In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin

Optical, noninvasive methods have become efficient in vivo tools in dermatological diagnosis and research. From these promising imaging techniques, only the confocal scanning laser microscopy (CSLM) provides visualization of subsurface skin structures with resolutions similar to those of light microscopy. Skin annexes, as well as cutaneous cells from different epidermal layers, can be distinguished excellently. Currently, two forms of application have been established in dermatological practice: the reflectance mode, predominantly in the clinical field, and the fluorescence mode in dermatological research. Differences in both methods exist in the preparative protocol, in maximum imaging depth and, particularly, in the gain of contrast extraction. The reflectance mode demonstrates naturally occurring tissue components, whereas the fluorescent CSLM achieves contrast by administering fluorescence dye, representing the dynamic distribution pattern of the dye's fluorescent emission. Therefore, the reflectance and fluorescent modes highlight various skin microstructures, providing dissimilar in vivo confocal images of the skin. This permits different predications and information on the state of the tissue. We report the advantages and disadvantages of both optical imaging modes. The comparison was drawn by scanning human skin in vivo. Representative images in varying depths were obtained and analyzed; preparation procedures are shown and discussed.     

In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy

Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo.     

Pilot study of semiautomated localization of the dermal/epidermal junction in reflectance confocal microscopy images of skin

Reflectance confocal microscopy (RCM) continues to be translated toward the detection of skin cancers in vivo. Automated image analysis may help clinicians and accelerate clinical acceptance of RCM. For screening and diagnosis of cancer, the dermal/epidermal junction (DEJ), at which melanomas and basal cell carcinomas originate, is an important feature in skin. In RCM images, the DEJ is marked by optically subtle changes and features and is difficult to detect purely by visual examination. Challenges for automation of DEJ detection include heterogeneity of skin tissue, high inter-, intra-subject variability, and low optical contrast. To cope with these challenges, we propose a semiautomated hybrid sequence segmentation/classification algorithm that partitions z-stacks of tiles into homogeneous segments by fitting a model of skin layer dynamics and then classifies tile segments as epidermis, dermis, or transitional DEJ region using texture features. We evaluate two different training scenarios: 1. training and testing on portions of the same stack; 2. training on one labeled stack and testing on one from a different subject with similar skin type. Initial results demonstrate the detectability of the DEJ in both scenarios with epidermis/dermis misclassification rates smaller than 10% and average distance from the expert labeled boundaries around 8.5 μm.     

Wavelength-resolved measurements of fluorescence lifetime of indocyanine green

We study fluorescence lifetime of indocyanine green (ICG) using femtosecond laser and sensitive detection based on time-correlated single-photon counting. A time-resolved multichannel spectral system is constructed and applied for determination of the fluorescence lifetime of the ICG in different solvents. Emission properties of ICG in water, milk, and 1% intralipid solution are investigated. Fluorescence of the fluorophore of different concentrations (in a range of 1.7-160 μM) dissolved in different solutions is excited by femtosecond pulses generated with the use of Ti:Sa laser tuned within the range of 740-790 nm. It is observed that fluorescence lifetime of ICG in water is 0.166 ± 0.02 ns and does not depend on excitation and emission wavelengths. We also show that for the diffusely scattering solvents (milk and intralipid), the lifetime may depend on the dye concentration (especially for large concentrations of ICG). This effect should be taken into account when analyzing changes in the mean time of arrival of fluorescence photons excited in ICG dissolved in such optically turbid media.     

In vivo and ex vivo virtual biopsy of the liver with near-infrared, reflectance confocal microscopy.

The assessment of liver architecture is an essential part of the understanding of its physiology and pathology. Current fluorescence confocal microscopy methods face numerous drawbacks, such as cytotoxicity, quenching effect, potential negative ino- and chrono-tropic effects and leaking of fluorescent agents through the sinusoid fenestrations. The recently developed, near-infrared reflectance confocal microscopy allows high-resolution optical sectioning through intact tissues, without employing fluorescent stains, while contrast between structures is provided by the natural refractivity of the tissue. The aim of this study is to assess the utility of near-infrared reflectance confocal microscopy in the evaluation of the hepatic microscopic architecture in vivo and ex vivo. Rat livers were noninvasively examined in vivo and ex vivo with near-infrared reflectance confocal microscopy. Two experimental contrast agents were subsequently used to enhance particular structures. Parenchymal and vascular structures are readily identified, as well as some intracellular details. Differences between in vivo and ex vivo states were also observed. The use of contrast agents also highlights certain morphologic structures. In conclusion, near-infrared reflectance confocal microscopy stands as a useful adjunct technique to the study of hepatic parenchyma offering details equivalent to, if not surpassing traditional light microscopy.     

