Limited Imaging Depth Research Articles

Reflectance confocal microscopy for the in vivo detection of Treponema pallidum in skin lesions of secondary syphilis

Secondary syphilis is a diagnostic challenge that relies on microscopic and laboratory tests. We sought to assess the usefulness of in vivo reflectance confocal microscopy (RCM) to detect Treponema pallidum in lesions suggestive of secondary syphilis. Macular and papular skin lesions from 3 patients clinically suggestive of secondary syphilis were imaged by RCM and confirmed by skin punch biopsy. In all lesions RCM demonstrated elongated small bright particles with a spiral shape intermingled with the keratinocytes. These features corresponded with immunohistochemical findings that revealed several spirochetes infiltrating the epidermis. Unlike immunohistochemistry, RCM did not visualize T pallidum in the dermis and vascular walls because of limited imaging depth. The specificity and sensitivity of this technique need to be assessed. RCM may be an effective diagnostic tool for in vivo real-time imaging of T pallidum in skin lesions of secondary syphilis, and seems to correlate well with immunohistochemistry.

Analysis Of Multiphoton Imaging Of Thick Biological Scattering Samples

Non linear optical scanning microscopy has became a useful tool for tissue imaging. Biological tissues are highly scattering media and this lead to an exponentially attenuation of the excitation intensity as the light moves into the sample. While performing imaging of biological scattering tissues in non linear excitation regime, the localization of the maximum 2PE intensity was found to shift closer to the surface [1] and the 2PE imaging depth limit appears strongly limited by near surface fluorescence [2]. In this work we computed the illumination and the photobleaching distribution [3] for different scattering coefficients in order to characterize the effects induced by scattering. An experimental test has been carried out by imaging, with medium numerical aperture objective (N.A.=0.8), thick scattering fluorescent immobile sample (polyelectrolyte gel). Results confirm that in this conditions no photobleaching effects due to scattering occur close to the surface.[1] J. P. Ying et al, Appl. Opt. 38, (1999).[2] P. Theer J. Opt. Soc. Am. A. 23, (2006).[3] D.Mazza et al, Appl. Opt. 46 (2007).

Virtual histology of the human heart using optical coherence tomography

Optical coherence tomography (OCT) allows for the visualization of micron-scale structures within nontransparent biological tissues. For the first time, we demonstrate the use of OCT in identifying components of the cardiac conduction system and other structures in the explanted human heart. Reconstructions of cardiac structures up to 2 mm below the tissue surface were achieved and validated with Masson Trichrome histology in atrial, ventricular, sinoatrial nodal, and atrioventricular nodal preparations. The high spatial resolution of OCT provides visualization of cardiac fibers within the myocardium, as well as elements of the cardiac conduction system; however, a limiting factor remains its depth penetration, demonstrated to be approximately 2 mm in cardiac tissues. Despite its currently limited imaging depth, the use of OCT to identify the structural determinants of both normal and abnormal function in the intact human heart is critical in its development as a potential aid to intracardiac arrhythmia diagnosis and therapy.

Noninvasive photoacoustic imaging of the thoracic cavity and the kidney in small and large animals

The internal organs of rats and rabbits were clearly imaged noninvasively using a deeply penetrating reflection-mode photoacoustic imaging system. This imaging system had previously been found to provide an imaging depth limit of approximately 38 mm. In the thoracic cavity, major blood vessels connecting to the heart were imaged, and the right atrium was imaged as deeply as approximately 8 mm. In the abdominal cavities, the kidney and vena cava inferior were also imaged in situ. The vena cava inferior approximately 13.7 mm deep was imaged. The kidney of a New Zealand white rabbit was also imaged. This study shows the deep internal organ imaging capability of the system in animals. This technology can potentially be used to study tumors in internal organs, and be adapted to clinical diagnosis.

Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics

This study describes the morphologic features of human non-melanoma skin cancer obtained using multiphoton laser scanning microscopy (MPLSM) on freshly excised specimens from 14 patients. Optical sectioning parallel to the tissue surface was performed, resulting in en face autofluorescence images of the epidermis and upper dermis, reaching tissue depths of 135 microm. The microscopy was carried out ex vivo using a femtosecond pulsed laser at 780 nm and a x 40/0.8 objective. The autofluorescence was detected in the range of 450-530 nm. Traditional histopathological criteria such as bowenoid dysplasia, multinucleated cells, or hyperkeratosis in squamous cell carcinoma in situ (SCCIS) (five specimens), and peripheral palisading of tumor cells in superficial basal cell carcinoma (SBCC) (six specimens) were clearly discerned. The morphologic features differed significantly between these lesions and perilesional skin. However, characteristic tumor aggregates were found in only one of the three investigated nodular basal cell carcinomas (NBCCs) due to limited imaging depth. In addition, speckled perinuclear fluorescence was observed in both lesions and normal perilesional skin. In conclusion, MPLSM could potentially be applied for non-invasive diagnostics of SCCIS and SBCC, whereas the ability to characterize NBCC is unclear at this point.

High-resolution imaging diagnosis and staging of bladder cancer: comparison between optical coherence tomography and high-frequency ultrasound

A comparative study between 1.3-microm optical coherence tomography (OCT) and 40-MHz high-frequency ultrasound (HFUS) is presented to enhance imaging of bladder cancers ex vivo. A standard rat bladder cancer model in which transitional cell carcinoma (TCC) was induced by intravesical instillation of AY-27 cells was followed independently with both OCT and HFUS, and the image identifications were compared to histological confirmations. Results indicate that both OCT and HFUS were able to delineate the morphology of rat bladder [e.g., the urothelium (low backscattering/echo) and the underlying lamina propria and muscularis (high backscattering/echo]. OCT differentiated inflammatory lesions (e.g., edema, infiltrates and vasodilatation in lamina propria, hyperplasia) and TCC based on characterization of urothelial thickening and enhanced backscattering or heterogeneity (e.g., papillary features), which HFUS failed due to insufficient image resolution and contrast. On the other hand, HFUS was able to stage large T2 tumors that OCT failed due to limited imaging depth. The results suggest that multimodality cystoscopy combining OCT and HFUS may have the potential to enhance the diagnosis and staging of bladder cancers and to guide tumor resection, in which both high resolution (approximately 10 microm) and enhanced penetration (> 3mm) are desirable.

