Antti Isomäki

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of- Concept Study

Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Investigation of protein distribution in solid lipid particles and its impact on protein release using coherent anti-Stokes Raman scattering microscopy.

The aim of this study was to gain new insights into protein distribution in solid lipid microparticles (SLMs) and subsequent release mechanisms using a novel label-free chemical imaging method, coherent anti-Stokes Raman scattering (CARS) microscopy. Lysozyme-loaded SLMs were prepared using different lipids with lysozyme incorporated either as an aqueous solution or as a solid powder. Lysozyme distribution in SLMs was investigated using CARS microscopy with supportive structural analysis using electron microscopy. The release of lysozyme from SLMs was investigated in a medium simulating the conditions in the human duodenum. Both preparation method and lipid excipient affected the lysozyme distribution and release from SLMs. Lysozyme resided in a hollow core within the SLMs when incorporated as an aqueous solution. In contrast, lysozyme incorporated as a solid was embedded in clusters in the solid lipid matrix, which required full lipolysis of the entire matrix to release lysozyme completely. Therefore, SLMs with lysozyme incorporated in an aqueous solution released lysozyme much faster than with lysozyme incorporated as a solid. In conclusion, CARS microscopy was an efficient and non-destructive method for elucidating the distribution of lysozyme in SLMs. The interpretation of protein distribution and release during lipolysis enabled elucidation of protein release mechanisms. In future, CARS microscopy analysis could facilitate development of a wide range of protein-lipid matrices with tailor-made controlled release properties.

Multimodal non-linear optical imaging for the investigation of drug nano-/microcrystal-cell interactions.

Drug nano-/microcrystals are being used for sustained parenteral drug release, but safety and efficacy concerns persist as the knowledge of the in vivo fate of long-living particulates is limited. There is a need for techniques enabling the visualization of drug nano-/microcrystals in biological matrices. The aim of this work was to explore the potential of coherent anti-Stokes Raman scattering (CARS) microscopy, supported by other non-linear optical methods, as an emerging tool for the investigation of cellular and tissue interactions of unlabeled and non-fluorescent nano-/microcrystals. Raman and CARS spectra of the prodrug paliperidone palmitate (PP), paliperidone (PAL) and several suspension stabilizers were recorded. PP nano-/microcrystals were incubated with RAW 264.7 macrophages in vitro and their cellular disposition was investigated using a fully-integrated multimodal non-linear optical imaging platform. Suitable anti-Stokes shifts (CH stretching) were identified for selective CARS imaging. CARS microscopy was successfully applied for the selective three-dimensional, non-perturbative and real-time imaging of unlabeled PP nano-/microcrystals having dimensions larger than the optical lateral resolution of approximately 400nm, in relation to the cellular framework in cell cultures and ex vivo in histological sections. In conclusion, CARS microscopy enables the non-invasive and label-free imaging of (sub)micron-sized (pro-)drug crystals in complex biological matrices and could provide vital information on poorly understood nano-/microcrystal-cell interactions in future.

Elucidation of Compression-Induced Surface Crystallization in Amorphous Tablets Using Sum Frequency Generation (SFG) Microscopy

PurposeTo investigate the effect of compression on the crystallization behavior in amorphous tablets using sum frequency generation (SFG) microscopy imaging and more established analytical methods.MethodTablets containing neat amorphous griseofulvin with/without excipients (silica, hydroxypropyl methylcellulose acetate succinate (HPMCAS), microcrystalline cellulose (MCC) and polyethylene glycol (PEG)) were prepared. They were analyzed upon preparation and storage using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM) and SFG microscopy.ResultsCompression-induced crystallization occurred predominantly on the surface of the neat amorphous griseofulvin tablets, with minimal crystallinity being detected in the core of the tablets. The presence of various types of excipients was not able to mitigate the compression-induced surface crystallization of the amorphous griseofulvin tablets. However, the excipients affected the crystallization rate of amorphous griseofulvin in the core of the tablet upon compression and storage.ConclusionsSFG microscopy can be used in combination with ATR-FTIR spectroscopy and SEM to understand the crystallization behaviour of amorphous tablets upon compression and storage. When selecting excipients for amorphous formulations, it is important to consider the effect of the excipients on the physical stability of the amorphous formulations.

