This PDF file contains the front matter associated with SPIE Proceedings Volume 8086, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The recent linkage between adaptive optics, a technique borrowed from astronomy and various imaging devices, has enabled to push forward their imaging capabilities by improving its contrast and resolution. A specific case is nonlinear microscopy (NLM) that, although it brings several inherent advantages (compared to linear fluorescence techniques) due to its nonlinear dependence on the excitation beam, its enhanced capabilities can be limited by the sample inhomogeneous structure. In this work, we demonstrate how these imaging capabilities can be enhanced by, employing adaptive optics in a two step correction process. Firstly, a closed-loop methodology aided by Shack-Hartman Wavefront sensing scheme is implemented for compensating the aberrations produced by the laser and the optical elements before the high numerical aperture microscope objective, resulting in a one-time calibration process. Then the residual aberrations are produced by the microscope objective and the sample. These are measured in a similar way as it is done in astronomy (employing a laser guide-star), using the two-photon excited fluorescence. The properties of this incoherent emission produced inside a test sample are compared to a genetically modified Caenorhabditis. elegans nematode expressing GFP showing that the emission of this protein (at 810nm) can be sensed efficiently with our WFS by modifying the exposure time. Therefore the recorded wavefront will capture the sample aberrations which are used to shape a deformable mirror in an open-loop configuration. This correction principle is demonstrated in a test sample by correcting aberrations in a "single-shot" resulting in a reduced sample exposure.

Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

In quantitative phase-based live cell imaging the intracellular refractive index represents an important parameter. On one hand decoupling of the refractive index and the cell thickness is required, e. g., for reliable investigations on the cell morphology. On the other hand the cell refractive index and its spatial distribution is related to the concentration of the intracellular content. We explored a method to determine the mean refractive index of the cytoplasm with digital holographic microscopy (DHM). Microspheres that have been incorporated by living cells are used as reference in quantitative DHM phase contrast images from which the refractive index is obtained by a two dimensional fitting procedure. As many cells show a phagocytic behavior the method may be used with a variety of different cell types. Furthermore, as no modification of the experimental setup is required, the principle prospects to be used with several existing quantitative phase contrast imaging techniques.

Quantitative Phase Imaging techniques such as DHM have emerged recently in life sciences and can be aimed at monitoring and quantifying non-invasively dynamic cellular processes modifying cell morphology and/or content. Concretely, the DHM phase signal depends on two cell parameters: cell thickness and integral refractive index. Consequently, due to its dual origin, the interpretation of the phase signal variations remain difficult. Since a net water flux across the cell membrane causes a variation of both parameters, the phase signal cannot be related directly to cellular RI or thickness variations, but must be understood as a coupled signal of these two parameters. We have developped a Dual-wavelength Digital Holographic Microscopy (DHM) setup to separately measure in a single shot fashion cellular thickness and integral RI of living cells. The method is based on the use of an absorbing dye that causes a high RI dispersion in the extracellular medium at the two recording wavelength. Consequently, the phase signals measured at the two wavelengths, differ significantly from each other. Practically, both cell RI and thickness can be univocally determined from the two phase measurements. Important biophysical parameters of living cells, including dry mass concentrations and water membrane permeability can be deduced.

Numerical managing and manipulation of complex wavefronts is performed to retrieve Differential Image Contrast (DIC) visualization starting from Digital Holographic (DH) recording. Dynamical Differential Holographic Image Contrast (DDHIC) is the name of the proposed technique. It dispenses from special optics and/or complex setup configurations with moveable components, as usually occurs in classical DIC. DDHIC presents as an appropriate method for investigating objects experiencing dynamic evolution during the measurement. In fact, it will be proved that it's useful for floating samples since it allows, from a single recording, to set a-posteriori the best conditions for DIC imaging. Dynamic representation of phase-contrast along all directions will be presented improving the specimen visualization. Furthermore, it is useful for making the proper choice of DIC key parameters as the amount of shear and the bias, with the aim to optimize the visualized phase-contrast imaging. Investigation is carried out on different biological samples.

We used human specimens of epithelial ovarian cancer (serous type) to test the feasibility of nonlinear imaging as complementary tools for ovarian cancer diagnosis. Classical hematoxylin-and-eosin stained sections were applied to combining two-photon excitation fluorescence (TPEF), second (SHG), and third (THG) harmonic microscopy within the same imaging platform. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with Hematoxylin & Eosin (H&E) stored for a very long time and that H&E staining enhanced the THG signal. We demonstrate using anisotropy and morphological measurements, that SHG and THG of stained optical sections allow reproducible identification of neoplastic features such as architectural alterations of collagen fibrils at different stages of the neoplastic transformation and cellular atypia. Taken together, these results suggest that, with our viable imaging system, we can qualitatively and quantitatively assess endogenous optical biomarkers of the ovarian tissue with SHG and THG microscopy. This imaging capability may prove to be highly valuable in aiding to determine structural changes at the cellular and tissue levels, which may contribute to the development of new diagnostic techniques.

