Ultrasound and magnetic resonance imaging for group stratification and treatment monitoring in the transgenic adenocarcinoma of the mouse prostate model.

BACKGROUND: The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a widely used genetically engineered spontaneous prostate cancer model. However, both the degree of malignancy and time of cancer onset vary. While most mice display slowly progressing cancer, a subgroup develops fast-growing poorly differentiated (PD) tumors, making the model challenging to use. We investigated the feasibility of using ultrasound (US) imaging to screen for PD tumors and compared the performances of US and magnetic resonance imaging (MRI) in providing reliable measurements of disease burden. METHODS: TRAMP mice (n = 74) were screened for PD tumors with US imaging and findings verified with MRI, or in two cases with gross pathology. PD tumor volume was estimated with US and MR imaging and the methods compared (n = 11). For non-PD mice, prostate volume was used as a marker for disease burden and estimated with US imaging, MRI, and histology (n = 11). The agreement between the measurements obtained by the various methods and the intraobserver variability (IOV) was assessed using Bland-Altman analysis. RESULTS: US screening showed 81% sensitivity, 91% specificity, 72% positive predictive value, and 91% negative predictive value. The smallest tumor detected by US screening was 14 mm3 and had a maximum diameter of 2.6 mm. MRI had the lowest IOV for both PD tumor and prostate volume estimation. US IOV was almost as low as MRI for PD tumor volumes but was considerably higher for prostate volumes. CONCLUSIONS: US imaging was found to be a good screening method for detecting PD tumors and estimating tumor volume in the TRAMP model. MRI had better repeatability than US, especially when estimating prostate volumes.

Labeling nanoparticles: Dye leakage and altered cellular uptake.

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP-dye retention is confirmed when no cellular fluorescence is detected at 4°C. Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing NP-based applications. © 2016 International Society for Advancement of Cytometry.

The effects of oil-in-water nanoemulsion polyethylene glycol surface density on intracellular stability, pharmacokinetics, and biodistribution in tumor bearing mice.

PURPOSE: Lipid-based nanoparticles are extensively studied for drug delivery. These nanoparticles are often surface-coated with polyethylene glycol (PEG) to improve their biodistribution. Until now, the effects of varying PEG surface density have been studied in a narrow and low range. Here, the effects of high and a broad range of PEG surface densities on the in vivo performance of lipid-based nanoparticles were studied. METHODS: Oil-in-water nanoemulsions were prepared with PEG surface densities of 5-50 mol%. Confocal microscopy was used to assess intracellular disintegration in vitro. In vivo pharmacokinetics and biodistribution in tumor bearing mice were studied using a small animal optical imager. RESULTS: PEG surface density did not affect intracellular nanoemulsion stability. Surprisingly, circulation half-lives decreased with increasing PEG surface density. A plausible explanation was that nanoemulsion with high (50 mol%) PEG surface density activated the complement in a whole blood assay, whereas nanoemulsion with low (5 mol%) PEG density did not. In vivo, nanoemulsion with low PEG surface density was mostly confined to the tumor and organs of the mononuclear phagocyte system, whereas nanoemulsion with high PEG density accumulated throughout the mouse. CONCLUSIONS: Optimal PEG surface density of lipid-based nanoparticles for tumor targeting was found to be below 10 mol%.

Optical investigation of osteoarthritic human cartilage (ICRS grade) by confocal Raman spectroscopy: a pilot study.

Biomolecular changes in the cartilage matrix during the early stage of osteoarthritis may be detected by Raman spectroscopy. The objective of this investigation was to determine vibrational spectral differences among different grades (grades I, II, and III) of osteoarthritis in human osteoarthritic cartilage, which was classified according to the International Cartilage Repair Society (ICRS) grading system. Degenerative articular cartilage samples were collected during total joint replacement surgery and were classified according to the ICRS grading system for osteoarthritis. Twelve cartilage sections (4 sections of each ICRS grades I, II, and III) were selected for Raman spectroscopic analysis. Safranin-O/Fast green was used for histological staining and assignment of the Osteoarthritis Research Society International (OARSI) grade. Multivariate principal component analysis (PCA) was used for data analysis. Spectral analysis indicates that the content of disordered coil collagen increases significantly during the early progression of osteoarthritis. However, the increase was not statistically significant during later stages of the disease. A decrease in the content of proteoglycan was observed only during advanced stages of osteoarthritis. Our investigation shows that Raman spectroscopy can classify the different stage of osteoarthritic cartilage and can provide details on biochemical changes. This proof-of-concept study encourages further investigation of fresh cartilage on a larger population using fiber-based miniaturized Raman probe for the development of in vivo Raman arthroscopy as a potential diagnostic tool for osteoarthritis.