Insights into Caco-2 cell culture structure using coherent anti-Stokes Raman scattering (CARS) microscopy

We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results.

Multimodal Nonlinear Optical Imaging for Sensitive Detection of Multiple Pharmaceutical Solid-State Forms and Surface Transformations

Two nonlinear imaging modalities, coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG), were successfully combined for sensitive multimodal imaging of multiple solid-state forms and their changes on drug tablet surfaces. Two imaging approaches were used and compared: (i) hyperspectral CARS combined with principal component analysis (PCA) and SFG imaging and (ii) simultaneous narrowband CARS and SFG imaging. Three different solid-state forms of indomethacin-the crystalline gamma and alpha forms, as well as the amorphous form-were clearly distinguished using both approaches. Simultaneous narrowband CARS and SFG imaging was faster, but hyperspectral CARS and SFG imaging has the potential to be applied to a wider variety of more complex samples. These methodologies were further used to follow crystallization of indomethacin on tablet surfaces under two storage conditions: 30 °C/23% RH and 30 °C/75% RH. Imaging with (sub)micron resolution showed that the approach allowed detection of very early stage surface crystallization. The surfaces progressively crystallized to predominantly (but not exclusively) the gamma form at lower humidity and the alpha form at higher humidity. Overall, this study suggests that multimodal nonlinear imaging is a highly sensitive, solid-state (and chemically) specific, rapid, and versatile imaging technique for understanding and hence controlling (surface) solid-state forms and their complex changes in pharmaceuticals.

Label-free visualization of cholesteatoma in the mastoid and tympanic membrane using CARS microscopy☆

Objective The present study aimed to evaluate the possibility of using coherent anti-Stokes Raman spectroscopy (CARS) microscopy to determine the specific molecular morphology of cholesteatoma by detecting the natural vibrational contrast of the chemical bonds without any staining. Materials and methods Specimens from the mastoid and tympanic membrane with and without cholesteatoma were analyzed using CARS microscopy, two-photon excited fluorescence (TPEF) microscopy, and the second harmonic generation (SHG) microscopy. Results In cholesteatoma tissues from the mastoid, a strong resonant signal at 2845 cm−1 was observed by CARS, which indicated the detection of the CH2 hydro-carbon lipid bonds that do not generate visible signals at 2940 cm−1 suggestive of CH3 bonds in amino acids. A strong resonant signal at 2940 cm−1 appeared in an area of the same specimen, which also generated abundant signals by TPEF and SHG microscopy at 817 nm, which was suggestive of collagen. In the tympanic membrane specimen with cholesteatoma, a strong resonant signal with corrugated morphology was detected, which indicated the presence of lipids. A strong signal was detected in the tympanic membrane with chronic otitis media using TPEF/SHG at 817 nm, which indicated collagen enrichment. The CARS and TPEF/SHG images were in accordance with the histology results. Conclusion These results suggest the need to develop a novel CARS microendoscope that can be used in combination with TPEF/SHG to distinguish cholesteatoma from inflammatory tissues.

Label-Free Imaging of Adipogenesis by Coherent Anti-Stokes Raman Scattering Microscopy

Label-free imaging technologies to monitor the events associated with early, intermediate and late adipogenic differentiation in multipotent mesenchymal stromal cells (MSCs) offer an attractive and convenient alternative to conventional fixative based lipid dyes such as Oil Red O and Sudan Red, fluorescent labels such as LipidTOX, and more indirect methods such as qRT-PCR analyses of specific adipocyte differentiation markers such as peroxisome PPARγ and LPL. Coherent anti-Stokes Raman scattering (CARS) microscopy of live cells is a sensitive and fast imaging method enabling evaluation of the adipogenic differentiation with chemical specificity. CARS microscopy is based on imaging structures of interest by displaying the characteristic intrinsic vibrational contrast of chemical bonds. The method is nontoxic, non-destructive, and minimally invasive, thus presenting a promising method for longitudinal analyses of live cells and tissues. CARS provides a coherently emitted signal that is much stronger than the spontaneous Raman scattering. The anti-Stokes signal is blue shifted from the incident wavelength, thus reducing the non-vibrational background present in most biological materials. In this chapter, we aim to provide a detailed approach on how to induce adipogenic differentiation in MSC cultures, and present our methods related to label-free CARS imaging of the events associated with the adipogenesis.