In minimally destructive SHG biomedical imaging (high resolution optical slicing) is greatly desirable to extract the maximum of information from the light matter interaction. Here we develop a 3-D biophysical model and a methodology, which extracts molecular information below the experimental resolution limit. Firstly, it provides the pitch angle (SHG effective orientation) of the SHG source helix of the sample. This information is used to characterize and categorize the SHG sources among them. And secondly, it provides the degree of organization of the SHG source molecules. This can be used as a quantitative imaging biomarker able to characterize the degree of organization (homeostasis) of the sample. Here we applied the model in dried and hydrated wheat starch granules. Our results show that the SHG source molecule in starch is amylopectin. We also conclude that under hydration, the amylopectin molecules are further organized but they do not change structure. This organization is reflected to the width of the pitch angles pixels' histograms' distributions. The shorter the width is, the more organized the amylopectin molecules in starch are.

Nonlinear imaging takes advantage of the localized nature of the interaction to achieve high spatial resolution, optical sectioning, and deeper penetration in tissue. However, nonlinear contrast (other than fluorescence or harmonic generation) is generally difficult to measure because it is overwhelmed by the large background of detected illumination light. Especially challenging to measure is the nonlinear refractive index - accessing this quantity would allow the extension of widely employed phase microscopy methods to the nonlinear regime. We have developed a technique to suppress the background in these types of measurements by using femtosecond pulse shaping to encode nonlinear interactions in background-free regions of the frequency spectrum. Using this individual pulse shaping based technique we have been able to measure self-phase modulation (SPM) in highly scattering environments, such as biological tissue, with very modest power levels. Using our measurement technique we have demonstrated strong intrinsic SPM signatures of glutamate-induced neuronal activity in hippocampal brain slices. We have also extended this measurement method to cross-phase modulation, the two-color analogue to SPM. The two-color approach dramatically improves the measurement sensitivity by reducing undesired background and associated noise. We will describe the nonlinear phase contrast measurement technique and report on its application for imaging neuronal activity.

Adipose-derived stem cells (ADSCs) are adult stem cells isolated from lipoaspirates. They are a good candidate for autologuous cell therapy and tissue engineering. For these applications, label-free imaging could be critical to assess noninvasively the efficiency of stem cell (SC) differentiation. We report on the development and application of a multimodal microscope to monitor and quantify ADSC differentiation into osteoblasts and adipocytes.

Alignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector; in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a 32×32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system. We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM).

Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. We present high speed 2D and 3D optically sectioned OPM imaging of live cells using a high NA water immersion lens.

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology of biopsy samples. Oral lesions are often flat and difficult to visualize under white light illumination. Moreover, histopathology is timeconsuming and there is a need to develop minimally invasive optical biopsy techniques to complement current techniques. Confocal laser endomicroscopy holds promise for virtual biopsy in disease diagnosis. This technique enables fluorescence imaging of tissue structures at microscopic resolution. We have developed a prototype real-time 3- dimensional (3D) imaging system using a laser endomicroscope interfaced with embedded computing. A Field- Programmable Gate Array computing platform has been programmed to synchronize cross-sectional image grabbing and Z-depth scanning, as well as automate acquisition of confocal image stacks. A PC was used for real-time volume rendering of the confocal image stacks. We conducted pre-clinical and pilot clinical studies to image the murine and human oral cavity. High quality volume renderings of the confocal image stacks were generated using 3D texture slicing. Tissue morphology and 3D structures could be visualized. The results demonstrate the potential of the system for diagnostic imaging of the oral cavity. This paves the way toward real-time virtual biopsy of oral lesions, with the aim to achieve same-day diagnosis in a clinical setting.

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation, revealed the development during cell migration processes and showed that an LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.