Single cell confocal Raman spectroscopy of human osteoarthritic chondrocytes: a preliminary study.

A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA) especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS) grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain. Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

Sonoporation Using Nanoparticle-Loaded Microbubbles Increases Cellular Uptake of Nanoparticles Compared to Co-Incubation of Nanoparticles and Microbubbles.

Therapeutic agents can benefit from encapsulation in nanoparticles, due to improved pharmacokinetics and biodistribution, protection from degradation, increased cellular uptake and sustained release. Microbubbles in combination with ultrasound have been shown to improve the delivery of nanoparticles and drugs to tumors and across the blood-brain barrier. Here, we evaluate two different microbubbles for enhancing the delivery of polymeric nanoparticles to cells in vitro: a commercially available lipid microbubble (Sonazoid) and a microbubble with a shell composed of protein and nanoparticles. Various ultrasound parameters are applied and confocal microscopy is employed to image cellular uptake. Ultrasound enhanced cellular uptake depending on the pressure and duty cycle. The responsible mechanisms are probably sonoporation and sonoprinting, followed by uptake, and to a smaller degree enhanced endocytosis. The use of commercial Sonazoid microbubbles leads to significantly lower uptake than when using nanoparticle-loaded microbubbles, suggesting that proximity between cells, nanoparticles and microbubbles is important, and that mainly nanoparticles in the shell are taken up, rather than free nanoparticles in solution.

Effect of Acoustic Radiation Force on Displacement of Nanoparticles in Collagen Gels.

Penetration of nanoscale therapeutic agents into the extracellular matrix (ECM) of a tumor is a limiting factor for the sufficient delivery of drugs in tumors. Ultrasound (US) in combination with microbubbles causing cavitation is reported to improve delivery of nanoparticles (NPs) and drugs to tumors. Acoustic radiation force (ARF) could also enhance the penetration of NPs in tumor ECM. In this work, a collagen gel was used as a model for tumor ECM to study the effects of ARF on the penetration of NPs as well as the deformation of collagen gels applying different US parameters (varying pressure and duty cycle). The collagen gel was characterized, and the diffusion of water and NPs was measured. The penetration of NPs into the gel was measured by confocal laser scanning microscopy and numerical simulations were performed to determine the ARF and to estimate the penetration distance and extent of deformation. ARF had no effect on the penetration of NPs into the collagen gels for the US parameters and gel used, whereas a substantial deformation was observed. The width of the deformation on the collagen gel surface corresponded to the US beam. Comparing ARF caused by attenuation within the gel and Langevin pressure caused by reflection at the gel-water surface, ARF was the prevalent mechanism for the gel deformation. The experimental and theoretical results were consistent both with respect to the NP penetration and the gel deformation.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels.

Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC (p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations (p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay (p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis.

Effect of Acoustic Radiation Force on the Distribution of Nanoparticles in Solid Tumors.

Acoustic radiation force (ARF) might improve the distribution of nanoparticles (NPs) in tumors. To study this, tumors growing subcutaneously in mice were exposed to focused ultrasound (FUS) either 15 min or 4 h after the injection of NPs, to investigate the effect of ARF on the transport of NPs across the vessel wall and through the extracellular matrix. Quantitative analysis of confocal microscopy images from frozen tumor sections was performed to estimate the displacement of NPs from blood vessels. Using the same experimental exposure parameters, ARF was simulated and compared with the experimental data. Enhanced interstitial transport of NPs in tumor tissues was observed when FUS (10 MHz, acoustic power 234 W/cm2, 3.3% duty cycle) was given either 15 min or 4 h after NP administration. According to acoustic simulations, the FUS generated an ARF per unit volume of 2.0×106 N/m3. The displacement of NPs was larger when FUS was applied 4 h after NP injection compared with after 15 min. This study shows that ARF might contribute to a modest improved distribution of NPs into the tumor interstitium.

Acoustic Cluster Therapy (ACT®) enhances accumulation of polymeric micelles in the murine brain.

The restrictive nature of the blood-brain barrier (BBB) prevents efficient treatment of many brain diseases. Focused ultrasound in combination with microbubbles has shown to safely and transiently increase BBB permeability. Here, the potential of Acoustic Cluster Therapy (ACT®), a microbubble platform specifically engineered for theranostic purposes, to increase the permeability of the BBB and improve accumulation of IRDye® 800CW-PEG and core-crosslinked polymeric micelles (CCPM) in the murine brain, was studied. Contrast enhanced magnetic resonance imaging (MRI) showed increased BBB permeability in all animals after ACT®. Near infrared fluorescence (NIRF) images of excised brains 1 h post ACT® revealed an increased accumulation of the IRDye® 800CW-PEG (5.2-fold) and CCPM (3.7-fold) in ACT®-treated brains compared to control brains, which was retained up to 24 h post ACT®. Confocal laser scanning microscopy (CLSM) showed improved extravasation and penetration of CCPM into the brain parenchyma after ACT®. Histological examination of brain sections showed no treatment related tissue damage. This study demonstrated that ACT® increases the permeability of the BBB and enhances accumulation of macromolecules and clinically relevant nanoparticles to the brain, taking a principal step in enabling improved treatment of various brain diseases.