In vivo confocal microscopy analysis of enamel defects after orthodontic treatment: A preliminary study

After orthodontic treatment with fixed appliances, bonded brackets and residual adhesive must be removed. This procedure should lead to restitutio ad integrum of the enamel or, at least, restore the enamel surface as closely as possible to its pre-treatment conditions. The purpose of this study is the in vivo assessment at a microscopic resolution of enamel surfaces after bracket debonding while avoiding the tooth extraction. Nine orthodontic patients who had brackets removed at the conclusion of orthodontic treatment were enrolled. In vivo reflectance confocal microscopy imaging of dental enamel surface after debonding was performed for each patient. Eighteen upper incisors were analyzed, 10 in which the enamel demineralization appeared after the treatment and 8 in which the demineralization was present before the treatment. RCM analyses showed some speckled or roundish dark areas within the enamel. Moreover enamel alterations were detected at different levels of depth. The present in vivo microscopic study allowed for highlighting structural features in dental enamel, after debonding, at a microscopic resolution in real-time and in a non-invasive way, without the need for extraction or processing of the samples.     

In situ fluorescence imaging of organs through compact scanning head for confocal laser microscopy

We develop a compact scanning head for use in laser confocal fluorescence microscopy for in situ fluorescence imaging of organs. The head, cylindrical in shape, has 3.5 mm diameter and 30 mm length, and is thus small enough to operate in a living rat heart. The lateral and axial resolutions, defined as full widths at half maximum (FWHM) of a point spread function (PSF), measures 1.0 and 5.0 microm, respectively, for 488-nm excitation and 1.0 and 5.4 microm, respectively, for 543-nm excitation. The chromatic aberration between 488- and 543-nm laser beams is well suppressed. We perform Ca2+ imaging in cardiomyocytes through the right ventricular chamber of a perfused rat heart in line-scan mode with 2.9-ms time resolution. We also carried out two-color imaging of a fixed mouse heart and liver with subcellular resolution. The compact head of the microscope equipped with a line-scan imaging mode and two-color imaging mode is useful for in situ imaging in living organs with subcellular resolution and can advantageously be applied to in vivo research.     

In vivo confocal and multiphoton microendoscopy

The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.     

Automated identification of epidermal keratinocytes in reflectance confocal microscopy

Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf(?). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6±2.8 μm and reflectance gradient b = 3.6±2.1 μm at the nuclear∕cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf(?) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf(?) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.     

Yield of fluorescence from indocyanine green in plasma and flowing blood

The purpose of this work was to obtain more quantitative knowledge about the yield of fluorescence from retinal vessles during indocyanine green angiography (ICG). The yield of fluorescence from blood was investigated for various shear rates, concentrations of ICG, and layer thicknesses. Measurements were performedin vitro on samples of human blood in a cone-plate shear chamber using frontal illumination as in scanning laser angiography. In blood and in plasma, the yield of fluorescence of ICG increased with concentration up to 0.05 and 0.1 mg/ml, respectively. At higher concentrations, the yield decreased for all layer thicknesses. For increasing layer thicknesses, both in plasma and in blood, the yield of ICG fluorescence increased nonlinearly for concentrations higher than 0.012 mg/ml. Saturation occurred for layers thicker than 200 μm in combination with ICG concentrations of 0.4 mg/ml and higher. Application of shear rates within the physiological range of the microcirculation (88/sec and 528/sec) increased the yield of fluorescence from the blood sample compared with stasis. The high transparency of blood for the excitation and emission light of ICG that was demonstrated will lead to superposition of fluorescence from superficial and deeper layers. This superposition precludes quantitative indocyanine angiography of ocular vessels.     

Molecular contrast optical coherence tomography: A pump-probe scheme using indocyanine green as a contrast agent

The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (approximately 190 kJ/cm2) is then used to permanently photobleach the ICG molecules--resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, phi(B,cum), defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that phi(B,cum) decreases with fluence as well as with increasing dye concentration. The quantity phi(B,cum) is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.     

In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy.

In vivo reflectance confocal microscopy is a novel technique for the noninvasive study and diagnosis of the skin. The aim of this study was to describe and characterize the cytological and architectural aspects of cell clusters in melanocytic lesions observed by confocal microscopy, and to correlate them with routine histopathology. A total of 55 melanocytic lesions comprising 20 melanomas, 25 acquired nevi and 10 Spitz nevi were studied by means of reflectance confocal microscopy, dermoscopy and routine histopathology. Three different types of cell clusters at confocal microscopy observation (dense, sparse cell and cerebriform clusters) were identified and correlated with histopathology. Dense clusters appeared characteristic for benign lesions, although present in 13 out of 20 melanomas. Sparse cell clusters were more frequently observable in melanomas, but also sporadically present in one Spitz nevus. Moreover, cerebriform clusters were exclusively observed in five out of 20 melanomas. Confocal microscopy allowed the in vivo characterization of aspects of melanocytic nests and their exact correlation with histopathology.     

In vivo degradation of processed dermal sheep collagen evaluated with transmission electron microscopy.

