Ulf Maeder

In line monitoring of the preparation of water-in-oil-in-water (W/O/W) type multiple emulsions via dielectric spectroscopy

Multiple emulsions offer various applications in a wide range of fields such as pharmaceutical, cosmetics and food technology. Two features are known to yield a great influence on multiple emulsion quality and utility as encapsulation efficiency and prolonged stability. To achieve a prolonged stability, the production of the emulsions has to be observed and controlled, preferably in line. In line measurements provide available parameters in a short time frame without the need for the sample to be removed from the process stream, thereby enabling continuous process control. In this study, information about the physical state of multiple emulsions obtained from dielectric spectroscopy (DS) is evaluated for this purpose. Results from dielectric measurements performed in line during the production cycle are compared to theoretically expected results and to well established off line measurements. Thus, a first step to include the production of multiple emulsions into the process analytical technology (PAT) guidelines of the Food and Drug Administration (FDA) is achieved. DS proved to be beneficial in determining the crucial stopping criterion, which is essential in the production of multiple emulsions. The stopping of the process at a less-than-ideal point can severely lower the encapsulation efficiency and the stability, thereby lowering the quality of the emulsion. DS is also expected to provide further information about the multiple emulsion like encapsulation efficiency.

Development of a skin phantom of the epidermis and evaluation by using fluorescence techniques

The aim of this project was to develop a skin phantom that resembles the epidermis including the lipid matrix of the stratum corneum and the dermis. The main intent was to achieve optical properties similar to skin tissue. Therefore, two compartments of the skin, dermis and epidermis, were examined regarding their optical properties. Based on these results, the skin phantom was designed using relevant skin components. The scattering coefficient was measured by using Reflectance-based Confocal Microscopy (RCM) and the fluorescence spectrum was detected via confocal laser-scanning microscopy (CLSM). Prospective, the skin phantom can be used to incorporate various fluorescing chemicals, such as fluorescent dyes and fluorescent-labeled drugs to perform calibration measurements in wide-field and laser-scanning microscopes to provide a basis for the quantification of skin penetration studies.

Evaluation and quantification of spectral information in tissue by confocal microscopy

Abstract. A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Feasibility of Monte Carlo simulations in quantitative tissue imaging

PURPOSE The feasibility of Monte Carlo simulations as a tool to facilitate quantitative image analysis is investigated by means of simulating light transport in skin phantoms. METHODS A Monte Carlo tool is used to compare if simulated fluorescent signals show agreement with measured data. The lipophilic fluorescent probe Nile Red and dedicated skin phantoms are also used in simulations to investigate the influence of the optical properties of the skin on the signal. RESULTS It is shown that the simulated and measured fluorescence signals show linear behavior up to a certain concentration of Nile Red. The simulations of the skin phantoms show the varying influence of single skin layers on the fluorescence signal. A calibration factor for quantitative analysis can be determined for the different skin layers. CONCLUSION Characterizing the influence of different media on imaging signals is a primary task in developing quantitative analysis methods. Monte Carlo simulations are a useful tool to investigate imaging properties of biological specimen where quantifying signals is important. However, detailed models must be provided.

Categorization of two-photon microscopy images of human cartilage into states of osteoarthritis.

OBJECTIVE The degeneration of articular cartilage is part of the clinical syndrome of osteoarthritis (OA) and one of the most common causes of pain and disability in middle-aged and older people(1). However, the objective detection of an initial state of OA is still challenging. In order to categorize cartilage into states of OA, an algorithm is presented which offers objective categorization on the basis of two-photon laser-scanning microscopy (TPLSM) images. METHODS The algorithm is based on morphological characteristics of the images and results in a topographical visualization. This paper describes the algorithm and shows the result of a categorization of human cartilage samples. RESULTS The resulting map of the analysis of TPLSM images can be divided into areas which correspond to the grades of the Outerbridge-Categorization. The algorithm is able to differentiate the samples in coincidence with the macroscopic impression. CONCLUSION The method is promising for early OA detection and categorization. In order to achieve a higher benefit for the physician the method must be transferred to an endoscopic setup for an application in surgery.