Non-linear optical imaging – Introduction and pharmaceutical applications

Nonlinear optical imaging is an emerging technology with much potential in pharmaceutical analysis. The technique encompasses a range of optical phenomena, including coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and twophoton excited fluorescence (TPEF). The combined potential of these phenomena for pharmaceutical imaging includes chemical and solidstate specificity, high optical spatial and temporal resolution, nondestructive and non-contact analysis, no requirement for labels, and the compatibility with imaging in aqueous and biological environments. In this article, the theory and practical aspects of nonlinear imaging are briefly introduced and pharmaceutical and biopharmaceutical applications are considered. These include material and dosage form characterization, drug release, and drug and nanoparticle distribution in tissues and within live cells. The advantages and disadvantages of the technique in the context of these analyses are also discussed. Introduction to Nonlinear Optics Nonlinear optics deals with processes where two or more photons interact with the sample material simultaneously. This is only possible when a high enough number of photons is confined in a small volume, i.e. the intensity of the incident light has to be much higher when compared to linear effects (normal absorption, refractive index). In practice this is made possible using ultrashort laser pulses with durations from picoseconds to femtoseconds. The narrow temporal width limits the interaction time between laser pulses and the target medium. Therefore, they offer a way to precisely probe the medium with reduced risk of optically induced damage. Even more importantly, the energy confined within the narrow optical pulse gives rise to very high light intensities and, thus, ultrashort pulses constitute ideal means for provoking a variety of nonlinear optical phenomena. Since the signal is produced selectively at the focus where the intensity is highest, nonlinear methods offer intrinsic axial sectioning property wellsuited for three-dimensional imaging. In optical microscopy, the two most commonly exploited phenomena are TPEF and SHG. CARS microscopy is yet another emerging nonlinear optical technique which is suitable for imaging various biological and pharmaceutical samples. The energy level diagrams of these processes are illustrated in Fig. 1. Figure 1. Energy level diagrams of two-photon excited fluorescence (left), second harmonic generation (middle), and coherent anti-Stokes Raman scattering (right). Two-photon excitation occurs through the absorption of two photons via intermediate virtual energy levels (dashed line). In SHG, two photons are combined similarly, but the emission occurs directly from a short-lived virtual level without excitation to the higher electronic states. In the CARS process, two photons stimulate a selected vibrational mode. Probing of this level with the third excitation photon results in the CARS emission. In the TPEF process (Fig. 1, left) a fluorophore molecule is excited by the simultaneous absorption of two photons in the infrared spectral range. In the conventional one-photon fluorescence the same transition to the higher energy level requires more energetic photons in the ultraviolet or visible range. The same fluorophores can often be used with both techniques. However, due to the fundamentally different quantum-mechanical processes, the excitation efficiencies are typically not equivalent. The fluorescence emission spectra are essentially the same in both cases. In TPEF, the longer incident wavelength leads to improved depth penetration in tissues, with reduced potential for photolytic damage [1, 2]. This has led to its increasing popularity in the biomedical setting, and the technique is commonly referred to as multiphoton imaging (this is because it is the most established nonlinear optical imaging method, even though other nonlinear phenomena also involve multiple photons). SHG is a nonlinear process where two photons interact with the target medium so that the energy from the incident laser beam is transferred to a beam with precisely double the original frequency (half the wavelength). This process does not involve the absorption of the photons, but relies on so-called virtual energy levels (Fig. 1, middle). SHG can only occur in materials that exhibit non-centrosymmetric structure. In biology, one such example is collagen, and most pharmaceutical crystals are also examples (whereas the amorphous form is not). One benefit of the technique is that it is straightforward to spectrally separate the SHG signal from other emission sources such as autofluorescence. In CARS microscopy, the signal originates from vibrational motion of the molecules in the sample. The process is based on a nonlinear phenomenon called four-wave mixing. In this process, three beams are interacting with the target medium to produce the fourth at a different wavelength (Fig. 1, right). The frequency difference between two of the input beams is selected to match the frequency of a vibrational energy state of the target molecule. When this resonant condition is fulfilled, the stimulating beams enhance the selected vibrational mode (e.g. stretching of a C-H molecular bond). Using the third beam, this motion can be probed. The resulting CARS signal is generated at the blueshifted (antiStokes) spectral range when compared to the input beams. Stimulated Raman scattering (SRS) is another variant of coherent nonlinear processes used to selectively probe molecular vibrations. When the diff erence frequency between two input beams matches a vibrational resonance, a small fraction (about one millionth) of the incident energy is transferred from one beam to the other. In order to measure the subtle variation, one of the input beams is intensitymodulated at high frequency. This modulation is transferred to the other beam and can be measured using sensitive photodetection. The main benefit of SRS over CARS is the lack of non-resonant background. Like conventional Raman microscopy (based on spontaneous Raman scattering), CARS and SRS are mostly used for imaging different chemical components without the need for labels. The major advantage of CARS and SRS compared to conventional Raman microscopy is the much faster imaging speed (orders of magnitude). This allows video rate imaging of dynamic processes, not to mention dramatically increasing analytical throughput. On the other hand, the spontaneous Raman microscopy typically gives richer spectral information which facilitates analysis of complex mixtures. The development of coherent Raman imaging systems capable of rapidly collecting rich spectral information is gradually overcoming this limitation. As a result, it is entirely possible that Raman microscopy based on spontaneous Raman scattering will largely be replaced with coherent Raman imaging in the pharmaceutical setting in the coming years. Nonlinear optical microscopes capable of TPEF and SHG (multiphoton imaging microscopes) are relatively widespread. CARS and SRS microscopes, which are also capable of TPEF and SHG imaging, are technically more complex but capable of richer sample analysis, especially when multiple nonlinear phenomena are simultaneously used (multimodal imaging). To illustrate the value of nonlinear optical imaging in the pharmaceutical setting, various examples are presented below. These include imaging drugs and dosage forms during the lifecycle of the product, from manufacturing to their fate in the body. Only label-free imaging examples are presented. Imaging Drugs and Dosage Forms Nonlinear optical imaging is slowly gaining interest in the area of drug and formulation imaging (Table 1). Some of the earliest work focused on using CARS to image the composition of emulsions [3]. In this work, the authors utilized CARS microscopy to image based on the C-H stretch vibrational region (2800-3200 cm-1). Later work conducted by Day et al. [4] used oil-in-water emulsions and investigated lipid digestion with CARS microscopy. CARS microscopy allowed them to discriminate between undigested oil and lipolytic products without the need for labeling. Table 1. Some publications for nonlinear optical imaging of pharmaceutical formulations