The in vivo degradation of hexamethylenediisocyanate-tanned dermal sheep collagen was studied with transmission electron microscopy. Discs of hexamethylenediisocyanate-tanned dermal sheep collagen were subcutaneously implanted in rats. Both an intra- and an extracellular route of degradation could be distinguished. In addition to normal components of a typical foreign body reaction, remarkable phenomena, such as locally deviant neutrophil morphology, infiltration of basophil-like cells, indications of foreign body multinucleate giant cells formed from different cell types, aluminium silicate accumulations and calcium phosphate depositions, were observed. Foreign body multinucleate giant cells intracellularly degraded hexamethylenediisocyanate-tanned dermal sheep collagen after internalization. Both internalized and cellularly enveloped hexamethylenediisocyanate-tanned dermal sheep collagen degraded by the detachment of fibrils. Another extracellular route of degradation was characterized by calcium phosphate depositions in large bundles of hexamethylenediisocyanate-tanned dermal sheep collagen. From 6 wk, the hexamethylenediisocyanate-tanned dermal sheep collagen implant was replaced by rat connective tissue, which was subsequently also degraded. After 15 wk, the presence of basophil-like foreign body multinucleated giant cells containing aluminium/silicon-crystalline accumulations still persisted. These phenomena were related to the specific nature of the material used and suggest cytotoxicity. They emphasize the need for detailed evaluation at the ultrastructural level of newly developed biomaterials before they can be used for medical applications.     

Clinical applicability of in vivo fluorescence confocal microscopy for noninvasive diagnosis and therapeutic monitoring of nonmelanoma skin cancer

Excisional biopsies and routine histology remains the gold standard for the histomorphologic evaluation of normal and diseased skin. However, there is increasing interest in the development of noninvasive optical technologies for evaluation, diagnosis, and monitoring of skin disease in vivo. Fluorescent confocal microscopy is an innovative optical technology that has previously been used for morphologic evaluation of live human tissue. We evaluate the clinical applicability of a fluorescent confocal laser scanning microscope (FLSM) for a systematic evaluation of normal and diseased skin in vivo and in correlation with routine histology. A total of 40 patients were recruited to participate in the study. Skin sites of 10 participants with no prior history of skin disease served as controls and to evaluate topographic variations of normal skin in vivo. Thirty patients with a suspected diagnosis of nonmelanoma skin cancer were evaluated, whereby FLSM features of actinic keratoses (AK) and basal cell carcinoma (BCC) were recorded in an observational analysis. Selected BCCs were monitored for their skin response to topical therapy using Imiquimod as an immune-response modifier. A commercially available fluorescence microscope (OptiScan Ltd., Melbourne, Australia) was used to carry out all FLSM evaluations. Common FLSM features to AK and BCC included nuclear pleomorphism at the level of the granular and spinous layer and increased vascularity in the superficial dermal compartment. Even though the presence of superficial disruption and mere atypia of epidermal keratinocytes was more indicative of AK, the nesting of atypical basal cells, increased blood vessel tortuosity, and nuclear polarization were more typical for BCC. All diagnoses were confirmed by histology. FLSM allowed a monitoring of the local immune response following therapy with Imiquimod and demonstrated a continuous normalization of diseased skin on repeated evaluations over time. This study illustrates potential applications of FLSM in clinical dermatology for the evaluation of dynamic skin conditions and monitoring of cutaneous response to noninvasive therapies. The findings are of preliminary nature and warrant further investigations in the future.     

Structure of dental glass-ionomer cements by confocal fluorescence microscopy and stereomicroscopy

The microstructure of four cements, setting by different mechanisms (acid-base, dual cure, triple cure), was studied. The porosity of unpolymerized materials was detected by stereomicroscopy. After polymerization and storage in water or lactic acid solution, the porosity, filler distribution and gel layer, which was formed at the filler/matrix interface of polymerized materials, were examined by confocal laser microscopy. For this purpose, the specimens were treated with fluorescent dye solution before the test. The results showed that hydrolytic degradation (pH 7) mainly involved the resin matrix, and the acid erosion (pH 3.5) involved the gel layer too. As regards the filler, materials with different setting mechanism released the glass particles in different times. The loss of the filler particles occurred quicker in acid-base setting cements, and slower in triple-cured material.     

吲哚菁绿荧光成像技术在胆道外科的应用进展

吲哚菁绿（ICG）荧光成像技术在胆道外科的临床应用逐渐凸显其作用。ICG荧光胆道成像在腹腔镜胆囊切除术、腹腔镜胆总管探查再次手术和肝胆管结石、肝内胆管癌、肝门部胆管癌、胆囊癌等胆道外科疾病行肝切除术,以及淋巴结清扫手术、肝移植术和防治胆管损伤、胆漏等方面均具有积极作用。本文主要阐述ICG荧光成像在上述胆道外科的应用进展。