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PubMedDate not supplied

High-speed addressable confocal microscopy for functional imaging of cellular activity.

Due to cellular complexity, studying fast signaling in neurons is often limited by: 1. the number of sites that can be simultaneously probed with conventional tools, such as patch pipettes, and 2. the recording speed of imaging tools, such as confocal or multiphoton microscopy. To overcome these spatiotemporal limitations, we develop an addressable confocal microscope that permits concurrent optical recordings from multiple user-selected sites of interest at high frame rates. Our system utilizes acousto-optic deflectors (AODs) for rapid positioning of a focused laser beam and a digital micromirror device (DMD) for addressable spatial filtering to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current system has an adjustable spatial resolution of approximately 0.5 to 1 microm. Furthermore, we show that recordings can be made at an aggregate frame rate of approximately 40 kHz. The system is capable of optical sectioning; this property is used to create 3-D reconstructions of fluorescently labeled test specimens and visualize neurons in brain slices. Additionally, we use the system to record intracellular calcium transients at several sites in hippocampal neurons using the fluorescent calcium indicator Oregon Green BAPTA-1.

Animals↗
PubMedDate not supplied

A novel skin penetration enhancer: evaluation by membrane diffusion and confocal microscopy.

PURPOSE: The aim of this study was to determine the in vitro transdermal efficacy of a Meyer Zall Laboratories (MZL) oil/water emulsion in two separate preparations containing the actives, coal tar and the non-steroidal anti-inflammatory drug, diclofenac sodium. METHOD: The release rate of the two active ingredients from MZL dermatological preparations, Exorex and Athru-Derm and four comparator products was determined using an enhancer cell system, whilst specific penetration characteristics of the MZL formulation were elucidated using confocal and electron microscopy. The latter properties were explored at both the organ level, using human skin, as well as at a cellular level using a melanoma cell line. RESULTS: While the in vitro release rates for all formulations was high, coal tar and diclofenac release from Exorex and Athru-Derm respectively was, at nearly all time intervals, significantly higher than from comparator products. Microscopy revealed the presence of spherical liposomal type structures in both the MZL lotion and a comparator gel. In the MZL lotion, the majority of these structures, referred to here as emzaloid particles, were in the order of magnitude of about 50 nm to 1 microm in diameter with a small minority exceeding these dimensions. After application of Athru-Derm to human skin, intact emzaloid particles of submicron dimensions were detected in the epidermis in association with the cell membranes. The affinity of the MZL lotion for cell membranes was further demonstrated with melanoma cells; in addition, the formulation was seen to penetrate even to the nucleus of viable cells. CONCLUSION: Overall the data suggest that the oil/water base in MZL formulations is a highly efficient transdermal vehicle able to transport a wide range of indication- specific actives to their site of action.

Administration, Topical↗
PubMed2000

Direct confocal microscopy studies of the bacterial colonization in vitro of a silver-coated heart valve sewing cuff.

The antimicrobial coating of prosthetic heart valve sewing cuffs has been considered a potentially effective method for preventing prosthetic valve endocarditis. Although traditional in vitro bacterial adherence studies are often useful as screening tools, they can be inadequate in examining the antiinfective efficacy of antimicrobial-coated devices. We conducted a pilot in vitro study to directly assess the antimicrobial activity of a silver-coated sewing cuff versus uncoated cuff using confocal scanning laser microscopy. Staphylococcus epidermidis adhered more to the surfaces of the silver-coated sewing cuff compared with the uncoated cuff. These pilot in vitro results cast a doubt on the antiinfective efficacy of silver-coated prosthetic heart valve sewing cuffs and suggest further assessment should be carried out using animal studies.

Anti-Bacterial Agents↗
PubMed2005

Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms.