Quantifying fluorescence signals in confocal image stacks deep in turbid media

When confocal depth stacks are taken, the collected signal (normally the fluorescence signal), decays dependent of the depth of the confocal slice in the turbid medium. This decay is caused by scattering and absorption of the exciting light and of the fluorescence light. As the attenuation parameters, i.e. scattering and absorption coefficients, are normally unknown when observing a new sample, a method is proposed to compensate for the attenuation of the involved light by correcting the fluorescence signal using the attenuation behavior of the sample measured directly on the spot where the fluorescence stack is taken. The method works without any a priori knowledge about the optical properties of the sample. Using this self-reference technique, a confocal fluorescence depth stack can be created where the signal intensity is not dependent on the scattering and absorption caused intensity decay. The proposed method is tested on fluorescent beads embedded in scattering and absorbing hydrogel phantoms.

Spectrally resolved visualization of fluorescent dyes permeating into skin

We present a spectrally resolved confocal imaging approach to qualitatively asses the overall uptake and the penetration depth of fluorescent dyes into biological tissue. We use a confocal microscope with a spectral resolution of 5 nm to measure porcine skin tissue after performing a Franz-Diffusion experiment with a submicron emulsion enriched with the fluorescent dye Nile Red. The evaluation uses linear unmixing of the dye and the tissue autofluorescence spectra. The results are combined with a manual segmentation of the skins epidermis and dermis layers to assess the penetration behavior additionally to the overall uptake. The diffusion experiments, performed for 3h and 24h, show a 3-fold increased dye uptake in the epidermis and dermis for the 24h samples. As the method is based on spectral information it does not face the problem of superimposed dye and tissue spectra and therefore is more precise compared to intensity based evaluation methods.

Fluorescent nanodiamonds as highly stable biomarker for endotoxin verification

Fluorescent nanodiamonds (ND) provide advantageous properties as a fluorescent biomarker for in vitro and in vivo studies. The maximum fluorescence occurs around 700 nm, they do not show photobleaching or blinking and seem to be nontoxic. After a pretreatment with strong acid fluorescent ND can be functionalized and coupled to endotoxin. Endotoxin is a decay product of bacteria and causes strong immune reactions. Therefore endotoxin has to be removed for most applications. An effective removal procedure is membrane filtration. The endotoxin, coupled to fluorescent ND can be visualized by using confocal microscopy which allows the investigation of the separation mechanisms of the filtration process within the membranes.

Studying skin penetration by NMR imaging

Skin penetration studies are an important part for the development of dermal drug carrier systems. As a novel approach a 7-tesla Magnetic Resonance Imaging (MRI) Scanner was used to obtain information about the penetration of agents into the skin. The main advantage of this method is, that the properties of the skin does not influence the signals. Compared to optical assessments the MRI method is not limited to imaging depth. Furthermore, it is possible to analyze fat and water components of the skin separately. The aim of this work was to evaluate, if this method is a promising analysis tool for the visualization of the transport of substances across the skin. Gadobutrol (Gadovist®1.0), respresenting a coventional contrast agent in MRI, was used as a model drug for the visualization of the skin penetration. These first promising results showed that Gadobutrol, incorporated in an oil-in-water emulsion, could be detected across the skin tissue compared to an aqueous solution. After 24 hours, the pixel intensity value was increased about 4-fold compared to an untreated tissue.

Calibration phantom for the quantification of fluorescent labels in deep skin tissue

A method to quantify fluorescent labels spatially resolved in scattering and absorbing samples is proposed and tested using a tissue phantom. The method works without any a priori knowledge about the optical properties of the sample. The scattering and absorption behavior of the sample is estimated by measuring reflectance from the sample simultaneously to the fluorescence. With this estimation, the attenuation of the fluorescence caused by scattering and absorption can be mathematically compensated. The method is planned to be used for evaluating skin penetrating drug carrier systems.