Intravital microscopy in window chambers: a unique tool to study tumor angiogenesis and delivery of nanoparticles.

Solid tumor growth is heavily dependant on angiogenesis. Tumor angiogenesis is the result of a complex interplay between tumor cells, endothelial cells, and other stromal cells. It has been found to be under strict control of a plethora of molecular factors that function as angiogenic up- and down-regulators; nevertheless, the identification of molecular and cellular players and their roles in angiogenesis is still ongoing. The microvasculature resulting from tumor angiogenesis lacks hierarchy and has a high permeability for macromolecules and nanoparticles, which offers significant potential for nanoparticulate tumor imaging and drug delivery platforms. However, improvements in the delivery to poorly vascularized regions and the distribution throughout the tumor interstitium are critical for nanoparticles to become more effective in the battle against cancer. A tool that has proven extremely valuable in both unraveling angiogenic pathways and characterizing in vivo nanoparticle behavior in solid tumors is intravital microscopy of tumors grown in window chamber preparations. In this review this technique is explained, several exciting examples illustrating its value in elucidating tumor angiogenesis are presented and the study of nanoparticle behavior in solid tumors using this approach is described. We conclude with a discussion of the potential value of intravital microscopy in window chambers in multimodality studies of tumor pathophysiology and nanoparticle dynamics.

Characterizing DNA condensation by structurally different chitosans of variable gene transfer efficacy.

Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.

Macromolecular diffusion in the extracellular matrix measured by fluorescence correlation spectroscopy.

Diffusion of therapeutic macromolecules through the extracellular matrix of tumor tissue is a crucial step in drug delivery. We use fluorescence correlation spectroscopy (FCS) to measure diffusion of IgG (150 kDa) and dextrans (155 kDa and 2 MDa) in solution, 5% gelatin hydrogel, and multicellular spheroids. Gel and spheroids are used as model systems for the extracellular matrix. The diffusion depends on the complexity of the environment, as well as on the size and structural shape of the diffusing molecules. The results based on one-photon FCS are in good agreement with diffusion coefficients obtained with two-photon fluorescence recovery after photobleaching (FRAP) using the same microscope (Zeiss LSM510 META/Confocor2). However, FCS reveals anomalous or multicomponent diffusion in gel and spheroids, which are not resolvable with FRAP. This study demonstrates that one-photon FCS can be used to study the extracellular transport of macromolecules in tumor tissue, and that FCS provides additional information about diffusion properties compared to FRAP.

Second-harmonic generation in collagen as a potential cancer diagnostic parameter.

The fibrillar collagen network in tumor and normal tissues is different due to remodeling of the extracellular matrix during the malignant process. Collagen type I fibers have the crystalline and noncentrosymmetric properties required for generating the second-harmonic signal. The content and structure of collagen were studied by imaging the second-harmonic generation (SHG) signal in frozen sections from three tumor tissues, osteosarcoma, breast carcinoma, and melanoma, and were compared with corresponding normal tissues, bone/femur, breast, and dermis/skin. The collagen density was measured as the percentage of pixels containing SHG signal in tissue images, and material parameters such as the second-order nonlinear optical susceptibility given by the d22 coefficient and an empirical anisotropy parameter were used to characterize the collagen structure. Generally, normal tissues had much more collagen than tumor tissues. In tumor tissues, a cap of collagen was seen at the periphery, and further into the tumors, the distribution of collagen was sparse and heterogeneous. The difference in structure was reflected in the two times higher d22 coefficient and lower anisotropy values in normal tissues compared with tumor tissues. Together, the differences in the collagen content, distribution, and structure show that collagen signature is a promising diagnostic marker.

Diffusion measured by fluorescence recovery after photobleaching based on multiphoton excitation laser scanning microscopy.