When bacteria assume the biofilm mode of growth, they can tolerate levels of antimicrobial agents 10 to 1,000 times higher than the MICs of genetically equivalent planktonic bacteria. The properties of biofilms that give rise to antibiotic resistance are only partially understood. Inhibition of antibiotic penetration into the biofilm may play a role, but this has not been proven directly. In this report, penetration of the glycopeptide antibiotic vancomycin into viable Staphylococcus aureus biofilms was analyzed by confocal scanning laser microscopy using a fluorescently labeled derivative of the drug. We found that while vancomycin bound to free-floating bacteria in water within 5 min, it took more than 1 h to bind to cells within the deepest layers of a biofilm. These results indicate that the antibiotic is transported through the depth of the biofilm but that the rate is significantly reduced with respect to its transport through flowing water. This suggests that, whereas planktonic bacteria were rapidly exposed to a full bolus of vancomycin, the bacteria in the deeper layers of the biofilm were exposed to a gradually increasing dose of the drug due to its reduced rate of penetration. This gradual exposure may allow the biofilm bacteria to undergo stress-induced metabolic or transcriptional changes that increase resistance to the antibiotic. We also investigated the role of poly-N-acetylglucosamine, an important component of the S. aureus biofilm matrix, and found that its production was not involved in the observed decrease in the rate of vancomycin penetration.

Anti-Bacterial Agents↗
PubMed1995

Chondrocyte deformation and local tissue strain in articular cartilage: a confocal microscopy study.

It is well accepted that mechanical forces can modulate the metabolic activity of chondrocytes, although the specific mechanisms of mechanical signal transduction in articular cartilage are still unknown. One proposed pathway through which chondrocytes may perceive changes in their mechanical environment is directly through cellular deformation. An important step toward understanding the role of chondrocyte deformation in signal transduction is to determine the changes in the shape and volume of chondrocytes during applied compression of the tissue. Recently, a technique was developed for quantitative morphometry of viable chondrocytes within the extracellular matrix using three-dimensional confocal scanning laser microscopy. In the present study, this method was used to quantify changes in chondrocyte morphology and local tissue deformation in the surface, middle, and deep zones in explants of canine articular cartilage subjected to physiological levels of matrix deformation. The results indicated that at 15% surface-to-surface equilibrium strain in the tissue, a similar magnitude of local tissue strain occurs in the middle and deep zones. In the surface zone, local strains of 19% were observed, indicating that the compressive stiffness of the surface zone is significantly less than that of the middle and deep zones. With this degree of tissue deformation, significant decreases in cellular height of 26, 19, and 20% and in cell volume of 22, 16, and 17% were observed in the surface, middle, and deep zones, respectively. The deformation of chondrocytes in the surface zone was anisotropic, with significant lateral expansion occurring in the direction perpendicular to the local split-line pattern. When compression was removed, there was complete recovery of cellular morphology in all cases. These observations support the hypothesis that deformation of chondrocytes or a change in their volume may occur during in vivo joint loading and may have a role in the mechanical signal transduction pathway of articular cartilage.

Animals↗
PubMed2004

Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells.

In mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIII Delta B) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER--where it colocalizes with calnexin--is transported to the Golgi complex in vesicular-tubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.

Animals↗
PubMed2004

Assessment of three-dimensional biofilm models through direct comparison with confocal microscopy imaging.

The mathematical modeling of spatial biofilm formation that provides the capability to predict biofilm structure from first principles has been in development for the past six years. However, a direct and quantitative link between model predictions and the experimentally observed structure formation still remains to be established. This work assesses the capability of a state-of-the-art technique for three-dimensional (3D) modeling of biofilm structure, individual based modeling (IbM), to quantitatively describe the early development of a multispecies denitrifying biofilm. Model evaluation was carried out by comparison of predicted structure with that observed from two experimental datasets using confocal laser scanning microscopy (CLSM) monitoring of biofilm development in laboratory flowcells. Experimental conditions provided biofilm growth without substrate limitation, which was confirmed from substrate profiles computed by the model. 3D structures were compared quantitatively using a set of morphological parameters including the biovolume, filled-space profiles, substratum coverage, average thickness and normalized roughness. In spite of the different morphologies detectable in the two independent short-term experiments analyzed here, the model was capable of accurate fitting data from both experiments. Prediction of structure formation was precise, as expressed by the set of morphology parameters used.

Bacteria↗
PubMed1993

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy.

