Milind Rajadhyaksha

Video-rate confocal scanning laser microscope for imaging human tissues in vivo

We have built a video-rate confocal scanning laser microscope for reflectance imaging of human skin and oral mucosa in vivo. Design and imaging parameters were determined for optimum resolution and contrast. Mechanical skin-holding fixtures and oral tissue clamps were made for stable objective lens-to-tissue contact such that gross tissue motion relative to the microscope was minimized. Confocal imaging was possible to maximum depths of 350 microm in human skin and 450 microm in oral mucosa, with measured lateral resolution of 0.5-1 microm and axial resolution (section thickness) of 3-5 microm at the 1064-nm wavelength. This resolution is comparable with that of conventional microscopy of excised biopsies (histology). Normal and abnormal tissue morphology and dynamic processes were observed.

Allergic contact dermatitis: Correlation of in vivo confocal imaging to routine histology

BACKGROUND Allergic contact dermatitis (ACD) is a common and often challenging clinical problem. In vivo near-infrared confocal reflectance microscopy (CM) is a new vital microscopy technique. OBJECTIVE CM was used to evaluate acute ACD. METHODS Patch testing by means of Finn Chambers technique was performed in 5 subjects to induce an acute allergic skin reaction. Noninvasive CM images from normal and eczematous skin were sequentially recorded before and after removal of the Finn Chambers. RESULTS The epidermis and papillary dermis were clearly seen in high resolution. Retention of nuclei in stratum corneum, epidermal edema with microvesicle formation, and transepidermal migration of inflammatory cells were observed in vivo. Isolated dendritic cells were present in the ACD sites of 2 subjects, with morphology, size, and location consistent with Langerhans cells. Dermal vasodilation was observed as well. CONCLUSION CM is a useful tool to study ACD and may be able to track Langerhans cell activation.

Noninvasive Imaging of Human Oral Mucosa in Vivo by Confocal Reflectance Microscopy

Objectives/Hypothesis: To study the microscopic anatomy of normal oral tissues in vivo using confocal reflectance microscopy (CRM). This novel and noninvasive imaging modality can define and characterize healthy oral mucosa and thus this work serves as the foundation for studying oral diseases in vivo.

Confocal reflectance imaging of folliculitis in vivo: correlation with routine histology.

Near‐infrared confocal reflectance microscopy (CM) is a high‐resolution non‐invasive imaging technique with promising future in dermatology. A pustular lesion from a 35‐year‐old male with a known history of folliculitis was non‐invasively viewed with CM and later biopsied. Optical sections were correlated with routine histology, This optical technique allows us to view non‐invasively transverse skin sections to a conirolled depth in real time. In the CM images, tissue can be visualized with cellular and subeellular detail as shown by imaging infiltrating neutrophils (PMNs) within the subcorneal pustule of a superficial folliculitis in vivo.

Confocal imaging of sebaceous gland hyperplasia in vivo to assess efficacy and mechanism of pulsed dye laser treatment

This case demonstrates, for the first time, the use of in vivo confocal imaging to assess the efficacy of laser treatment of a skin lesion with a vascular component.

IN VIVO CONFOCAL MICROSCOPY IN DERMATOLOGY

Confocal microscopy is an optical imaging tool that allows for high resolution, noninvasive imaging in vivo. Thin sections of human tissue can be imaged allowing visualization of cellular and nuclear detail without biopsy. This technique recently has been used to image benign and malignant pigmented skin lesions, nonmelanoma skin cancer, inflammatory skin conditions, and dynamic skin processes.

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.

In vivo fluorescence imaging of the transport of charged chlorin e6 conjugates in a rat orthotopic prostate tumour

SummaryPolymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorin e6 conjugates (derived from polymers of average Mr 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454–528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the larger Mr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates.

In vivo abnormal keratinization in Darier-White's disease as viewed by real-time confocal imaging

Darier‐Whites disease is a rare autosomal‐dominant disorder of keratinization. The underlying pathology for the clinical presentation is acantholysis, and various types of dyskeralosis and acanthosis. In this study, we utilized a non‐invasive optical imaging modality, confocal reflectance microscopy, to identify specific histologic features of Darier‐Whites disease in vivo. Micro graphic findings in the confocal images were corps ronds, suprabasal clefts, acantholytic suprabasal keratinocytes, and villi. Real‐time confocal images are illustrative and can be well correlated with known light microscopic phenomena, particularly in the case of keratinization abnormalities in Darier‐Whites disease.

Near-infrared confocal laser scanning microscopy of bladder tissue in vivo

OBJECTIVES To assess the potential of a near-infrared confocal laser scanning microscope (CLSM) for imaging bladder tissue in vivo. METHODS Confocal images of the exposed bladder of male Sprague-Dawley rats were obtained with a CLSM. To minimize tissue motion, the bladder was placed in light contact under an objective lens housing, and the top surface was lightly flattened with a coverslip. Images were obtained from the outer and inner layers of the bladder wall with a lateral resolution of 0.5 to 1 microm and an axial resolution (section thickness) of 3 to 5 microm. The confocal images were later correlated with routine histologic studies. RESULTS The CLSM allows imaging of the urothelium, the superficial and deep portions of the lamina propria, the muscularis propria, and the serosa of the bladder wall in vivo. Urothelial cells, collagen bundles and fibers, muscle, and circulating blood cells in capillaries and larger blood vessels are easily visualized. The confocal images correlated well with the histologic studies. CONCLUSIONS Confocal microscopy allows real-time, high-resolution, high-contrast imaging of cellular and structural morphologic features to a maximal depth of 300 microm within the bladder wall in vivo. Artifacts caused by tissue motion can be minimized with a bladder-objective lens contact housing.