Optical tweezers is a technique to trap and to manipulate micron sized objects under a microscope by radiation pressure force exerted by a laser beam. Optical tweezers has been utilized for single-molecular measurements of force exerted by molecular interactions and for cell palpation. To extend applications of optical tweezers we have developed a novel optical tweezers system combined with a pulse laser. We utilize a pulse laser (Q-switched Nd: YAG laser, wavelength of 1064 nm) to assist manipulations by conventional optical tweezers with a continuous wave (CW) laser. The pulse laser beam is introduced into the same optics for conventional optical tweezers. In principle, instantaneous radiation force is proportional to instantaneous power of laser beam. As a result, pulse laser beam generates strong instantaneous force on an object to be manipulated. If the radiation force becomes strong enough to get over an obstacle structure and/or to be released from adhesion, the object will be free from these difficulties. We investigate the effect of pulse laser assistance with changing pulse duration of the laser. We report optimum pulse duration of 100 ns to 200 ns deduced from motion analysis of a particle in a beam spot. Our goal is to realize in-vivo manipulation and operation of a cell. For this purpose we need to reduce light energy of pulse laser beam and to avoid laser induced breakdown caused by strong light field. So we have developed a pulse laser with 160-ns pulse duration and have confirmed that availability on manipulation of living cells.

The analysis of particle size and structure without any manipulation by exogenous markers is essential for investigating biological cells and tissues. For this purpose we built up a scattering microscope and already proofed that spectral resolved scattering microscopy is suitable to detect differences in sphere diameters of a few nanometers. Using an angular resolved scattering microscope permits to distinguish diameters of single polystyrene spheres with a standard deviation of less than 1 %. The setup consists of an inverse microscope with a reflected darkfield illumination that is realized by a collimated beam with a well-defined angle. A supercontinuum laser in combination with an acousto-optic tunable filter allows wavelength tuning of free choice. For validation we measured single polystyrene beads at different wavelengths and determined the diameters by correlating measurements of the scattered light with the theoretical angular distribution based on Mie theory. The average diameter of a single polystyrene bead was determined with a relative standard deviation of 0.65%.

The mean diameter and the standard deviation of diluted polystyrene beads are determined by a collimated transmission setup. A significant amount of these beads is analyzed separately by a spectral resolved scattering microscope. The mean diameters of both methods differ by 10 nm which is equivalent to a systematic error of 0.24 %.

Optical-based microscopy plays an important role in various scientific fields such as physics, chemistry and biology. Second harmonic generation (SHG) microscopy has become one of the indispensable tools for biomedical imaging for the last decade because the signal generated from SHG is sensitive to the objective structure and this amazing non-invasive method can also directly observe the objective without using extra fluorescent labels, especially for collagen molecules. As the most abundant protein in animals, collagen is responsible for a number of important structural and functional roles in vertebrates. For certain diseases, it has been shown that collagen fiber diameter has a significant variation and thus as a vital symptom for diagnosis. Moreover, collagen diameter is also a key parameter for fibrogenesis studying. Therefore, the determination of collagen fiber diameter is important for studying biophysical processes and identifying bioengineering applications. In this study, we investigated various collagen fibril thicknesses and the corresponding forward (FSHG) and backward (BSHG) second harmonic signal intensity variation. Our result exhibits that SHG intensity can quantify describe the relative collagen fibril thickness alteration, which also indicates the coherent effect difference between FSHG and BSHG. This approach demonstrates the capability of SHG imaging in providing collagen mechanical information and that may be applied in the evaluation of advancing collagen issues in vivo.

Lung efficiency as gas exchanger organ is based on the delicate balance of its associated mucosal immune system between inflammation and sterility. In this study, we developed a dynamic imaging protocol using confocal and twophoton excitation fluorescence (2PEF) on freshly harvested infected lungs. This modus operandi allowed the collection of important information about CX3CR1⁺ pulmonary cells. This major immune cell subset turned out to be distributed in an anisotropic way in the lungs: subpleural, parenchymal and bronchial CX3CR1⁺ cells have then been described. The way parenchymal CX3CR1⁺ cells react against LPS activation has been considered using Matlab software, demonstrating a dramatic increase of average cell speed. Then, interactions between Bacillus anthracis spores and CX3CR1⁺ dendritic cells have been investigated, providing not only evidences of CX3CR1⁺ cells involvement in pathogen uptake but also details about the capture mechanisms.

Due to low light scattering, bacteria are difficult to detect using lensless imaging systems. In order to detect individual bacteria, we report a method based on a thin wetting film imaging that produces a micro-lens effect on top of each bacterium when the sample dries up. The imaging using a high-end CMOS sensor is combined with an in-line holographic reconstruction to improve positive detection rate up to 95% with micron-sized beads at high density of ~10³ objects/mm². The system allows detecting from single bacterium to densely packed objects (10³ bacteria/μl) within 10μl sample. As an example, E.coli, Bacillus subtilis and Bacillus thuringiensis, has been successfully detected with strong signal to noise ratio across a 24mm² field of view.

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 real tissue. 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.