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

Animals↗
PubMed1999

Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy.

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.

Animals↗
PubMed2000

A vehicle for photodynamic therapy of skin cancer: influence of dimethylsulphoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin IX accumulation determined by confocal microscopy.

Topical application of 5-aminolevulinic acid (5-ALA) followed by light irradiation is a new concept of photodynamic therapy (PDT) of skin cancers. 5-ALA is a prodrug that can be converted by the heme biosynthetic pathway into protoporphyrin IX, an effective photosensitizer. In the present work we propose the enhancement of 5-ALA-induced protoporphyrin IX accumulation by dimethylsulphoxide (DMSO) and ethylenediamine-tetraacetic acid disodium salt (EDTA). The presence of 20% DMSO (w/w) in oil-in-water emulsions increased the in vitro permeation of 5-ALA through hairless mouse skin. In vivo studies demonstrated a significant increase in the amount of protoporphyrin IX extracted from healthy hairless mouse skin after 3 h treatment with an oil-in-water emulsion containing 10% 5-ALA (w/w), 3% EDTA (w/w) and 20% DMSO (w/w). By confocal scanning laser microscopy imaging, an observed increase in red fluorescence, at 476 nm excitation and emission detected longer than 590 nm, in skin that had received this treatment, was attributed to protoporphyrin IX accumulation. Although no effect of EDTA on short-term protoporphyrin IX accumulation in skin was detected, this chelator could protect 5-ALA from decomposition during prolonged topical administration. The results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 5-ALA to the skin in the topical PDT.

Aminolevulinic Acid↗
PubMedDate not supplied

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas.

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Animals↗
PubMed1999

Different microcirculatory and interstitial matrix patterns in idiopathic dilated cardiomyopathy and Chagas' disease: a three dimensional confocal microscopy study.

OBJECTIVE: To analyse the morphological aspects of the extracellular matrix and microcirculation to clarify whether chronic Chagas' cardiopathy (CCC) is an accurate model to study the pathogenesis of idiopathic dilated cardiomyopathy (IDCM). DESIGN: Thick histological myocardial sections were prepared to analyse collagen, and microcirculation was examined during confocal laser and light microscopy. SETTING: The specimens were prepared at the pathology service of the Heart Institute of São Paulo, Brazil. PATIENTS: Nine control hearts, eight IDCM hearts, and 10 CCC hearts were studied after necropsy. MAIN OUTCOME MEASURES: The number of collagen struts per 100x field, the area of fibrosis (%), and the diameters of arterioles and capillaries were measured in each heart to establish outcome. RESULTS: A smaller number (mean (SD)) of collagen struts was seen in the hearts in the IDCM group (9.1 (4.1)) than in the control (22.4 (3.2)) (p < 0.05) or CCC (15.7 (7.4)) (p > 0.05) groups. Fibrosis was greater in the CCC hearts (13.8 (10.5)%) than in the IDCM hearts (5.9 (6.6)%) (p > 0.05). Major increases in arteriole (65.4 (9.9) microm) and capillary (9.9 (1.7) microm) diameters were seen in the CCC hearts but not in the IDCM hearts (arteriole diameter 40.3 (7.9) microm; capillary diameter 7.9 (1.3) microm). CONCLUSIONS: Hearts demonstrating CCC and IDCM present different extracellular and microvessel alterations. This suggests that distinct pathogenic mechanisms are responsible for each condition and that CCC is not an effective model to study IDCM.

Adolescent↗
PubMed1998

Intracellular fluorescent probe concentrations by confocal microscopy.

A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.

3T3 Cells↗
PubMed2005

Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D.

BACKGROUND: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. METHODS: Nuclear DNA was stained in 14 microm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of x40 or x100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. RESULTS: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at x60 or x100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. CONCLUSIONS: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

Adrenal Glands↗
PubMed1995

Activity-related calcium dynamics in lamprey motoneurons as revealed by video-rate confocal microscopy.

