUSION: CSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and were comparable to standard histologic sections of the same tissue. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images. Additional studies are needed to 1.) establish correlation of the confocal and traditional histologic images for the various diseases of the breast; 2.) validate diagnostic use of CSLM and; 3.) further define features of borderline lesions such as well-differentiated ductal CIS vs. atypical hyperplasia.

Modulation transfer function measurement of scanning reflectance microscopes.

Real-time medical imaging systems such as reflectance confocal microscopes and optical coherence microscopes are being tested in multiple-patient and multiple-center clinical trials. The modulation transfer function (MTF) of these systems at any given time influences the image information content and can affect the interpretation of the images. MTF is difficult to measure in real-time scanning systems when imaging at the Nyquist limit. We describe a measurement technique similar to the electronic imaging resolution standards ISO-12233 (electronic cameras) that can be applied to scanned spot imaging systems with asynchronous pixel clocks. This technique requires the acquisition of a single image of a reflective stripe object. An asynchronous pixel clock induces subpixel jitter in the edge location. The jitter is removed using a Fourier method, and an oversampled edge response function is calculated using algorithms developed in MATLAB. This technique provides fast, simple to use, and repeatable full-width at half maximum lateral resolution and MTF measurements based on only one test image. We present the results for reflectance confocal microscopes operating at 0.9 numerical aperture.

Confocal reflectance theta line scanning microscope for imaging human skin in vivo.

A confocal reflectance theta line scanning microscope demonstrates imaging of nuclear and cellular detail in human epidermis in vivo. Experimentally measured line-spread functions determine the instrumental optical section thickness to be 1.7 +/- 0.1 microm and the lateral resolution to be 1.0 +/- 0.1 microm. Within human dermis (through full-thickness epidermis), the measured section thickness is 9.2 +/- 1.7 microm and the lateral resolution is 1.7 +/- 0.1 microm. An illumination line is scanned directly in the pupil of the objective lens, and the backscattered descanned light is detected with a linear array, such that the theta line scanner consists of only seven optical components.

Detectability of contrast agents for confocal reflectance imaging of skin and microcirculation.

Confocal reflectance microscopy of skin and other tissues in vivo is currently limited to imaging at the cellular, nuclear and general architectural levels due to the lack of microstructure-specific contrast. Morphologic and functional imaging at specific organelle and microstructure levels may require the use of exogenous contrast agents in small (nontoxic) concentrations, from which weakly backscattered light must be detected in real time. We report an analysis based on Mie theory to predict detectability, in terms of signal-to-background and signal-to-noise ratios, of reflectance contrast agents within skin and microcirculation. The analysis was experimentally verified by detectability of (a) intravenously injected polystyrene microspheres that enhance the contrast of dermal microcirculation in Sprague-Dawley rats, and (b) acetic acid-induced compaction of chromatin that enhances nuclear morphology in normal and cancerous human skin. Such analyses and experiments provide a quantitative basis for developing the opto-biochemical properties and use of contrast agents and for designing confocal instrumentation to enable real-time detectability in vivo.

Noninvasive imaging, treatment, and microscopic confirmation of clearance of basal cell carcinoma.

BACKGROUND: The diagnosis of basal cell carcinoma (BCC) is generally established by skin biopsy followed by tissue preparation and microscopic analysis. Treatment of BCC is often accomplished by surgical excision. OBJECTIVE: To confirm the presence of BCC with a noninvasive imaging technique, to treat the patient with a topical immune response modifier, and to confirm the clearance of BCC noninvasively. METHODS: Confocal microscopy (CM) is a noninvasive technique for real-time imaging of skin in vivo. Imiquimod, an immune response modifier, is applied topically by the patient to the skin lesion. RESULTS: The presence of BCC was confirmed with CM. Posttreatment CM imaging confirmed the clearance of BCC from the entire treatment field. Both the pretreatment and the posttreatment CM findings were confirmed by invasive biopsy. CONCLUSION: The ability to use CM to image in real time without discomfort to the patient makes it a powerful tool to assist in the diagnosis of skin disease.