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.
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 skin's 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.
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.
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.
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
A 4D confocal microscopy (xyzλ) method for measuring the drug distribution in skin samples after a permeation study is
investigated. This approach can be applied to compare different drug carrier systems in pharmaceutical research studies.
For the development of this detection scheme phantom permeation studies and preliminary skin measurements are
carried out. The phantom studies are used to detect the permeation depth and the localization of the external applied
fluorescent dye naphthofluorescein that is used as a model agent. The skin study shows the feasibility of the method for
For the differentiation of tissue/phantom and the dye, spectral unmixing is performed using the spectral information
detected by a confocal microscope. The results show that it is possible to identify and localize external dyes in the
phantoms as well as in the skin samples.
Cryogenic procedures are fundamental tools in modern biology, e.g. for conservation or purification of biological
materials. The processes occurring in biological cells and tissues during freezing and thawing are subject to ongoing
research. Optimization of cell survival rates demands the development and evaluation of exactly defined temperature
profiles. 4D-DMD-microscopy is capable of imaging these highly dynamic processes with high spatial and temporal
resolution, utilizing well established staining procedures for differentiating structures of interest.
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.
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
We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast
and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through
a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets,
resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8x8 multi
anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each
column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two
scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly
shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral
information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and
orange peel cells.