In lamprey spinal cord, intracellular calcium ([Ca2+]i) plays a key role in mechanisms regulating neuronal activity in the segmental network for locomotion. In this report, measurements of [Ca2+]i with fluo-3 in various regions of motoneurons in the intact spinal cord were obtained on a high speed confocal microscope following electrical stimulation. Likewise, rhythmic calcium fluctuations within dendrites and axons were seen during "fictive swimming" and were directly correlated with electrical activity. Antidromic stimulation of motoneuron axons induced large calcium transients and revealed spatially restricted "hot spots," both of which required external calcium and were blocked by nickel, but not by known calcium channel antagonists. These results suggest that lamprey spinal cord axons may possess a pharmacologically novel class of calcium channel.

Aniline Compounds↗
PubMed1996

Single nuclear pores visualized by confocal microscopy and image processing.

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.

3T3 Cells↗
PubMed2004

Effects of repeated sunbed exposures on the human skin. In vivo measurements with confocal microscopy.

BACKGROUND: Ultraviolet (UV) lamps used in commercial sunbeds are usually defined as UVA sources. Although it is well accepted that sunbed exposure significantly increases melanin pigmentation, its capacity to induce epidermal thickening is discussed controversially. OBJECTIVES: The aim of this study was to assess non-invasively the effects of repeated sunbed exposures on epidermal thickness, cell size, and pigmentation by means of confocal laser-scanning microscopy (CLSM) in vivo. METHODS: Eight volunteers had sunbed exposures six times in a 3-week period (cumulative dose: 126 J/cm(2) UVA). During irradiation, a small site (2 cm x 2 cm) on the lateral aspect of the inner forearm was covered with a UV-opaque sheet (non-exposed site). CLSM was performed with the Vivascope (Lucid, Henrietta, NY, USA) 24 h after the last UVA exposure on non-exposed sites and UVA-exposed sites that were on the medial aspect of the inner forearm at a distance of 2 cm to the non-exposed measurement site. The following parameters were assessed: thickness of the horny layer (DSC), minimal thickness of the epidermis (E(min)), minimal thickness of the viable epidermis (VE(min)), cell size of the granular layer (A(gran)), and the epidermal melanin content (MI). Additionally, colorimetric measurements have been carried out on non-exposed and UVA-exposed sites. RESULTS: DSC of the UVA-exposed skin was significantly higher than the one of non-exposed sites (mean+/-SD: 15+/-2.9 microm vs. 12.8+/-3 microm). Although E(min) was significantly higher in UVA-exposed sites (mean+/-SD: 40.4+/-3.6 microm vs. 39+/-2.9 microm), a slight but not statistically significant (P>0.05) decrease of VE(min) was observed (25.5+/-2.1 microm vs. 26.2+/-2.4 microm). The median of cell size of the granular layer (A(gran)) significantly (P=0.008) differed between non-exposed (752.1 microm(2)) and UVA-exposed sites (600 microm(2)). MI was significantly (P=0.014) higher for the UVA-exposed skin (1.12 vs. 1.34). Accordingly, colorimetry revealed significantly (P< 0.01) lower skin brightness for UVA-exposed sites (L*=60.2+/-4.3) as compared with non-exposed sites (L*=63.4+/-3.9). CONCLUSIONS: Sunbed exposures seem to induce photoadaptation not only by skin pigmentation but also by epidermal thickening that is predominantly due to an increase in thickness of the horny layer. Moreover, our data indicate that UVA radiation has an influence on the cell size of the granular layer. CLSM is a promising tool for photobiological studies in vivo.

Adaptation, Physiological↗
PubMed2006

Axially resolved complete mueller matrix confocal microscopy.

We introduce a technique that is capable of obtaining complete polarization-sensitive three-dimensional images that could reveal unknown anatomical conditions of living tissue that possess polarization-dependent signatures. Previously, the 16 Mueller coefficients were measured independently only by use of two-dimensional imaging techniques. We also present the experimental combination of a depth-resolved confocal imaging system with a complete Mueller matrix polarimeter. To calibrate the system, a double-pass method had to be implemented. We also indicate, experimentally, that the confocal sectioning of the system has a degrading effect on axially resolved Mueller matrix measurements.

Algorithms↗