In general, second-harmonic generation (SHG) microscopy is used to image highly ordered structures in biological samples, like starch, collagen, myosin and tubulin. In an effort to expand the possible targets for SHG microscopy, a number of new fluorescent probes with high performance in SHG imaging were designed and synthesized. The design is based on an electron-rich carbazole template, functionalized with pyridinium-like acceptors, resulting in cyanine-like dyes. In this paper, we report on the linear and nonlinear optical characterization of one of these dyes and its applicability in microscopy using two-photon excited fluorescence (2PEF) and SHG to visualize the specificity of the dyes in HeLa cells.

We present chromatic confocal microscopy as a technique to axially scan the sample by spectrally encoding depth information to avoid mechanical scanning of the lens or sample. We have achieved an 800 μm focal shift over a range of 680-1080 nm using a hyperchromat lens as the imaging lens. A more complex system that incorporates a water immersion objective to improve axial resolution was built and tested. We determined that increasing objective magnification decreases chromatic shift while improving axial resolution. Furthermore, collimating after the hyperchromat at longer wavelengths yields an increase in focal shift.

In microscopy, several different phase contrast methods (e.g. Zernike contrast, differential interference contrast) have been developed to image phase objects. For these methods specialized microscopic equipment (modified microscope objectives, filters, etc.) is needed. The static elements within such microscopes are a trade-off, because phase contrast imaging depends strongly on the object and the information to be visualized. We show results of an implementation of many different phase contrast methods using a high resolution phase-only spatial light modulator (SLM) in the pupil plane. All implemented methods are realized by software. Therefore, it is not only feasible to change the different phase contrast methods in real time, but it is also possible to optimize the parameters. Images obtained from different settings are combined digitally to improve the final image quality. Furthermore, completely new phase contrast filters can be tested easily because the phase of each pixel can be changed arbitrarily. We use this method to implement a new phase contrast filter that is obtained by combining a focused and a defocused point spread function. We will present theoretical as well as experimental results of this vertical differential interference contrast filter.

We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4 pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids. A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends on the composition of the membrane and the chemical structure of the peptide. The peptide we studied is the beta amyloid A_β which is a major component of the amyloid plaques deposited on neuronal membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides. In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4] respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures. The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (A_β) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.

A novel digital scanning microscope (DSM) for observing cellular fluorescent micro-images is proposed and manufactured in this study. DSM applied in the biomedical field has been designed based on a concept of fast access time of an optical pick-up head (PUH) in optical disc devices; hence, DSM has been developed based on a blue-ray PUH module with a triaxial scanning actuator (TSA) system. High-resolution and high-speed scanning is effectively realized by TSA system instead of utilizing high-precision transpose stage mechanism. In consequent, a PUH module can work with a time-correlated single photon counting (TCSPC) module and serve as DSM for detecting fluorescent signals on samples.

The aim of this work is to obtain improved sectioning ability in our slit-scanning microscope. The experimental setup is based on a linear sensor and different illumination modes were evaluated. MTF measurements from USAF target images showed the great benefit of using slit illumination in comparison to widefield. Experimental determination of Strehl ratios of 0.62 and 0.96 for wide-field and slit-illumination configurations, respectively, is depicted. Also MTF degradation with defocus is fairly established as stated by Strehl ratio decrease from 0.68 to 0.59 in 2 μm defocus using widefield configuration. Experimental measurement of axial response showed good accordance to numerically simulated curves modeled for the same parameters as the experimental setup. Linear sensor microscopy shows its advantage in comparison to simple slit microscopy particularly for slit illumination. Imaging for details running in sensor direction is accomplished for parallel illumination and detection slits as well as effective axial response for right-angled slits. These results indicate that linear sensor microscopy should be able to surpass lateral resolution asymmetry of slit microscopy. Preliminary results from tests to develop a reconstruction method that combine algorithms to improve lateral resolution isotropy of 2D images and those to build 3D images will be presented.

Device for registration of bacterial cells it is possible to examine as an information task which is characterized by a priori vagueness of moment of appearance of prototype system in the area of their registration. These reasons do not allow to provide exactness measuring on the base of measuring charts, functioning of which is provided structurally, and require the program hardware methods of measuring. An effective decision of this task is possible subject to condition application of the adaptive, program-driven methods, realized on the base of the fast-acting specialized charts of high integration. A new device for registration of bacterial cells in liquid medium after the changes of them size distributing is described. Procedure of registration of bacterial cells consists in dissociating of signal from noises, amplitude and duration measurement of the proper electric impulses. The process of registration particles in real-time imaging the screening on monitor of the personal computer. The developed software allows estimating the size distributing of the explored objects and their concentration in a liquid medium in 3D-imaging. There possibilities of device from registration and visualization of bacterial cells of different nature are presented.

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.