Fluorescence recovery after photobleaching (FRAP) is a widely used method to measure diffusion. The technique is normally based on one-photon excitation, which limits diffusion to two dimensions due to extended photobleaching in the axial direction. Multiphoton excitation, on the other hand, creates a well-defined focal volume. In the present work, FRAP based on a scanning laser beam and two-photon excitation is used to measure diffusion of macromolecules in solution and gels, as well as in the extracellular matrix in multicellular spheroids and tumor tissue in dorsal chambers. The bleaching profile is determined experimentally in immobilized gels, and for small scanning areas (approximately twice the lateral radius of the laser beam) a Gaussian bleaching distribution is found. In addition, the bleaching profile is determined theoretically based on the convolution of the Gaussian point spread function and a circular scanning area. The diffusion coefficient is determined by fitting a mathematical model based on a Gaussian laser beam profile to the experimental recovery curve. The diffusion coefficient decreases with increasing complexity of the sample matrix and increasing the amount of collagen in the gels. The potential of using two-photon laser scanning microscopes for noninvasive diffusion measurements in tissue is demonstrated.

Sonopermeation to improve drug delivery to tumors: from fundamental understanding to clinical translation.

INTRODUCTION: Ultrasound in combination with microbubbles can make cells and tissues more accessible for drugs, thereby achieving improved therapeutic outcomes. In this review, we introduce the term 'sonopermeation', covering mechanisms such as pore formation (traditional sonoporation), as well as the opening of intercellular junctions, stimulated endocytosis/transcytosis, improved blood vessel perfusion and changes in the (tumor) microenvironment. Sonopermeation has gained a lot of interest in recent years, especially for delivering drugs through the otherwise impermeable blood-brain barrier, but also to tumors. AREAS COVERED: In this review, we summarize various in vitro assays and in vivo setups that have been employed to unravel the fundamental mechanisms involved in ultrasound-enhanced drug delivery, as well as clinical trials that are ongoing in patients with brain, pancreatic, liver and breast cancer. We summarize the basic principles of sonopermeation, describe recent findings obtained in (pre-) clinical trials, and discuss future directions. EXPERT OPINION: We suggest that an improved mechanistic understanding, and microbubbles and ultrasound equipment specialized for drug delivery (and not for imaging) are key aspects to create more effective treatment regimens by sonopermeation. Real-time feedback and tools to predict therapeutic outcome and which tumors/patients will benefit from sonopermeation-based interventions will be important to promote clinical translation.

Multi-modal characterization of vasculature and nanoparticle accumulation in five tumor xenograft models.

Preclinical research has demonstrated that nanoparticles and macromolecules can accumulate in solid tumors due to the enhanced permeability and retention effect. However, drug loaded nanoparticles often fail to show increased efficacy in clinical trials. A better understanding of how tumor heterogeneity affects nanoparticle accumulation could help elucidate this discrepancy and help in patient selection for nanomedicine therapy. Here we studied five human tumor models with varying morphology and evaluated the accumulation of 100 nm polystyrene nanoparticles. Each tumor model was characterized in vivo using micro-computed tomography, contrast-enhanced ultrasound and diffusion-weighted and dynamic contrast-enhanced magnetic resonance imaging. Ex vivo, the tumors were sectioned for both fluorescence microscopy and histology. Nanoparticle uptake and distribution in the tumors were generally heterogeneous. Density of functional blood vessels measured by fluorescence microscopy correlated significantly (p = 0.0056) with nanoparticle accumulation and interestingly, inflow of microbubbles measured with ultrasound also showed a moderate but significant (p = 0.041) correlation with nanoparticle accumulation indicating that both amount of vessels and vessel morphology and perfusion predict nanoparticle accumulation. This indicates that blood vessel characterization using contrast-enhanced ultrasound imaging or other methods could be valuable for patient stratification for treatment with nanomedicines.

Quantification of the second-order nonlinear susceptibility of collagen I using a laser scanning microscope.

Characteristic changes in the organization of fibrillar collagen can potentially serve as an early diagnostic marker in various pathological processes. Tissue types containing collagen I can be probed by pulsed high-intensity laser radiation, thereby generating second harmonic light that provides information about the composition and structure at a microscopic level. A technique was developed to determine the essential second harmonic generation (SHG) parameters in a laser scanning microscope setup. A rat-tail tendon frozen section was rotated in the xy-plane with the pulsed laser light propagating along the z-axis. By analyzing the generated second harmonic light in the forward direction with parallel and crossed polarizer relative to the polarization of the excitation laser beam, the second-order nonlinear optical susceptibilities of the collagen fiber were determined. Systematic variations in SHG response between ordered and less ordered structures were recorded and evaluated. A 500 microm-thick z-cut lithiumniobate (LiNbO(3)) was used as reference. The method was applied on frozen sections of malignant melanoma and normal skin tissue. Significant differences were found in the values of d(22), indicating that this parameter has a potential role in differentiating between normal and pathological processes.