Understanding dissolution and crystallization with imaging: a surface point of view

The tendency for crystallization during storage and administration is the most considerable hurdle for poorly water-soluble drugs formulated in the amorphous form. There is a need to better detect often subtle and complex surface crystallization phenomena and understand their influence on the critical quality attribute of dissolution. In this study, the interplay between surface crystallization of the amorphous form during storage and dissolution testing, and its influence on dissolution behavior, is analyzed for the first time with multimodal nonlinear optical imaging (coherent anti-Stokes Raman scattering (CARS) and sum frequency generation (SFG)). Complementary analyses are provided with scanning electron microscopy, X-ray diffraction and infrared and Raman spectroscopies. Amorphous indomethacin tablets were prepared and subjected to two different storage conditions (30 °C/23% RH and 30 °C/75% RH) for various durations and then dissolution testing using a channel flow-through device. Trace levels of surface crystallinity previously imaged with nonlinear optics after 1 or 2 days of storage did not significantly decrease dissolution and supersaturation compared to the freshly prepared amorphous tablets while more extensive crystallization after longer storage times did. Multimodal nonlinear optical imaging of the tablet surfaces after 15 min of dissolution revealed complex crystallization behavior that was affected by both storage condition and time, with up to four crystalline polymorphs simultaneously observed. In addition to the well-known α- and γ-forms, the less reported metastable ε- and η-forms were also observed, with the ε-form being widely observed in samples that had retained significant surface amorphousness during storage. This form was also prepared in the pure form and further characterized. Overall, this study demonstrates the potential value of nonlinear optical imaging, together with more established solid-state analysis methods, to understand complex surface crystallization behavior and its influence on drug dissolution during the development of amorphous drugs and dosage forms.