In vivo reflectance confocal microscopy of mycosis fungoides: A preliminary study

Mycosis fungoides (MF) is a diagnostic challenge, frequently needing multiple and sequential biopsies to establish the diagnosis. Our aim was to evaluate lesions suggestive of MF using in vivo reflectance confocal microscopy (RCM) and to correlate confocal features with histopathologic findings. A total of 8 lesions from 7 patients either with a history of biopsy-proven MF or with lesions clinically suggestive of MF were imaged with RCM followed by a skin punch biopsy. These 8 lesions were confirmed to be MF by histopathology: patch type (n = 3), plaque type (n = 4), and tumor type (n = 1). Under RCM, epidermal findings in patch lesions were subtle, as on histopathology, while the most prominent changes were observed in plaque type MF. At the level of the epidermis, weakly refractile oval to round structures within the spinous layer were observed in all MF lesions, but were difficult to distinguish from surrounding keratinocytes; these structures corresponded to epidermotropic lymphocytes on histopathology. In plaque-type lesions, vesicle-like dark spaces filled with collections of monomorphous weakly refractile oval to round cells were clearly elucidated by RCM; these structures corresponded to Pautrier's microabscesses on histopathology. RCM was also able to demonstrate spongiosis in the MF lesions, with findings of epidermal architectural disarray, areas with thickened and blurred intercellular demarcations, and epidermal cells with elongated nuclei. At the dermoepidermal junction, the basal cells surrounding the dermal papillae appeared as only faintly refractile rings on RCM. This feature corresponded with histopathologic findings of basal layer infiltration by tumor cells with permeation of rete ridges, thus, obscuring the dermoepidermal interface. Examination of the dermis under RCM for all the MF lesions showed weakly refractile structures, but was limited by loss of detail and contrast below the dermoepidermal junction. Because of limited imaging depth, RCM did not visualize dermal infiltration by tumor cells in tumor-type MF. Epidermotropic lymphocytes appeared weakly refractile under RCM and were difficult to distinguish from surrounding keratinocytes as a result of minimal difference in contrast. Other limitations on RCM include some similarity in findings with spongiotic and lichenoid dermatitides, and an inability to distinguish specific cell types. Moreover, this study did not address the inherent heterogeneity of MF lesions, but was primarily focused on correlating RCM and hematoxylin-eosin histopathology of the included cases. Features correlating well to histopathology are observed on RCM of MF lesions; however, the specificity of these findings needs to be assessed.

On the fundamental imaging-depth limit in two-photon microscopy

We have analyzed how the maximal imaging depth of two-photon microscopy in scattering samples depends on properties of the sample and the imaging system. We find that the imaging depth increases with increasing numerical aperture and staining inhomogeneity and with decreasing excitation-pulse duration and scattering anisotropy factor, but is ultimately limited by near-surface fluorescence with slight improvements possible using special detection strategies.

Interstitial Doppler optical coherence tomography

Doppler optical coherence tomography (OCT) can image tissue structure and blood flow at micrometer-scale resolution but has limited imaging depth. We report a novel, linear-scanning, needle-based Doppler OCT system using angle-polished gradient-index or ball-lensed fibers. A prototype system with a 19-guage (diameter of approximately 0.9 mm) echogenic needle is constructed and demonstrates in vivo imaging of bidirectional blood flow in rat leg and abdominal cavity. To our knowledge, this is the first demonstration of Doppler OCT through a needle probe in interstitial applications to visualize deeply situated microcirculation.

Adaptive ranging for optical coherence tomography

At present, optical coherence tomography systems have a limited imaging depth or axial scan range, making diagnosis of large diameter arterial vessels and hollow organs difficult. Adaptive ranging is a feedback technique where image data is utilized to adjust the coherence gate offset and range. In this paper, we demonstrate an adaptive optical coherence tomography system with a 7.0 mm range. By matching the imaging depth to the approximately 1.5 mm penetration depth in tissue, a 3 dB sensitivity improvement over conventional imaging systems with a 3.0 mm imaging depth was realized.

Simulation-based investigation of partially parallel imaging with a linear array at high accelerations.

Partially parallel imaging strategies such as SMASH, SENSE, and PILS rely on the sensitivity distribution of phased array RF coils to reduce MRI imaging time. Using an N-element phased array, these techniques allow maximum accelerations, L, such that L < or = N, with acceleration defined as the factor by which scan time is reduced in comparison to traditional, fully gradient encoded acquisitions. As N increases in modern MRI facilities or using special hardware extensions, its role as the primary limitation in partially parallel imaging will be reduced and other limiting factors will become dominant. Two such factors include available SNR and the variation of sensitivity distributions with imaging depth. Simulations have been conducted to evaluate the impact of slice depth and noise on partially parallel reconstructions for the case of a square linear array of overlapped elements that are parallel to the imaging plane. Results indicate that even when sensitivity distributions are exactly known, the linear surface array can only provide high accelerations over a limited imaging depth due to changing suitability of the sensitivity distributions for partially parallel reconstruction. This work emphasizes the importance of simulations for target-based partially parallel array design.